Molecularly Imprinted Polymers for the Analysis of Protein Phosphorylation and the Role of Htra2/Omi Protein in Parkinson's Disease

Molecularly Imprinted Polymers for the Analysis of Protein Phosphorylation and the Role of HtrA2/Omi Protein in Parkinson's Disease

by Jing Chen

Dissertation

Submitted to the Faculty of Chemistry and Biochemistry

In Candidacy for the Degree of

Doctor Rerum Naturalium (Dr. rer. nat)

Accomplished at Medizinisches Proteom-Center

Ruhr-Universität Bochum, Germany

03. 2015, Bochum

Statement in Lieu of Oath

I hereby declare that I have accomplished the thesis independently and did not submit to any other faculty or refer to more than the publications listed in the references. The digital figures contain only original data and no modification was added. There are altogether 5 identical copies of my dissertation.

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Jing Chen

Referee: Prof. Dr. Katrin Marcus

Co-referee: Dr. Dirk Wolters

Acknowledgement

I would like to express my deep and sincere gratitude to Prof. Dr. Katrin Marcus, director of the Medizinische Proteom-Center, for her friendly invitation to the working group, for the great opportunity working in the interesting research field, for her dedication in supervising of my project execution and her unconditional help at the end of my Ph.D.

I am very grateful to Dr. Dirk Wolters for his kind acceptance of attending and co- judging my dissertation.

I owe my sincere gratitude to Dr. Stefan Helling, for his outstanding mentoring to this work. His valuable advice is deciding. Hadn’t for his endeavor in discussing and clearing my confusion at all times, I wouldn’t have managed to accomplish the work.

I know Prof. Dr. Börje Sellergren, my collaboration partner at biomedical science in Malmö University, Sweden the longest. It was he who recommended me to work in MPC as a Ph.D candidate. I want to express my sincere gratitute to him for his constructive suggestions thoughout my Ph.D study. Dr. Sudhirkumar Shinde from Malmö University was very kind and helpful at work too. I want to thank him for the intensive discussion and his polymer material synthesis and characterization.

Prabal Subedi contributed a lot in my research results discussion and dissertation correction. Kathy Pfeiffer performed many Western blotting for me. I am deeply grateful that she helped me so much during the stressful thesis-writing phase. I also want to thank Thilo Lerari, Sara Galozzi, Katalin Barkovits, Sarah Plum, Markus- Hermann Koch, Maike Ahrens, Caroline May, Jale Stoutjesdijk, Kathrin Barlog and all other MPC colleagues for reliable and whole-hearted assistance in my research, all Marie Curie PEPMIP members, for the discussions at project meetings and conferences.

I thank Prof. Karl Mechtler from the Institute of Molecular Pathology (Vienna, Austria) and Mr. Ingo Feldmann from ISAS (Dortmund, Germany) for providing self- synthesized peptides, Rejko Krüger from Hertie-Institute for Clinical Brain Research at University Tuebingen for providing mouse brain and cell line sample material and David Just for his assistance raising cells in the cell culture.

III

In the end, I’d like to thank my beloved parents far away in China with all my heart. After years of striving in Germany I finally come this far. Without their support I couldn’t have made it. My boyfriend Judong Yang has been with me all these years by my side and supporting me so much from all aspects of life. Without him I am nowhere. I am truely grateful to have him experiencing those ups and downs in life together with me.

Content Table 1. Introduction ...... 1

1.1 Role of HtrA2/Omi in neurodegeneration and Parkinson's disease (PD) ...... 1

1.1.1 HtrA2/Omi protein ...... 1

1.1.2 HtrA2/Omi and its mutations found in PD patients ...... 3

1.1.3 Mouse model studies in relation to PD ...... 4

1.1.4 Cell stress model in relation to PD ...... 6

1.2 Protein phosphorylation and techniques for its analysis ...... 7

1.3 MIPs for phosphoanalysis ...... 10

1.4 Proteomics and applied MS techniques ...... 13

1.5 Aims ...... 19

2. Materials and Methods ...... 20

2.1 Materials ...... 20

2.1.1 Instruments, expendable items and chemicals ...... 20

2.1.2 Buffers and reagents ...... 24

2.1.3 Antibodies ...... 25

2.1.4 Abbriviation list ...... 25

2.2 Methods ...... 26

2.2.1 Samples (cells, mouse brains, CSF) ...... 26

2.2.2 Cell culture and cell stress experiment ...... 28

2.2.3 Sample preparation ...... 28

2.2.4 Protein and peptide concentration determination ...... 30

2.2.5 Western blots ...... 31

2.2.6 Phosphopeptide enrichment ...... 32

2.2.7 Instrumentation based analytical methods ...... 37

2.2.8 Label-free quantification with spectra counting ...... 40

2.2.9 Data processing, databank search and pathway analysis ...... 41

2.2.10 Phosphopeptide motif-x analysis ...... 42

3. Results ...... 44

3.1 Efficiency comparison of pY-MIP, TiO2 and anti-pY antibodies for tyrosine phosphorylated peptides enrichment ...... 45

3.1.1 Method development and optimization ...... 45

3.1.2 Comparison results of PETRA and EDMA pY-MIPs ...... 49

3.1.3 Comparison results of pY-MIP, TiO2 and anti-pY antibodies (standard peptides) ...... 50

1. Results of TiO2 SPE phosphopeptide enrichment ...... 50

2. Results of phosphopeptide enrichment via three anti-pY antibodies ...... 51

3. Deeper understanding of the conventional anti-pY antibodies ...... 53

4. Results of IP with combination of anti-pY antibodies ...... 55

3.1.4 Comparison results of pY-MIP, TiO2 and anti-pY antibodies (spiking experiment) ...... 55

1. Results of pY-MIP performance ...... 56

2. Results of TiO2 SPE performance ...... 56

3. Results of IP performance using 3 anti-pY antibodies ...... 57

3.2 Results of pS-MIP phosphopeptide enrichment from biological samples ...... 59

3.2.1 Template rebinding test ...... 59

3.2.2 Results of pS-MIP SPE sample loading and elution condition test ...... 61

3.2.3 Further probing for phosphopeptide recognition using pS-MIP ...... 63

3.2.4 Results of pS-MIP SPE targeting the single peptide spiked in mouse brain matrix ...... 64

3.2.5 Results of pS-MIP SPE application in human cell samples and comparison with TiO2 SPE ...... 70

3.2.6 Results of pS-MIP SPE application in clinical relevant samples ...... 74

3.3 Analysis of HtrA2/Omi model studies with novel methods in relation to PD ...... 76

3.3.1 Results from global proteomics analysis of transgenic mouse brain samples ...... 76

1. LC-MS/MS identified proteins with different ways of regulation ...... 76

2. G399S mutant HtrA2/Omi over-expression effect leading to 18 differential proteins in 6- month-old mouse brains, and 12 differential proteins in 12-month-old mouse brains...... 77

3. WT HtrA2/Omi effect leading to 8 proteins differential in 6-month-old mouse brains, and 8 differential proteins in 12-month-old mouse brains...... 79

4. Protein over-expression effect leading to 17 proteins regulated in mouse brains at 6 months old and 2 at 12 months old ...... 81

5. Age effect regulated proteins in 3 types of mouse brain samples ...... 84 VI

6. Validation of protein candidate ubiquitin-conjugating enzyme E2L3 (UBE2L3) identified by LC-MS/MS ...... 87

3.3.2 Results from global proteomics analysis of human HtrA2/Omi WT and mutation G399S transfected neuroblastoma SH-SY5Y cell lines...... 89

1. Stress induced SH-SY5Y cell apoptosis ...... 89

2. Staurospoine induced cell stress effect on the phophorylation state of HtrA2/Omi at serine 400 ...... 91

3. LC-MS/MS identified proteins with different ways of regulation in SH-SY5Y cell samples...... 92

4. G399S mutant HtrA2/Omi effect resulting in 95 proteins differential (protein list in the Attachment 3) ...... 93

5. WT HtrA2/Omi effect resulting in 72 proteins differential (protein list in the Attachment 4) ...... 93

6. Transfection effect resulting in 15 proteins differential (protein list in the Attachment 5)94

7. Stress effect for V, WT and G399S SH-SY5Y cell line ...... 94

8. Validation of protein candidate DNA replication licensing factor MCM4 identified by LC- MS/MS ...... 95

4. Discussion ...... 98

4.1 Development of novel imprinted polymer based phosphoenrichment techniques ...... 98

4.1.1 Efficiency of pY-MIP, TiO2, and anti-pY antibodies enrichment of phosphopeptides ...98

4.1.2 Efficiency of phosphopeptide enrichment using pS-MIP ...... 101

4.2 Global proteomics study for mouse brain and cell models ...... 105

4.2.1 Advantages and disadvantages in protein validation using Western blot ...... 105

4.2.2 Discussion on different effect cases based on the proteomics data in mouse model study ...... 106

4.2.3 Discussion on pathway analysis for regulated proteins in mouse brain study ...... 107

1. Networks and canonical pathways identified by proteins having G399S HtrA2/Omi over- expression effect ...... 108

2. Networks and canonical pathways identified by proteins having WT HtrA2/Omi over- expression and protein over-expression effect ...... 109

3. Networks and canonical pathways identified by proteins having age effect in NT, WT and G399S HtrA2/Omi over-expressing mouse brains ...... 110 VII

4. Short review of the validated protein in mouse brain proteomics study ...... 112

4.2.4 Discussion on pathway analysis for regulated proteins in cell model study ...... 114

4.2.5 Discussion on pathway analysis for common proteins in mouse and cell model studies ...... 116

4.2.6 Discussion on down regulation of DNA replication licensing factor 4 MCM4 ...... 118

4.2.7 Discussion on down-regulation of three key enzymes in purine nucleotide pathway . 119

1. PURA2—adenylosuccinate synthetase isozyme 2 ...... 119

2. AMPD2--AMP deaminase 2 ...... 120

3. ADSL--adenylosuccinate lyase ...... 121

4.2.8 Discussion on the role of phosphor-S400 HtrA2/Omi ...... 123

5. Conclusion ...... 125

6. References ...... 127

7. Attachment ...... 138

8. Curriculum Vitae ...... 173

VIII

Introduction______

1. Introduction

1.1 Role of HtrA2/Omi in neurodegeneration and Parkinson's disease (PD)

1.1.1 HtrA2/Omi protein

HtrA2/Omi is a mitochondrial serine protease with a great extent of homology to bacterial high temperature requirement A protease (HtrA) (Faccio et al. 2000). The human HtrA family is composed of four serine protease members HtrA1-4, which are characterized by the combined presence of a trypsin-like catalytic domain with at least one PDZ interaction domain. PDZ is abbreviated for three proteins — post synaptic density protein (PSD95), Drosophila disc large tumor suppressor (Dlg1) and zonula occludens-1 protein (zo-1) — which were first discovered to share the domain. Proteins with PDZ domains help interactions, either they bind to C-terminals or they dimerize with other PDZ domains, and assemble the proteins into large complex that can initiate signal transduction (Fanning et al. 1996). The main functions of the HtrA family members are key aspects of protein quality control and they combine the dual activities of molecular chaperons and proteases (Clausen 2002). HtrAs are indicated in important cellular processes including maintenance of the photosynthetic apparatus, proliferation, cell migration and cell fate. (Huesgen et al. 2009 and Chien et al.2009). The loss of mammalian HtrA activity is considered to be related to diseases such as cancer and age-related macular degeneration, Parkinson’s disease (PD) and Alzheimer’s diseases (Coleman et al. 2008, Walle et al. 2008, Grau et al. 2005). The gene is located on chromosome 2p13 and contains 458 amino acids (Gray et al. 2000) and the protein HtrA2 is expressed in human tissues and body fluids as indicated in the HtrA2/Omi expression map (Wilhelm et al. 2014).

Figure 1-1: The domain organization of human HtrA2 protein. MTS: mitochondrial targeting signal domain, TM: transmembrane domain, Proteolytic: trypsin-like domain, PDZ: 1

Introduction______

PDZ domain, AVPS: amino acids of the IAPs binding motif (numbers indicate the position of the given residue in the polypeptide chain), proposed by Yun et al. 2008.

Figure 1-2: HtrA2/Omi expression map of human tissue. Data from the “Proteomics DB” (Wilhelm et al. 2014). The expression degree in different body tissue is indicated here with color code from dark red (high expression in e.g. muscle) to grey (medium expression e.g. in the brain) and green (low expression in e.g. lung)

The human HtrA2/Omi is known to be a quality control factor in the mitochondrial intermembrane. HtrA2/Omi protein has a transmembrane domain (TM), a trypsin-like proteolytic domain, and a PDZ domain, located among amino acids 105-121, 182- 330 and 390-445, respectively. The IAP (inhibitor apoptosis protein) binding motif (AVPS) binds to IAPs and relieves its inhibitory effect on caspases (Faccio et al. 2000).

The IAP binding motif binds to the inhibitor apoptosis proteins therefore inhibits the caspase-inhibitory effects of the IAPs (Faccio et al. 2000). The PDZ and protease domain play important roles in HtrA2 activity regulation. The HtrA2 precursor protein with the transmembrane domain localized behind the N-terminal mitochondrial targeting signal domain is anchored in the inner membrane, while the proteolytic

Introduction______domain and the PDZ domain exposed into the intermembrane space undergoing proteolytic maturation (Martin et al. 2002). During maturation, the first 133 amino acids from N-terminus are cleaved thus exposing the inhibitor of apoptosis protein (IAP)-binding motif (Lin et al. 2011).

HtrA2/Omi has been found as an IAP-binding protein and is released into the cytoplasm from the mitochondrial intermembrane space during apoptosis and cleaves proteins that block apoptosis, such as X-linked inhibitor of apoptosis proteins (XIAP) (Clausen et al. 2011) and also mediating caspase-independent death through its serine protease activity (Martins et al. 2004). Mutations of either the N-terminal alanine or mature HtrA2/Omi or the catalytic serine residues could diminish the ability of HtrA2 therefore promoting cell death (Verhagen et al. 2002).

1.1.2 HtrA2/Omi and its mutations found in PD patients

Parkinson’s disease (PD) results primarily from the death of dopaminergic neurons in the substantia nigra. (Dauer, W & Prozdborski, S. 2003). Both mitochondrial dysfunction and ubiquitin-proteasome system damage have been proposed as possible mechanisms leading to dopaminergic neuronal degeneration (Lin and Beal 2006). There are at least nine genes identified as causing PD or affecting PD risk: α- synuclein, parkin, ubiquitin carboxy-terminal hydrolase L1, DJ-1, phosphotase and tensin homologue (PTEN)-induced kinase 1 (PINK1), leucine-rich-repeat kinase 2 (LRRK2), the nuclear receptor NURR1, HTRA2 and tau out of which the α-synuclein (PARK2), parkin, DJ-1, PINK1, LRRK2 and HTRA2 directly or indirectly involve mitochondria (Lin and Beal 2006). The HtrA2/Omi protein was found to interact with the Alzheimer's disease-associated amyloid beta Aß 1-42 in HEK 293 cells (Park et al. 2004). In mice, targeted deletion of HtrA2/Omi causes mitochondrial dysfunction involved in PINK1 pathway leading to a neurodegenerative disorder with Parkinsonian features (Plun-Favreau et al. 2007). PINK1 dependent phosphorylation of HtrA2/Omi might regulate its proteolytic activity, therefore increasing the cell resistance to mitochondrial stress. HtrA2 may multifunction in the central nervous system and be particularly relevant in PD neurodegeneration (Bogaerts et al. 2008).

Introduction______

HtrA2 mutations among patients with PD and controls were assessed in Taiwan and results show that Pro143Ala in HtrA2 contributes to PD by inducing hyperphosphorylation of HtrA2 protein in mitochondria (Lin et al. 2011). According to literature, functionally important phosphorylation sites are A141S and G399S in HtrA/Omi protein. The highly conserved mutation G399S found in sporadic Parkinson’s disease (PD) reportedly results in reduced serine protease activity of HtrA2/Omi (Yun et al. 2008, Strauss et al. 2005). Moreover, the mitochondrial serine protease HtrA2/Omi mutated on G399S was identified in a Turkish kindred with essential tremor and PD (Gulsuner et al. 2014). Another mutation A141S polymorphism was identified and considered as associated with PD (Strass et al. 2005). HEK 293 and SH-SY5Y cells overexpressing S399 mutant (G399S mutant) of HtrA2/Omi were more sensitive to stress-induced cell death than wild-type (Strauss et al. 2005). A141S and G339S mutations localize to the N-terminal portion of the mature form of HtrA2/Omi and the PDZ domain respectively. Another HtrA2/Omi mutation was identified in PD patients as Arg404Trp within the PDZ domain and was predicted to freeze HtrA2 in an inactive form (Bogaerts et al. 2008).

1.1.3 Mouse model studies in relation to PD

HtrA2/Omi has been implicated involved in neurodegenerative disorders such as Parkinson’s and Alzheimer’s disease (Jones et al. 2003, Park et al. 2004) because of its neuroprotective role inside mitochondria. The HtrA2 came into focus in relation to PD, when mnd2 (motor neuron degeneration 2) mutation found in mouse was dicovered as a homozygous missense mutation (Ser276Cys) in the protease domain of the nuclear-encoded HtrA2 (Yacobi-Sharon et al. 2013). The mnd2 mutant mouse is considered to be an animal model of PD and both the features of necrosis and apoptosis were observed in these mice. Adding to this mouse model study, a previous research observed that homozygous deletion of HtrA2 gene has led to similar phenotype with early death due to loss of striatal neurons similar as mnd2 mice. These HtrA2/Omi knockout mice died of a parkinsonian phenotype syndromes within 30 days (Martins et al. 2004). The studies in mnd2 and HtrA2 knockout mice provided evidence relating HtrA2 loss-of-function to early onset neurodegeneration. Nevertheless, these results couldn't distinguish whether the cause of neurodegeneration in mice was due to the loss of HtrA2 activity in neurons

Introduction______themselves or other tissue types (Kang et al. 2013). To answer this question, Kang et al. introduced a HtrA2 crossed mnd2 mice—a mnd2 mouse line with expression of HtrA2 in neuron cells, to investigate the rescue potential from the neurodegenerative phenotype by re-introducing WT HtrA2/Omi in mnd2 mice and they could demonstrate that transgenic expression of human HtrA2/Omi in the central nervous system of mnd2 mice rescues them from neurodegeneration and prevents premature death (Kang et al. 2013) and adult transgenic mnd2 mice were reported to develop aging phenotypes, such as weight loss, hair loss, reduced fertility, curvature of the spine and death by 12-17 months of age. This study proved the neuroprotective role of HtrA2/Omi and revealed the role of HtrA2/Omi in aging.

In cooperating with Functional Neurogenomics Laboratory, Hertie-Institute for Clinical Brain Research, Eberhard Karls Universität Tuebingen, 35 mouse brain samples (Chapter 2.2.2) were analyzed in this work to try to reveal the WT HtrA2/Omi and its G399S mutant overexpressing effect caused cellular proteome change in mouse brains and try to further understand whether overexpression of WT HtrA2/Omi or G399S mutant has a toxic effect leading to cell death.

In the mouse model study, Tuebingen generated mouse models overexpressing the WT HtrA2/Omi and its G399S mutant transgenes in all regions of mouse brain which could more closely reflect the pathological pattern observed in PD and maybe more advantegous compared to models based on very restricted expression such as the research conducted by Kang et al. 2013 (Poonam dissertation, Tübingen). One previous research (Liu et al. 2007) was performed on examining the biological relevance of WT HtrA2/Omi overexpression in mice using the phenomenolotical data (organ weights, hair phenotype or pregnancy abnormalities) but found no malfunction. In our study, the overexpression of WT HtrA2/Omi will be explained further in proteome alteration for more insights. It has been shown that G399S mutant HtrA2 displays a reduced serine protease activity in vitro following activating stimuli in favor of a dominant negative effect of the G399S mutation in relation to PD (Strauss et al., 2005). It was also proven that mice carrying the G399S HtrA2/Omi mutation could shown significant reduction in S400 phosphorylation in the brain and the phosphorylation of HtrA2/Omi is known to be deregulated in neurodegenerative disorders (Fitzgerald et al. 2012). Based on the fact that the mutation G399S found

Introduction______in sporadic Parkinson’s disease (PD) reportedly leading to reduced serine protease activity of HtrA2/Omi (Yun et al. 2008, Strauss et al. 2005), and that S400 phosphoryation reduction was observed in mice carring G399S mutation, we expect more toxic effect due to G399S overexpression in both mouse model and cell model reflected by protein regulation in the proteomics perspective.

1.1.4 Cell stress model in relation to PD

All metazoan cells are able to activate programmed cell death for the elimination of unwanted or potentially hazardous cells during the development and homeostasis of organism. A defect of this process is related with the pathogenesis of a variety of diseases such as cancer and neurodegerative disorders (White 2006, Bredesen 2008, Fuchs and Steller 2011). It is considered that kinase inhibitors currently are composed of up to 30% of drug-discovery projects in pharmaceutical industry and small molecule inhibitors have been applied by scientists to perform basic research to comprehend protein signal transduction (Cohen 1999 and Zachary et al. 2005). Staurosporine is a protein kinase inhibitor to induce cell stress and apoptosis and has already been applied to cell lines for this purpose (Karamann et al. 2008, Curci et. al 2014, Shao et al. 2014 and Sodja et al. 2014). The bacterial alkaloid compound staurosporine has been shown to inhibit many protein kinases and can induce apoptosis in nearly all cell types including neurons (Pong et al. 2001).

In this work, we established a cell model SH-SY5Y neuroblastoma cell lines using vector control, WT HtrA2/Omi and its G399S mutant overexpression by inducing the cell stressor staurosporine to activate the neuronal apoptosis. In this way, the role of WT HtrA2/Omi and its G399S mutant will be investigated by the proteome change of neuron cell lines under neurodegenerative/stressed condition.

Biochemically, apoptosis is demonstrated by the activation of a unique family of cysteine proteases called caspases (Degterev et al. 2003). Caspases play a key role in the signaling and execution of apoptosis and their activation is controlled by activating and inhibitory proteins (Budihardjo et al. 1999; Salvesen and Dixit1999; Bader and Steller, 2009). The markers used for apoptosis in mouse model study in Tübingen were cleaved caspase-9 and caspase-3 (Poonam Dissertation, Tübingen). 6

Introduction______

From the literature it is known that two confirmed phosphorylation serines of HtrA2 are adjacent to two of the mutation sites of HtrA2 in PD patients-- A141S and G399S, which were considered as functionally important. Along with phosphorylated HtrA2/Omi, there are also many other phosphorylated proteins involved in neurodegenerative disease study. However, the identification of the protein phosphorylation sites in general by mass spectromety based proteomics study is notoriously difficult and all phosphorylation enrichment strategies (Chapter 1.2) are far from perfect, we aim to develop a new method for further clinical research application.Therefore, methodology study for phosphoenrichment will be carried out intensively within this work.

1.2 Protein phosphorylation and techniques for its analysis

Phosphorylation and dephosphorylation of proteins serve as a key regulatory mechanism in biological processes such as metabolism, transcriptional and translational regulation, proliferation, and differentiation amongst others (Bononi et al. 2011). It is one of the most understood protein modification and probably most diverse and influential one (Lehmann 2010). It is estimated that about 30% of the human protein is phosphorylated and 500 protein kinases and a third that number of protein phosphotases are encoded by the human genome (Cohen 2001). Mutations in certain protein kinases and phosphotases can cause disorders in neurodegeneration (Brady et al. 2012). Genomic mutations misleading a signal transduction pathway to uncontrolled dysregulation of protein kinases such as mitogen-activated protein kinase (MARK) of growth factors such as EGF (epidermal growth factor receptor) and PDGF (platelet-derived growth factor) can cause cancer (Lehmann 2010). Protein kinase inhibitors have been used clinically since 1990 as drug treatment to cancer patients, chronic inflammatory diseases, diabetes and stroke. Therefore, the investigation of protein phosphorylation is of high importance in clinical research and drug investigation.

The development and application of sensitive and selective methods capable of analyzing the phosphorylation state of proteins is urgently needed in proteomics, drug discovery, and diagnostics. Mass spectrometry based identification of the phosphorylation sites in proteins is often inadequate without a prior enrichment of

Introduction______phosphorylated proteins or peptides (Palma et al. 2013). There are several reasons: 1). Phosphate groups attached to serine and threonine are labile due to β- elimination; 2). have low ionization efficiency due to the acidic phosphate group and suppression by more abundant non- phosphorylated species (i.e. ion suppression phenomenon) during mass spectrometry analysis; 3). Phosphorylated species are low abundant due to dynamic kinase and phosphatase activity; 4). Phospho-groups make peptides more hydrophilic, hence making reverse phase HPLC separation and purification difficult, adversely affecting subsequent MS or other detection methods; 5). Phospho-groups have affinity to metals like aluminum and iron causing sample loss. Especially difficult is the tyrosine phosphorylation (pY) analysis, which is a type of O-phosphorylation with the lowest abundance (approximately 0.05% of all phosphorylated proteins in mammals) among all three phosphorylation modifications. Therefore, high throughput analysis using mass spectrometry to elucidate the tyrosine-phosphorylated proteome requires sensitive and efﬁcient enrichment procedures before or after an enzymatic digestion. In principle, it is now possible to perform affinity purification experiments using anti tyrosine-phosphorylation (pY) antibodies where tyrosine-phosphorylated proteome is enriched and its dynamic changes are monitored (Blagoev et al. 2004; Schmelzle et al. 2006). Three clones of phosphotyrosine antibodies are commonly used: 4G10, PY20 and PY100 and are proven to exhibit substantial sequence selectivity (Tinti et al 2012). According to their peptide profiling data, phosphotyrosine peptides with proline at +3 and leucine at -1 were overall enriched by all three antibodies, whereas each antibody was still observed with difference sequence preferences. Another bottleneck is that an efficient affinity purification of tyrosine-phosphorylated peptides/proteins is only possible with sufficient amount of expensive antibodies and sample material (Palma et al. 2013).

The enrichment of phosphoserine/threonine-containing proteins has not been routinely possible. Antibodies that specifically recognized serine/threonine phosphorylated proteins have become available in recent years. However, due to the lower immunogenicity of the phosphoserine and phosphothreonine side chains, it is impossible to use these antibodies for enrichment purposes (Gronborg et al. 2002 & Oda et al. 2001). The Gronborg group demonstrated several antibodies that failed in immunoprecipitation experiments. Three antibodies were observed with improved

Introduction______results when immunoprecipitated with HeLa cells against pSer/Thr or pThr or against phosphorylated threonine only in the co-presence of proline (pThr-Pro). However, their large scaled purification of serine/threonine phosphorylated proteins via immunoprecipitation was able to enrich and identify only 7 proteins after the cells treated with the phosphotase inhibitor calyculin A. Regarding a global phosphoproteome mapping, we should expect a large number of identified phosphoproteins.

Chemical derivatization following ß-elimination of pSer and pThr to identify serine and theronine phosphorylated peptides is an alternative. The main disadvantage of this method lies in that the current chemistries require high amount of protein or peptide for identification by MS. In addition, the selectivity of these methods has not been confirmed yet (Delom and Chevert 2006).

Figure 1-3: The most commonly used phospho-specific enrichment strategies There are three groups of phosphoenrichment strategies in general: immunoprecipitations, affinity chromatography and chemical derivatisation. (Thingholm et al. 2009)

Other frequently reported alternatives to immunologic methods, chemoaffinity protocols such as IMAC (Immobilized Metal Affinity Chromatography) or TiO2 (titanium dioxide) on the other hand are widely used in phophoproteomics. IMAC

Introduction______uses metal ions (Fe3+, Al3+, Ga3+ or Zr4+, Co2+) to chelate to nitrilotriacetic acid (NTA) or iminodiacetic acid (IDA) coated beads, forming a stationary phase to bind netagively charged phosphopeptides in certain mobile phase (Thingolm et al. 2009).

After protein digestion, the peptides can be applied to IMAC or TiO2 and a large number of phosphopeptides are expected in the LC-MS/MS measurement. The subsequent data bank search with peptide spectra matching will reveal the information about the identified phosphoproteins. A flaw of IMAC is often high level of nonspecific bindings and that the applied complex protein digest amount for efficient enrichment is considerably high up to the milligram range (Thingholm et al. 2006, Villen et al. 2008, Zarei et al. 2011 and Palma et al. 2013). With improved protocol for TiO2 phosphopeptide enrichment, low non-specific bindings are observed however, obvious bias towards the acidic amino acids (glutamic acid and aspartic acid) discussed in this work should raise the attention from phosphoproteomics researchers to scrutinize and decide for their own phosphoenrichment strategies regarding different research aims.

1.3 MIPs for phosphoanalysis

“Intelligent polymers”, “polymers with memory” and “artificial antibodies” are often used to describe molecularly imprinted polymers (MIPs) (Sellergren 2000). The year 1972 is considered as the beginning of molecular imprinting technology, when the laboratories of Wulff and Klotz (Guyot 1988 and Sherrington 1993) independently reported the preparation of organic polymers with predetermined ligand selectivity. The molecular imprinting idea is developed based on the antibody formation theories. The targeted molecule is decided and template designed, with functional monomers that assemble around the template then cross-linked to each other to form the polymer as shown in Figure 1-2. After template removal, the MIP is able to recognize and rebind the template molecule.

Introduction______

Figure 1-4: Principle of molecular imprinting (Alexander et al. 2006)

MIPs are used for the determination of trace chemicals such as acrylamide (Xu et al. 2012) or chemical warfare agents (Boyd et al 2004). They are used in versatile analytical fields, e.g. small molecule enzyme inhibitors imprinted MIP was also suggested to reach sufficient affinity and selectivity (Sellergren 2010). Solid phase extraction (SPE) is widely used in analytical chemistry in fields such as environmental and pharmaceutical analysis where cleaning and concentration of samples take place (Olsen et al. 1998). The first MIP-SPE application with recognition sites for the antiprotozoal drug determination was reported by Sellergren in 1994. A biological urine sample with spiked pentamidin was applied to the MIP. The result could show with a clean extraction of the drug via MIP SPE. Another MIP SPE application for bioanalysis (Muldoon et al. 1997) was performed to enrich 0.005-0.500 ppm spiked herbicide atrazine from 10 g beef liver homogenate extracted with chloroform followed by direct analysis by HPLC and ELISA. The extraction solvent chloroform was optimized for recovery and high specificity. Another example of the MIP SPE application was performed in environmental analysis (Matsui et al. 1997), where atrazine was enriched from distilled water containing simazine and impurities.

While MIPs have been widely used to enrich low molecular weight analytes, they have had little success in enriching biological macromolecules. One difficulty to target macromolecules such as proteins lies in using water as the recognition conditions. This has hindered success in imprinting technology for proteins (Turner et al. 2006). Another source of difficulty is the steric accessibility within the separation material pore structure when a native protein is imprinted: both diffusion out of the highly

Introduction______cross-linked polymer network and diffusion through the tight polymer network to reach the imprinted binding sites (Suedee 2013). Nevertheless, recent reports have shown that MIPs can protentially enrich peptides and proteins, and moreover, now can challenge and exceed the performance of antibodies (Emgenbroich et al. 2008).

Following the work using MIP to target peptides, the epitope Fmoc-phosphotyrosine and Fmoc-phosphoserine imprinted polymers (pY-MIP, pS-MIP Figure 1-5 and 1-6) with designed host monomers were used in this work to prepare phosphotyrosine (pY) and phosphoserine (pS) imprinted polymers, respectively, that were capable of selective enrichment of pY or pS peptides resulting in MIPs displaying binding affinities in the range of pY antibodies (Helling et al. 2011). Till now this is the only example of MIPs that are capable of recognizing post-translational modifications of peptides.

Figure 1-5: Solubilization of bis-PMP salt of Fmoc-pTyr-OEt imprinted pY-MIP by urea monomers and the proposed pY-MIP-pY peptide binding complex (Emgenbroich et al. 2008).

Introduction______

Figure 1-6: Solubilization of bis-PMP salt of Fmoc-pSer-OEt imprinted pS-MIP by Urea monomers shows that formation of strong non-covalent complex in dry THF.

The preparation of pY-MIP and pS-MIP were carried out in TU-Dortmund, Dortmund, Germany. It was observed that both polymers (pY-MIPs and pS-MIPs) exhibited significant performance in template recognition and rebinding.

The template rebinding tests were performed on a HPLC system (Chapter 3.2.1), both polymers have exhibited significant enrichment performance in the template recognition and rebinding. The MIP particles were packed into HPLC columns using the templates mixture as pushing solvent. Since the primary tests of both MIPs have demonstrated promising features in distinguishing and rebinding their template respectively, we proceeded further material tests with standard peptide mixture test, then a spiking experiment and, finally trypsinized proteins from biological sample application in the form of micro-column solid phase extraction during this doctoral research.

1.4 Proteomics and applied MS techniques

The term “proteomics” was originally defined 15 years ago by Wilkins et al. in 1996 The term “proteome” refers to the protein and and genome combined by Marc Wilkins in 1994 during his Ph.D period (Wilkins et al. 1996). Proteomics study refers to the analysis in large-scale proteins in a cell, tissue, or entire organism with defined conditions. Although there is only one definitive genome of an organism, it is encoded in multiple possible proteomes due to protein changes under certain 13

Introduction______conditions and is the result of combined factors in protein transcription, translation, turnover and posttranslational modifications (Abdallah et al. 2012). Compared to the static genome, proteome is dynamic and inclines to change due to the phase of development, environmental differences, infections, diseases or other influential factors such as oxidative cell stress or knockout/over-expression of a certain gene.

Use of mass spectrometry for analysis of complex protein samples is on the rise. MS- based proteomics has been made possible by the availability of genome sequence databases and technical advances in instrumentation, of which the discovery and development of protein ionization was recognized by the 2002 Nobel Prize in Chemistry (Aebersold and Mann 2003). The top-down MS method to sequence intact proteins and post-translational modifications is not yet a high-throughput method and mainly a technique for analyzing single purified proteins (Allison Doerr 2008). Another obstacle is the difficulty to predict from the sequence of a mature and modified protein to the measured protein mass. Moreover, the mass spectrometer is more efficient at obtaining sequence information from peptides that are less than 20 amino acids than from whole proteins (Mørtz et al. 1996, Horn et al 2000, Taylor et al. 2003). Recent researches have made significant progress and is able to apply top-down MS approach to quantify single intact protein in post–mortem brain tissue (Kellie et al. 2014) or in pre-treated (proteins with molecular weight < 30 kDa) yeast lysate for quantitative proteomics analysis (Ntai et al. 2015). Another proteomics study could identify ca. 80 proteins in CSF with top-down approach with the discovery of potential biomarkers in brain tumor (Desiderio et al. 2012).

The well established large-scale MS-based proteomics analysis is known as bottom- up proteomics, where a mixture of proteins is digested into short peptides before MS analysis. In principle, it is composed of a few practical steps, of which the crucial one is to extract proteins during tissue or cell lysis. The second step is to use enzymes such as trypsin to digest the proteins into peptides. Finally, the trypsinized peptides are separated by high pressure liquid chromatography (HPLC) systems and analyzed by mass spectrometers (MS). Softwares using protein data bank searches are necessary to facilitate the peptide sequence matching to tandem MS experiments for protein identification. According to our in house study using bottom-up proteomics, the scale of identified and quantified proteins in cell or tissues via this approach can

Introduction______be over 2000, while in CSF body fluid 700. In this work, two differential studies (global proteomics analysis) of SH-SY5Y cell lines and mouse brain samples were carried out with the bottom-up proteomics analysis procedure as described above. Further data validation can be achieved e.g. via 1) Western blot using antibodies for protein detection and densitometric quantification; 2) by the application of immunohistochemical staining with attention to the antigen-antibody reaction, tissue fixation and processing considerations (Ramos-Vara and Miller 2014), 3) targeted MS approach PRM (parallel reaction monitoring), or SRM/MRM (single reaction monitoring/multiple reaction monitoring), where the chosen peptide precursor ions out of the complex background are monitored and their MS/MS fragments quantified.

Mass spectrometers differentiate each other from ionization, mass analyzers and detection methods. The MS ionization source determines the classes of analytes to be measured, whereas it is the combination of mass analyzer and the detector that determines the quality and reliability of analysis (Zubarev and Makarov 2013). The widely used MS analyzers are classified as quadrupole, magnetic sector, ion trap, time-of-flight (TOF), or Fourier transform ion cyclotron resonance (FTICR). For large biomolecule analysis, electrospray ionization has been used since its invention in 1989 (Fenn and Mann 1989) which led to a share of the Nobel Prize for chemistry in 2002.

In the proteomics studies presented here, digested peptides as analytes are all dissolved in solution as highly complex samples. To obtain thousands of proteins identified in such complex biological matrix, high performance liquid chromatographic (HPLC) separation is always applied prior to the MS measurement. The HPLC system is coupled with a nano electrospray ionization (ESI) source, referring to a Q Exactive mass spectrometer (Figure 1-7) in this work. After the chromatographic separation, the analytes and the LC mobile phases are firstly dispersed by electrospray into small droplets and vaporized with the help of nitrogen as an inert gas into aerosol at the ion source. The solvent evaporates, firstly forming charged droplets until the original droplet exploding into much smaller and more stable gas phased ions, which pass through the transferring vacuum stages of S lenses and the quadruple mass filter. The MS scanning event takes place in the quadruple mass filter. The scanned ions are collected and ramped in the C-trap then ejected into the high collision dissociation cell (HCD) for further MS/MS fragmentation. The masses of 15

Introduction______precursor ions and the MS/MS fragments are trapped and analyzed in the orbitrap mass analyzer, while the image current from the trapped ions is converted into mass spectra via Fourier transform (FT) of the generated frequency signals.

Figure 1-7: MS instrumental structure of Orbitrap Q Exactive (Thermo Scientific) The peptide structure of a molecule is determined by the masses of the fragments generated in the collision cell. During peptide fragmentation, the fragment ions a, b, c, x, y, z ions are generated (Figure 1-8). Depending on research purposes, MS spectrum analysis — in this work referring to protein data bank search - requires software assisted interpretation. The peptide-sequencing data obtained from the MS measurement will be searched against theoretical fragments via the search engines Mascot (Perkins et al. 1999), a probability-based matching, which evaluates the theoretically predicted fragments for all peptides in the database and calculates the identification scores starting with the most intense peaks such as b- and y- ions (Steen and Mann 2004).

Figure 1-8: ions generated via MS/MS fragmentation (Steen and Mann 2004).

Introduction______

Another soft ionization mass spectrometry used in this work is matrix assisted laser dissociation ionization called MALDI-TOF/TOF-MS (Figure 1-9). Analytes are mixed with excessive amount of ultra-violet absorbing matrix with low molecular weight aromatic acid. When UV-laser is provided, the matrix molecules and analytes will be ablated therefore form a hot plume flow (Knochenmuss 2006). The desorbed and excited matrix molecules are protonated (primary ions) and they then transfer the protons to the analytes. The ionized analytes, also called secondary ions, will fly through the magnetic field time of flight (TOF) mass analyzer into the detector.

Figure 1-9: Matrix assisted laser dissociation ionization (Steen and Mann 2004)

Figure 1-10: Reflector and linear detector in the time-of-flight (TOF) analyzer (Suckau et al. 2003)

Introduction______

The TOF mass analyzer has the advantage of recording a mass spectrum in short time. The upgraded full TOF/TOF provides improved sensitivity. However, the major physical factor limiting the mass resolution is a spread of initial velocities of the flying ions. With the help of a non-magnetic mass-reflector (Mamyrin 1973) as in Figure 1- 10, the kinetic energy distribution of ions can be corrected so that all ions are focused at one point of the detector, leading to high resolution mass analysis.

Introduction______

1.5 Aims

In method development, it should be focused on the application of plastic antibodies for phosphopepitde enrichment. Optimized methods using self-made plastic antibody- -epitope imprinted polymer powder pY-MIP for phosphotyrosine peptides enrichment and pS-MIP polymer for phosphoserine peptides enrichment in biological complex matrix are to be established.

Paralell, the role of WT HtrA2/Omi and its G399S mutant should be investigated on the proteome change in two model studies

1. Transgenic mouse study with WT HtrA2/Omi and its G399S mutant over- expressed in all areas of mouse brains. In a heterogeneous brain tissue composed of many types of cells, the discovery of differential protein candidates may better reflect PD pathologies. Combined with the mouse behavioral study and PD pathylogical phenotype observations achieved from collaboration partner in Tübingen, the proteomics data in our work should further contribute to the understanding of the link between HtrA2/Omi and PD.

2. WT HtrA2/Omi and its G399S mutant transfected human neuroblastoma SH-SY5Y neuronal cell lines under induced cell stress are to be analyzed. Using this experimental setup, we aim to reveal either a toxic effect or neuroprotective role of WT HtrA2/Omi and G399S mutant through the discovery of protein regulation in neuronal cellular proteome change with the help of novel bottom-up proteomics approach. The identified proteins in both proteomics studies should be validated.

Materials and Methods______

2. Materials and Methods

2.1 Materials

2.1.1 Instruments, expendable items and chemicals

Instrument Name Producer SCALTEC Instruments, balance Scaltec SBA 31 Heiligenstadt, Deutschland Owl HEP-1 Semi Dry Thermo Fisher Scientific, blotting chamber Electroblotting System Rockford, USA Eppendorf AG, Hamburg, centrifuge 5415 R Germany Thermo Scientific, centrifuge Heraeus Multifuge X3 Langenselbold, Germany Tecan Austria GmbH, Grodig, fluorescence-reader Tecan infinite M200 Pro Austria OdysseyR- ImagingTM LI-COR Biotechnology GmbH, fluorescence-scanner System Bad Homburg, Germany XCell SureLock™ Mini- Gibco®, InvitrogenTM, gel chamber Cell Electrophoresis Karlsruhe, Germany System HPLC UltiMate 3000 LC system Dionex, Idstein, Germany Ecotron incubator shaker Infors AG, Bottmingen, Swizerland Inkubationsschüttler OLYMPUS Corporation, Tokio, light microscope CKX31SF Japan magnetrührer RH basic 2 IKAMAGR IKAR, Staufen, Deutschland Q Exactive™ Hybrid Quadrupole-Orbitrap Thermo Fisher Scientific, mass spectrometer Mass Spectrometer; Rockford, USA

Ultraflex II MALDI TOF/TOF Bruker Daltonics, mass spectrometer Mass Spectrometer Bremen, Germany 384 Anchor Chip target plate with Bruker Daltonics, MALDI target Transponder Technology Bremen, Germany Hamilton, Bonaduz, GR, Microliter syringe 250 µl Switzerland NanoDropR ND-1000 Thermo Fisher Scientific, Nanodrop photometer UV/Vis- Rockford, USA Spektralphotometer pH meter Mettler Toledo Sigma-Aldrich Chemie GmbH, pH-meter Quattro MP220 Basic Schnelldorf, Germany Heidolph Instruments GmbH & platform shaker Duomax 1030 Co. KG, Schwabach, Germany PowerPac universal BIO-RAD Laboratories GmbH, power supply (PAGele) power supply München, Germany Electrophoresis power GE Healthcare, New Jersey, power supply (blots) supply eps 3501 XL USA 20

Materials and Methods______

Progenesis LC-MSTM ver. 4.0.4265.42984, Nonlinear Dynamics Ltd Newcasle upon Tyne, UK

Martin Christ rotational vacuum SpeedDry RVC 2-25 Gefriertrocknungsanlagen concentrator CDplus GmbH, Osterode, Germany Heareus HERAsafe Typ Kendro Laboratory Products, sterile bentch 12 Hanau, Germany Eppendorf AG, Hamburg, thermomixer Thermomixer® comfort Germany UHPLC (AAA) ACQUITY UPLC System Waters, Milford, USA Bandelin SONOREX Bandelin electronic, Berlin, ultrasonic bath SUPER RK 52 Germany Ultraschallreiniger Bibby Scientific Ltd. San vortex-mixer Vortex MixerStuart SA7 Francisico, USA ChemiDoc™ MP Bio-Rad Laboratories GmbH, Western blot scanner Imaging System München, Germany TKA Water Purification Thermo Electron LED GmbH, Water apparatus Systems Niederelbert, Germany Gesellschaft für Labortechnik water bath Wasserbad Typ 1008 mbH, Borgwedel, Germany

Expendable iteml Specifications Producer gel cassette 1 mm und 1,5 mm Invitrogen®, Carlsbad, USA Eppendorf AG, Hamburg, gel loader-tips 2-200 µl Germany Nunc-Immuno™ Sigma-Aldrich Chemie GmbH, microwell plate Microwell™ 96 well Schnelldorf, Germany nitrocellulose membran Protran BA 300 mm x 3 Whatman® GmbH, Dasel, m Nitrocellulose Germany BD Biosciences, San Jose, falcon tube 15 ml; 50 ml USA 0,2 ml; 0,5 ml; 1,5 ml; Eppendorf AG, Hamburg, Eppendorf tube 2 ml Germany SARSTEDT AG & Co., glass pipette 5 ml, 10 ml, 25 ml Nümbrecht, Germany Chromabond adapter 1,3,6 ml PP-column Macherey-Nagel, Düren, Germany

titanium dioxide TiO2 beads 5 µm GL-Sceince, Torrance, USA Life Technologies, Darmstadt, magnetic beads Dynabeads Germany C8 membrane -- 3M, Düsseldorf, Germany 5 µm BioBasic SCX HPLC column Thermo Scientific™,USA

Ø 10 cm, 6-well,12-well, 24-well, TPP Techno Plastic Products cell culture dishes 96-well AG, Trasadingen, Switzerland HyperSep C18 SpinTips Thermo Fisher Scientific, 1-10 µl Rockford, USA 21

Materials and Methods______

C18 trap column 100 μm × 2 cm, 5 µm, Thermo Fisher Scientific, 100 Å Rockford, USA C18 separation column 75 μm × 50 cm, 2 μm, Thermo Fisher Scientific, 100 Å Rockford, USA

Chemical Producer 3-(N-Morpholino)- Propansulfonic acid (MOPS) AppliChem GmbH, Darmstadt, Germany Acetonitril (LC/MS grade) Biosolve b.v., Valkenswaard, Netherlands formic acid „LC/MS grade“ J.T. Baker, Baker Inc. Deventer, Netherlands Sigma-Aldrich Chemie GmbH, Schnelldorf, ammoniumhydrogencarbonate Germany Sigma-Aldrich Chemie GmbH, Schnelldorf, ammonium persulfate (APS) Germany BIO-RAD Protein Assay Dye BIO-RAD Laboratories GmbH, München, Reagent Concentrate Germany Bis(2-hydroxyethyl)aminotris (hydroxymethyl)methan AppliChem GmbH, Darmstadt, Germany (BisTris) Sigma-Aldrich Chemie GmbH, Schnelldorf, bovine serum albumin (BSA) Germany

calcium chloride (CaCl2) J. T. Baker, Griesheim, Germany complete, EDTA-free protease Roche Diagnostics GmbH, inhibitor cocktail Tablette Mannheim, Germany Coomassie® Brillantblau G-250 AppliChem GmbH, Darmstadt, Germany Dithiothreitol (DTT) AppliChem GmbH, Darmstadt, Germany Dulbecco’s Modified Eagle Medium(DMEM) Gibco®, Invitrogen™, Karlsruhe, Germany Phosphate-Buffered Saline (PBS) Gibco®, Invitrogen™, Karlsruhe, Germany ethanol AppliChem GmbH, Darmstadt, Germany ethylenediaminetetraacetic acid Merck KGaA, Darmstadt, Germany (EDTA) fetal bovine serum (FBS) Gibco®, Invitrogen™, Karlsruhe, Germany glycerin 80 % Carl Roth GmbH & Co. KG, Karlsruhe, Germany urea J.T. Baker, Griesheim, Germany potassium chloride (KCl) J. T. Baker, Griesheim, Germany Sigma-Aldrich Chemie GmbH, Schnelldorf, N-(Tri(hydroxymethyl)methyl)glycin (Tricin) Germany BIO-RAD Laboratories GmbH, München, N,N,N’,N’-Tetramethylethylendiamine (TEMED) Germany sodium chlorid (NaCl) J. T. Baker, Griesheim, Germany

sodium dihydrogen phosphoate(NaH2PO4) J. T. Baker, Griesheim, Germany

sodium hydrogen phosphate(Na2HPO4) J. T. Baker, Griesheim, Germany VWR International GmbH, Langenfeld, sodium hydroxide (NaOH) Germany Penicillin/Streptomycin Gibco®, Invitrogen™, Karlsruhe, Germany Carl Roth GmbH & Co. KG, Karlsruhe, hydrochloric acid (HCl) Deutschland sodium dodecyl sulfate (SDS) AppliChem GmbH, Darmstadt, Deutschland StartingBlock blocking buffer Thermo Fisher Scientific, Rockford, USA

Materials and Methods______

thiourea AppliChem GmbH, Darmstadt, Germany Sigma-Aldrich Chemie GmbH, Schnelldorf, trifluoroacetic acid (TFA) Germany Sigma-Aldrich Chemie GmbH, Schnelldorf, tris(hydroxymethyl)-aminomethane (Tris) Germany Sigma-Aldrich Chemie GmbH, Schnelldorf, TritonTM X-100 Germany SERVA Electrophoresis GmbH, trypsin Heidelberg,Germany Germany Sigma-Aldrich Chemie GmbH, Schnelldorf, ε-Aminocapronsäure Germany Sigma-Aldrich Chemie GmbH, Schnelldorf, ammonium hydroxide Germany

phoshoric acid J. T. Baker, Griesheim, Germany

Tween 20 AppliChem GmbH, Darmstadt, Deutschland

Sigma-Aldrich Chemie GmbH, Schnelldorf, dimethyl pimelimidate dihydrochloride Germany Sigma-Aldrich Chemie GmbH, Schnelldorf, triethanolamine Germany

methanol(MeOH) J. T. Baker, Griesheim, Germany

Sigma-Aldrich Chemie GmbH, Schnelldorf, staurosporine Germany N-(9-fluorenylmethoxycarbonyloxy)succinimide (Fmoc- Sigma-Aldrich Chemie GmbH, Schnelldorf, OSu) Germany dimethylformamide (DMF) Sigma-Aldrich Chemie GmbH, Schnelldorf, Germany Sigma-Aldrich Chemie GmbH, Schnelldorf, 2,5-dihydroxybenzoic acid (DHB) Germany Sigma-Aldrich Chemie GmbH, Schnelldorf, glycolic acid Germany

Pierce Amino Acid Standard Thermo Scientific, Bremen, Germany

Life technologies, Thermo Scientific, Bremen, Penicillin Streptomycin (Pen Strep) Germany Life technologies, Thermo Scientific, Bremen, G418 Geneticin Germany

Dimethyl formamide (DMF) Riedel-deHaen, Seelze, Germany

1,2,2,6,6-pentamethylpiperidine (PMP) salt Fluka, Bucks, Switzerland

Pentaerythritol triacrylate (PETRA) Sigma-Aldrich, Steinheim, Germany

N,N´-Azo-bis-(2,4-dimethyl)valeronitrile (ABDV) Wako Chemicals GmbH, Neuss, Germany

Materials and Methods______

Smart Protein Layers SPL Kit for Western blot NH DyeAgnostics GmbH, Halle, Germany

2.1.2 Buffers and reagents

phosphotase inhibitors 10 mM sodium orthovanadate, 9.5 mM sodium fluoride, and 0.5 µM okadaic acid (dissolved in DMSO)

DIGE buffer without CHAPS containing 7M urea, 30 mM tris(hydroxymethyl)aminomethane and 2M thiourea, pH 8.5

PBS phosphate buffered saline (80 mM NaH2PO4; 20 mM Na2HPO4; 100 mM NaCl), pH 7.4

cell medium DMEM, FBS 15%, G418 1% and PS 1%

MALDI matrix solution 40 mg DHB (2,5-dihydroxybenzoic acid), in MeCN : Water (1:1 v/v, 1 ml) with 1% phosphoric acid and 0.1% TFA.

IP immunoprecipitation buffer 73 mM sodium chloride, 2.7 mM potassium chloride, 0.02 % Tween 20, 25 mM tris[hydroxymethyl]aminomethane and pH 7.4 adjusted by hydrochloric acid (HCl).

TBS buffer 73 mM sodium chloride, 2.7 mM potassium chloride, 25 mM tris[hydroxymethyl]aminomethane and pH 7.4 adjusted by hydrochloric acid (HCl).

acrylamide gel recipe acrylamid (30 %) 3.33 ml, Bis-Tris (7 x) 1.14 ml, TEMED 3 µl, water 3,53 ml, APS (40 %) 12 µl

cell lysis buffer PBS (80 mM NaH2PO4, 20 mM Na2HPO4 ,100 mM NaCl), 0.1% Triton X-100 and protease inhibitor) pH 7.4 The phosphotase inhibitors concentration is in the table. gel washing buffer A 10 mM ammonium hydrogen carbonate, pH 7.8

gel washing buffer B 50% gel washing buffer A and 50% MeCN

LDS sample loading buffer 73 mM LDS, 869 mM glycerol, 141 mM TrizmaBase, 106 mM TrizmaHCl, 5.13 mM EDTA , pH 8.5

Materials and Methods______

2.1.3 Antibodies

anti-xx antibody/clone application Producer number or dilution degree

pTyr/PY100 Cell Signalling Technology, USA

pTyr/PY20 peptides immunoprecipitation Biozol,Germany

pTyr/4G10 pTyr-peptides immunoprecipitation Merck KGaA, Germany

cleaved caspase 3 Cell Signalling Technology, USA (1:1000)

ß-actin (1:5000) Sigma Aldrich, Germany

pS400 HtrA2/Omi Produced in Tübingen University

(1:500) Western blot UBE2L3 (1:1000) GeneTex (Biozol Diagnostica Vertrieb, Echling, Germany) MCM4 (1:5000) Abcam, Cambridge, UK

HtrA2/Omi (1:5000) BD Transduction Laboratories, Franklin Lakes, USA ADSS (1:500) Sigma Aldrich, Germany

2.1.4 Abbriviation list

HPLC : high performance liquid chromatography MS : mass spectrometer

MIP : molecularly imprinted polymer TiO2 : titanium dioxide

IP : immunoprecipitation PBS : phosphate buffered saline

SCX : strong cation exchange EDTA : ethylendiamintetraacetic acid

APS : ammonium persulphate MeOH : methanol

Pen Strep : Penicillin Streptomycin SDS : sodium dodecyl sulphate

SPE : solid phase extraction NaOH : sodium hydroxide

PMP : 1,2,2,6,6-pentamethylpiperidine DMF : dimethyl formamide

PETRA : pentaerythritol triacrylate ABCDV : N,N´-Azobis-(2,4-dimethyl)valeronitrile

LDS : lithium dodecyl sulphate AAA : amino acid analysis

DMEM : dulbecco’s modified eagle medium Tris : tris(hydroxymethyl)-aminomethane

DTT : dithiothreitol DHB : 2,5-dihydroxybenzoic acid

Materials and Methods______

TFA : trifluoroacetic acid MeCN : acetonitrile

MALDI : matrix assisted laser desorption ionization ESI : electrospray ionization

FBS : fetal bovine serum TEMED : tetramethylethylenediamine

KCl : potassium chloride pT: phosphor-threonine

BSA : bovine serum albumin pTyr, pY : phosphor-tyrosine pS: phosphor-serine CSF : cerebrospinal fluid

NT 6: non-trnasgenic 6 months NT 12: non-transgenic 12 months

WT 6: transgenic wild type HtrA2/Omi overexpressing WT 12: transgenic wild type HtrA2/Omi 6 months overexpressing 12 months

G399S 6: transgenic G399S mutant HtrA2/Omi G399S 12: : transgenic G399S mutant HtrA2/Omi overexpressing 6 months overexpressing 12 months

VC 0; VC 1;VC 3; VC 6 : SH-SY5Y cell vector control WT0;WT1;WT3;WT6 : wild type HtrA2/Omi after stress 0,1,3,6 hours transfected SH-SY5Y cell after stress 0,1,3,6 hours

G399S0; G399S1; G399S3; G399S6 : G399S mutant PD: Parkinson's Disease HtrA2/Omi transfected SH-SY5Y cell after stress 0,1,3,6 hours

2.2 Methods

2.2.1 Samples (cells, mouse brains, CSF)

Cells

To understand the influence of HtrA2/Omi and HtrA2/Omi mutation on the entire proteome, human wild type overexpressing (WT) and G399S (G399S) mutant HtrA2 as well as vector control (VC) SH-SY5Y cell samples were generated from the cell culture. The SH-SY5Y cell samples collected from cell stress experiment (Table 2-4 and 2.2.3) were measured on nHPLC-MS/MS for further investigation of regulated protein candidates in the context of neurodegeneration.

Table 2-4: SH-SY5Y sample list

sample type Time after stressor/h number of biological replication VC 0; 6 5 WT 0; 6 5 G399S 0; 6 5

Materials and Methods______

Mouse brains

Mouse brain samples were delivered from Functional Neurogenomics Laboratory, Hertie-Institute for Clinical Brain Research, Eberhard Karls Universität Tübingen.

To further understand the influence of HtrA2/Omi and HtrA2/Omi mutation on the entire proteome, transgenic mice overexpressing human wild type (TG WT) and G399S (TG G399S) mutant HtrA2 as well as non-transgenic (NG) mouse brain samples were generated in Tübingen while the global proteomic study was carried out at Medizinische Proteom-Center, Ruhr-University Bochum. The samples analyzed are listed in Table 2-5.

Table 2-5: mouse brain sample list

number of biological replication sample type age/months

NT 6 6 6 NT 12 12 6 WT 6 6 6 WT 12 12 6 G399S 6 6 5 G399S 12 12 5

Here the non-transgenic mouse brains samples with the age of 6 months or 12 months were abbriated as NT 6 or NT 12. Transgenic mice overexpressing wild type HtrA2/Omi with 6 months or 12 months old were named as WT 6 or WT 12. The G399S mutant HtrA2/Omi transgenic mouse brains with 2 different ages were named as G399S 6 and G399S 12 respectively. The samples were provided in biological replicates.

CSF sample

The study of human cerebrospinal fluid was approved by the ethics committee at the Georg-August-University Goettingen, Germany with the request number 36/7/02 and 9/7/04. The CSF sample was pooled from 4 different patients as a test sample to check if we could enrich and identify phosphoproteins from clinical relevant samples by introducing pS-MIP approach.

Materials and Methods______

2.2.2 Cell culture and cell stress experiment

Three different types of stable transfected SH-SY5Y cell (vector control), wild type HtrA2/Omi wild type over-expressed (WT) SH-SY5Y and G399S mutated HtrA2/Omi (G-mutant) over-expressed SH-SY5Y were delivered from University Tübingen and further cultivated in the cell culture at 37 °C inside the incubator in 10 cm dish until 90% confluence was reached. Humidity was controlled at 95% with 5% CO2. Cell medium was changed every second day. Cells were then splitted into 6 wells and cultivated. The stressor staurosporine was applied when cells reached the confluence of 70%. The medium was removed first and cells were washed with PBS buffer 3 times. Previously in DMSO dissolved staurosporine with the concentration of 1 µM in 2 ml medium was added to the cells. Staurosporine caused cell stress to three cell lines SH-SY5Y vector control (VC), wild type HtrA2/Omi stably transfected SH-SY5Y cells (WT) and G399S mutant stably transfected SH-SY5Y (G-mutant) was performed by adding 1 µM staurosporine in the cell medium and harvesting the cells after 0,1,3,6 hours of stress induction. Cells were then harvested after one, three, six hours of incubation with 5 replicates of each condition and were further analyzed either via Western blot or via nanoLC-ESI-MS/MS for protein identification and quantification.

2.2.3 Sample preparation

Cell lysis

Human embryo kidney 293T (HEK 293T) cells or transfected human neuroblastoma cells SH-SY5Y cells were dounced with potter in 1.5 ml Eppendorf tube in lysis buffer (2.1.2 Buffers and reagents ). After homogenization, the cell lysate was sonicated six times for 10 seconds with 10 seconds intervals in ice to avoid sample heating. Centrifugation at 16.000 x g for 15 minutes was followed and the supernatant collected. The lysis process was repeated once and two supernatant fractions were collected in one Eppendorf tube. Protein concentration was determined by Bradford protein assay.

Mouse brain tissue lysis

For studying the SPE behavior in a complex enviroment, pY, pS and non phosphoryrlated peptides were spiked in a trypsin digested 6-month-old mouse brain

Materials and Methods______lysate from mouse brain differential analysis. Mouse brain tissue was lysed with a Dounce-Homogenisator and a tight pestle with 30 ups and downs in DIGE buffer without CHAPS in a glass tube on ice. The tissue lysis was carried out the same as cell lysis further. The mouse brain sample analysis using pS-MIP was prepared the same way with phosphotase inhibitors omitting any spiking. The protein concentration of mouse brain was determined by Bradford assay as explained in Chapter 2.2.5.

CSF sample preparation

A CSF sample was prepared in three different ways for the SCX/pS-MIP application in preliminary tests to check the method applicability to clinical relevant samples. Hereto we used (i) in solution tryptic digested (Stoop et al. 2010) 200 µg protein from CSF, (ii) 200 µg CSF for depletion of the 14 most abundant proteins via SEP010 Seppro IgY14 Spin Columns (Sigma) combined with in solution digestion and (iii) in- gel digestion of 150 µg of protein, separated 2 cm by SDS-PAGE (short gel, see following paragraph) and sequestered by the prominent albumin band.

Short gel and tryptic in-gel digestion

Prior to enzymatic protein digestion from either cell lysate or mouse brain tissue lysate, a gel-based method was performed to remove the detergent from the sample. Hereto, 25 µg lysate was mixed with LDS loading buffer, heated at 95 °C for 5 minutes and then loaded on a 12% acrylamide gel. Gel electrophoresis was performed with 50 V for 15 min. After gel electrophoresis, the gel was stained with Coomassie blue for 10 minutes then destained with water overnight. In CSF sample preparation (iii), the short-gel was running at 50 V for ca. 25 minutes until the most abundant albumin was separated then removed by cutting. The advantage over albumin removal (ii) was that the proteins in CSF were partially separated during gel electrophoresis then removed, avoiding the loss of interacting proteins bound to albumin. We also performed CSF in-solution digestion (i) as described by Stoop et al. in 2000 without depletion of any proteins.

The visible protein band of the gel was then cut into small pieces and washed with washing buffer A and B for in 10 minutes alternative for 6 times until the dye was removed. The gel pieces were dried for 1 hour in the rotational vacuum concentrator. Trypsin was added to the gel pieces, (trypsin:protein 1:20) and gel pieces were incubated at 37 °C for 16 hours. The digestion was stopped by adding 29

Materials and Methods______

(MeCN/H2O:50/50 (0.1% TFA) and using ultrasound for 15 minutes twice. The supernatant was collected into a glass vial then dried in the microcentrifuge, and finally re-dissolved in 20 µl water 0.1% TFA. The digested peptide concentration was determined by amino acid analysis.

2.2.4 Protein and peptide concentration determination

The limit of quantification of Bradford protein concentration determination is as sensitive as 5 µg/ml protein (Bradford, 1976). The determination of protein concentration by optical density of the dye Coomassie-Brillantblue G250 at 595 nm with the fluorescence multimode microplate reader (Infinite M200 PRO, Tecan Trading AG, Switzerland). Coomassie-Brillantblue G250 binds to the cationic and non-polar, hydrophobic side-chains of protein. In house, the measured protein concentration was compared with standard concentration of BSA (bovin serum albumin) calibration curve with 8 concentrations from 0 µg/µl, 0.25 µg/µl, 0.5 µg/µl, 0.75 µg/µl, 1 µg/µl, 1,25 µg/µl, 1,75 µg/µl to 2 µg/µl, so that the concentration of unknown samples within this range could be determined.

The peptide or protein concentration was measured by amino acid analysis using AccQ TagTM derivatization of amino acids and subsequent separation and detection with an ACQUITYTM UPLC system. Hereto, the protein or peptide was hydrolysed with HCl so that all amino acids were released. The next step is to derivate the amino acids since the amino acids do not have chromophor and are not able to be detected either under UV or florescence. Therefore, AccQ tag (6-aminoquinoyl-N-hydroxy- succinimidyl-carbamate reagenz) was used for the derivatization to facilitate the UHPLC UV detection later. Before the derivatization, commercial internal standard "Norvaline" was added so that the system error (personal handing during derivatization, pipetting before UHPLC measurement) could be minimized. Concentration of each type of amino acids were determined later based on the spiked internal standard, therefore the total peptide or protein concentration could be calculated based on the amino acid concentration measured on the UHPLC. Alternatively, if the peptides were desalted using HyperSep C18 SpinTips (1-10 µl, Thermo Scientific) according to the manufacturer’s instructions, peptide concentration was easily determined on Nanodrop.

Materials and Methods______

2.2.5 Western blot

To separate the proteins of a cell lysate, 12 % acrylamide gels were used (Table 2.1.2). The polymerization process was initiated by adding the APS and gently inverting the falcon tube twice. The solution was poured into gel cassettes and a comb was used to make wells.

The samples for gel electrophoresis were prepared in a ratio 1:3 by by mixing the sample with LDS buffer (Table 2.1.2). The samples were mixed and heated briefly in a thermomixer for 5 min at 95 °C and 300 rpm. After short centrifugation, each sample contains 25-30 µg protein was applied to the gel wells. The proteins were separated firstly at 50 V for 15 minutes and then at 180 V for 60 min.

For further analysis, the acrylamide gel separated proteins were blotted on nitrocellulose membrane by semi-dry western blotting. Hereto, two pieces of 2 mm thick blotting paper and a single piece of nitrocellulose membrane were cut in size of slightly larger than the size of gel (64 cm2) to be blotted under the current. The membrane and one of the blotting paper were soaked in anode buffer and other paper in cathode buffer. The orientation of the blots was as follows: anode - blotting paper (anode buffer) - nitrocellulose membrane - Gel (briefly soaked in anode buffer) - blotting paper (cathode buffer) - cathode. The air bubbles between the layers were removed by rolling with a 25 ml pipette on the top layer. The blotting was done with 2 mA per cm2 of gel size for one hour.

After blotting, the membrane was incubated in the TBS Starting Block buffer (Table 2.1.1) for 1 hour with gentle shaking at room temperature. The primary antibody was diluted in 5 ml of starting block buffer with recommended dilution degrees (Table 2.1.3). The blot was then put into 50 ml falcon tube containing the diluted primary antibodies (cleaved caspase-3, ß-actin and pS400 HtrA2/Omi) and incubated overnight at 4 °C. After incubation, the blot was washed 3 times with 1 x TBS for 10 minutes on a shaker at room temperature. The secondary antibody (1µg/15 ml) was diluted in 15 ml of 1x TBS and incubated with blot in the dark box for 1 hour. After incubation with secondary antibody, the blot was washed 3 times with 1x TBS for 10 minutes on a shaker at room temperature. After washing, the Western blot membrane was scanned with Licor Biosciences scanner using 700 or 800 nm scanning channels dependent on the secondary antibody types.

Materials and Methods______

Protein validation of UBE2L3 and MCM4 were performed using Smart Protein Layers (SPL Kit) for Western blot for whole protein labeling as the loading control. The protein amount applied was 5 µg – 10 µg each lane. Later, the Wester blot quantification was normalized on both loading control and an internal protein standard according to the product information of SPL Kit introduction.

2.2.6 Phosphopeptide enrichment

The phosphotyrosine peptide enrichment study was carried out based on the comparison with well established methods such as TiO2 and immunoaffinity-based immunoprecipitation using real antibodies to P2 pY-MIP SPE approach. In the study, the comparison was made as demonstrated in Figure 2-11 using standard peptides or spiked biological samples.

Figure 2-1: Schematic representation of all the three applied phosphopeptide enrichment strategies. The work tyrosine-phosphorylated peptide enrichment performance will be demonstrated in a comparison study where pY-MIP, TiO2 and anti-pY antibodies are compared.

Materials and Methods______

A mixture of standard peptides was used as sample to probe and compare all three methods for tyrosine-phosphorylated peptides enrichment. The standard peptide sample amount used was the same for all three methods. Elution fraction of pY-MIP,

TiO2 SPE and immunoprecipitation were measured on MALDI-TOF/TOF-MS.

Another comprehensive phosphopeptide enrichment method using pS-MIP was demonstrated with different types of biological samples without any spiking as shown in Figure 2-12.

Figure 2-2: Work flow of phosphoproteomic analysis of harvested HEK 293T cells, mouse brain or CSF using SCX fractionation followed by pS-MIP/TiO2 enrichment. A mass of 10 µg of tryptic digests of the cell lysates and the CSF sample were loaded directly or after prefractionation with SCX onto pS-MIP or TiO2 columns for phosphor-specific enrichment

Materials and Methods______

MIP solid phase conditioning

20 mg pS-MIP or pY-MIP in a 1.5 ml Eppendorf tube was firstly washed at room temperature with methanol (0.1% TFA) in a thermomixer for 5 min once and supernatant disgarded, then washed for 2 h with acidified methanol again and followed by overnight agitation to remove any remaining template. MIP sample was then centrifuged at 16.000 × g sedimentation and supernatant discarded after each washing. The MIP conditioning was carried out by 10 minute followed by 2 h agitation with MeCN and 0.1% TFA in the thermomixer. For every washing and conditioning step, the slurry was centrifuged and supernatant discarded.

MIP SPE

Solid phase extraction is an analytical separation method using a solid phase and solvents to separate one or more types of analyte from a solution in either a cartridge or micro-column depending on the sample scale. It is widely applied for clean-up or enrichment purpose to extract the analyte from a liquid sample, such as soil extract, liquid waste or biological cellular extracts for further quantification purposes. Typically, an SPE analysis involves four steps: solid phase conditioning, sample loading, column washing and analyte eluting. In this work, solid phase extraction experiments were performed in a micro SPE tip column format to enrich the phosphopeptides from either a standard peptide mixture or a digests of complex biological samples.

A 2-200 µl pipette tip was filled with a C8 membrane plug at the bottom. The conditioned MIP material in 500 µl MeCN (0.1% TFA) was vortexed, of which 15 µl of the slurry containing ~ 0.8 mg MIP was quickly pipetted into the pipette tip. After a few minutes of sedimentation, the remaining solvent was removed by air pressure with a 20 ml syringe equipped with a chromabond adapter. Ten µg trypsinized biological sample or a standard peptide mixture was loaded in the loading solution consisting of 95% MeCN, 0.1% TFA and water on the self-made SPE column, and sedimented for 1 min, followed by centrifugation at 200 x g for 5 minutes. The loading procedure was repeated once. The column was washed with the same solution twice, and the sample eluted firstly with 90% MeOH, 0.1% TFA and then 50% MeOH 0.1% TFA. Two eluted fractions were collected separately and dried in rotational vacuum concentrator (speedvac). The dried eluate was re-dissolved in water 0.1% TFA and

Materials and Methods______ready for the HPLC-MS or MALDI measurement. The SPE procedure is illustrated in Figure 2-13.

Figure 2-3: MIP solid phase extraction work flow. Flow through after loading and two eluate fractions were measured on MALDI Ultraflex II when standard peptide mixture was used as the sample. Later on, when further complex sample was used, nano HPLC-ESI- MS/MS Orbitrap Q Exactive was used for detection.

TiO2 SPE

TiO2 beads were washed twice with loading buffer (80% MeCN, 5% TFA and 1M glycolic acid) then packed in a 2-200 µl tip column as shown in Figure 2-1 MIP SPE tips. Sample was loaded twice onto the TiO2 beads, incubated for 3 minutes statically and supernatant removed by short centrifugation. According to the protocol provided by Thingholm group (Thingholm et al. 2009), the optimum ratio between sample and

TiO2 amount is 1:6. Tip column was washed twice with loading solution, twice with 80% MeCN, 1% TFA then twice with 10% MeCN, 0.1 % TFA. Three eluting steps were carried out using 50 µl elution solvents as follow: elution 1 (E1): 250 mM ammoniumhydrogencarbonate (NH4HCO3) and ammoniumhydroxide NH4OH pH 9.1;

Materials and Methods______

elution 2 (E2): 125 mM NH4HCO3 phosphoric acid 14 µl and NH4OH pH 10.5; elution

3 (E3): NH4OH pH 11.3.

After the three eluting steps, remaining membrane bound peptides were washed with 2 µl 30% acetonitrile. The eluting fractions were collected together and then acidified with 15 µl formic acid (100%). All washing and eluting procedures were performed by centrifugation at 1500 x g, ca. 2 min. After the samples were desalted with C18 self- made tips, they were dried in rotational vacuum concentrator, finally dissolved in 5 µl of 0.1% TFA. The 5 µl sample mixture was mixed with 5 µl MALDI matrix solution, and was ready for MALDI target spotting. In case of complex samples the dried sample was re-dissolved in 0.1% TFA/ water just before LC-MS/MS measurement.

Affinity based immunoprecipitation using anti-pY antibodies

Peptide mixture sample was reconstituted in IP buffer. Mangnetic beads conjugated with M-280 sheep anti-mouse secondary antibody was incubated with anti-pY immunoglubin G (IgG) primary antibody for 3 hours under constant rotation at 4°C and then cross-linked by 0.2 M dimethyl pimelimidate dihydrochloride in triethanolamine buffer (pH 8.2, 20 °C) for 30 minutes to avoid co-elution of pY- antibody with the peptides. The crosslinking of lgG primary antibody and secondary antibody was stopped by adding 1 ml of 50 mM 2-amino-2-(hydroxymethyl)-1,3- propanediol (pH 7.5, adjusted by HCl), incubated for 15 minutes under strong agitation (crosslinking protocol of lgG and magnetic beads by Dynabeads, Life Technologies).The conjugated lgG and mangnetic beads (prewashed three times with IP buffer) were added to sample and incubated overnight at 4°C under constant rotation. After incubation, the beads were washed five times with 1ml IP buffer (Table 2.1.2) (without Tween 20) followed by twice washing with 1 ml water. Peptides were eluted twice with 0.15% TFA at 20 °C on a thermomixer with strong agitation for 30 minutes, the supernatant was dried in rotatuibak vacuum concentrator, then redissolved in 5 µl of 0,1% TFA and then mixed with 5 µl MALDI matrix solution.

Materials and Methods______

2.2.7 Instrumentation based analytical methods

SPE fractions were collected in glass vials separately and dried in Speedvac. After 10 minutes of drying, the two elution fractions of each sample were collected in one vial and rotated until complete dryness. The dried samples were dissolved in 5 µl 0.1% TFA acidified water and mixed with 5 µl of MALDI matrix solution with additional ultrasonication for 10 minutes. 2 µl of this mixture was deposited on the MALDI target plate and dried at room temperature.

MALDI-TOF/TOF analysis

Mass spectrometric measurement of the fractions collected in standard peptide MIP /TiO2 SPE and IP experiments were performed with MALDI TOF/TOF MS with a reflector time of flight mass analyzer. Hereto, fractions were collected in LC insert vials and dried in the rotational concentrator. The dried samples were re-dissolved in 5 µl of 0.1% TFA and mixed with 5 µl of MALDI matrix solvent with additional ultrasonication for 10 minutes. Two µl of this mixture was deposited in triplicates on the MALDI target plate (384 Anchor Chip target plate with Transponder Technology, Bruker Daltonik) and dried at 25°C under the hood. The data was processed using the Flex Control software (Bruker Daltonics). Data collection, in terms of the laser and reflector voltage conditions and number of the scans, was performed identically for all samples unless otherwise noted. The spectra were collected by accumulating 400 laser shots under reflector mode and further analyzed with the Flexanalysis 3.0 software (Bruker Daltonics) and Microsoft Excel.

To consolidate the selectivity of the pS-MIP material the SpS peptide AVPSPPPApSPR was added to a complex matrix of 1.68 µg tryptic digested mouse brain lysate in concentrations ranging from 2 pmol down to 5 fmol. MALDI TOF/TOF MS analysis detected the SpS peptide ion in elution fractions down to 0.5 pmol, given by the ion of m/z=1155.556 Da. The peptide identity was assigned by subsequent fragmentation experiments. Hereto, the precursor MS/MS spectrum of the 2 pmol eluate was compared with previous characterisations of the pure peptide. Lower concentrations of 0.25 and 0.1 pmol were not detectable by MALDI-TOF/TOF-MS. Therefore for concentrations below 0.5 pmol (100 fmol, 50 fmol, 10 fmol, 5 fmol)

Materials and Methods______elution fractions were measured by nanoLC-ESI-MS/MS on an UltiMate 3000 RSLC nano LC system online coupled to an Orbitrap Q-Exactive mass spectrometer.

Reversed phase nanoLC-ESI-MS/MS analysis

For the LC-MS/MS analysis, ca 200 - 300 ng peptides were dissolved in 16 µl water 0.1% TFA.The nano-HPLC analysis was performed on an UltiMate 3000 RSLC nano LC system using the solvent system: (A) 0.1% FA; (B) 84% MeCN, 0.1% FA. Samples were firstly loaded on a trap column with a flow rate of 30 μl/min in 0.1% TFA acidified water. After washing, sample trapped in the pre-column was serially eluted to a separation C18 column and the peptides were separated with a flow rate of 400 nl /min with the solvent gradient of 4% to 40% B for 95 min then a washing step at 95% B (for 5 min) and finally an equilibration step from 95% to 4% B.

In the ESI-MS/MS analysis (Orbitrap Q-Exactive), full MS spectra were scanned between 350 and 1400 m/z with a resolution of 70,000 at 200 m/z (AGC target 3e6, 80 ms maximum injection time). The spray voltage was 1600 V (+) and the capillary temperature 250 °C. Lock mass polydimethylcyclosiloxane (m/z 445.120) was used for the internal recalibration. The m/z values initiating MS/MS were set on a dynamic exclusion list for 30 s and the top ten most intensive ions (charge state +2, +3, +4) were selected.

MS/MS fragments were generated by high energy collision induced dissociation (HCD) in which ion dissociation was performed at normalized collision energy (NCE) of 27%, fixed first mass 130.0 m/z and isolation window 2.2 m/z. The fragments were analyzed in an orbitrap analyser with 35,000 resolution at 200 m/z (AGC 1e6, maximum injection time 120 ms).

Pre-fractionation using Strong Cation Exchange (SCX) chromatography

The SCX stationary phase was composed of sulfonic acid cation-based exchange ligand which is covalently bound to the peptide friendly polymer coated silica as shown in Figure 2-14. The adsorption of the molecules to the solid support is driven by the ionic interaction between the oppositely charged ionic groups in the analyte and in the functional ligand sulfonic acid ligand on the support.

Materials and Methods______

Figure 2-4: Strong Cation Exchange chromatography stationary phase. The phosphate on the analyte peptide interacts with the sulfonic acid ligand of the polymer.

When the salt concentration (KCl) increases, the peptide analytes with weakest ionic interactions start to elute from the SCX column first. The positively charged peptides bound stronger to the SCX column were eluted by increasing cation concentration in the mobile phase. Phosphopeptides having a phosphate group and hence are negatively charged at pH = 2.7, are retained much shorter than the non- phosphopeptides. This results in early elution as shown in Figure 2-5.

SCX chromatographic separations were performed on a Dionex Ultimate3000 HPLC System using a 5 µm SCX HPLC column. Hereto, 160 µg of tryptic digested HEK 293T, SH-SY5Y, mouse brain lysate or trypsinized CSF was lyophilized over-night and re-dissolved in 16 µl of SCX Solvent A (25% MeCN, 5 mM KH2PO4, pH 2.7) and then loaded to the SCX Solvent A equilibrated column. After 5 minutes, peptides were eluted with SCX Solvent B (25% MeCN,5 mM KH2PO4, 500 mM KCl, pH 2.7) using a gradient from 0% to 50% B in 75 min followed by 50% to 100% B in 5 min and then maintained at 100% B for 10 min at a flow rate of 50 µl/min. To obtain a cationic compound depleted fraction, the flow through was collected from 4th to 40th minute of the gradiant, lyophilized and desalted by HyperSep C18 SpinTips (Thermo Scientific product introduction sheet). Afterwards, the peptide concentration was determined by amino acid analysis (see above) before application to MIP or TiO2 based phosphopeptide enrichment. The UV chromatogram of the SCX was shown in Figure 2-5.

Materials and Methods______

phosphopeptides fraction

Figure 2-5: UV spectrum of SCX lysate pre-fractionation. Fraction is collected from the 4th-40th minute of the SCX gradient

2.2.8 Label-free quantification with spectra counting

Spectra counting is a method where the PSMs (peptide spectra matches) identified for a unique peptide that belongs to a certain protein in different samples is counted. The amount of PSMs typically increases in relation to the peptide/protein concentration so that the sample identified protein can be quantified and compared within different samples. The spectral counting analysis was performed with the help a local software PIA (protein interference algorithms https://github.com/mpc- bioinformatics/pia) with FDR rate of MS/MS spectra at 1%.

The choice of using only unique peptides—able to be assigned to only one protein, was based on the fact that more than one proteins may share certain sequence of peptide, and therefore some identified peptides could be assigned to more than one protein. Using only unique peptide identified for proteins can avoid the ambiguity assignment.

We applied spectra counting to analyze two proteomics studies here in this work: mouse brain proteomics and neuron cells SH-SY5Y proteomics studies. To minimize the technical variation from LC-MS measurement, each peptide spectra number was normalized to the total counts of the peptide spectra in one sample.

Materials and Methods______

To make sure that the identified protein candidates were not due to "one-hit-wonder", being identified only with 1 peptide during the LC-MS measurements, further filtering strategies in cell proteomics study include: 1). Minimum peptide spectra found from one sample type contain at least 4 spectra in the cell study; 2). The regulated proteins should be identified in all experiments and no less than 4 sample groups (total replicate: 5 for each sample types). Regarding the mouse brain study, the further filtering focused on the fact that regulated proteins should be identified in all experiments and no less than 5 sample replicates for NT,WT (total replicate: 6 for each group) and no less than 4 samples for G399S mouse types (total replicate: 5 for both ages).

With the statistic test like ANOVA (analysis of variance), the significance between different conditions can be located. Hereto, only the proteins with p ≤ 0.05 were considered. Anova test will tell that the protein is significantly regulated, but not able to tell between which groups of comparison is the significance. In order to pinpoint the significant difference, comparison in pair groups was conducted using Tukey's post hoc test. The Tukey post hoc test was performed to check the variance homogeneity within the participating sample groups. Practically, multiple comparisons will be performed in pair. Proteins were removed with p value bigger than 0.05. In this way, the possible stress-regulated or transfection-specific proteins for the proteomics changes in each comparison can be identified.

2.2.9 Data processing, databank search and pathway analysis

For database searches, the raw files were analyzed with the ProteomDiscoverer 1.4 (Thermo Fisher Scientific) software using the Mascot V.2.3 search algorithm (Matrixscience, London UK) against the Uniprot/Swissprot database using human taxonomy (released 2013/10, 541,561 sequences in the whole database and 20,352 for human) or mouse (released 2014/05, 545,388 sequences in the whole database and 16676 for mouse). The software-implemented Percolator was used to determine the false discovery rate (FDR), and the PhosphoRS 3.1 tool was applied to locate the phosphorylation sites. The following search parameters were used: peptide precursor mass ion selective range of 400-10,000 Da with mass tolerance of 5 ppm; fragment mass tolerance of 20 mmu; two allowed missed cleavages; two dynamic

Materials and Methods______modifications at oxidation of methionine and phosphorylation at serine, threonine or tyrosine. The filter cut-off for the identified peptides was set at a targeted FDR of 1%. The Uniprot oncology analysis was performed using the Uniprot website. Hereto, the “gene oncology” analysis including biological processes, molecular function, cellular component and interacting partner options were chosen for all proteins and the FASTA file was downloaded from Uniprot. The oncology of protein candidates identified in the LC-MS/MS study was then compared with all proteins in the FASTA and sorted out using VLOOKUP (S-Verweis).

The protein cellular distribution and also the pathway analysis was performed on IPA (Ingenuity Pathway Analysis, QIAGEN). Differential proteins identified in cell and mouse brain proteomics study was uploaded into IPA so that networks or canonical pathways of identified proteins can be analyzed by the software according to the user manual. Each analysis was performed separately to identify interesting network or canonical pathways related to neurological disease. The detailed reports are in the attachment.

2.2.10 Phosphopeptide motif-x analysis

This analysis was performed together with Markus-Hermann Koch a PhD student in the bioinformatics group at MPC. From a total of 952 peptides we identified 274 peptides exclusively when using the three different TiO2 methods and 476 exclusively when using the two pS-MIP methods for enrichment. In order to examine the phyisco- chemical properties of these exclusive sets and for distinguishing them against each other we applied the online tool motif-x (Schwartz et al. 2005 and Chou et al. 2011). Motif-x finds repetitive motifs in a foreground database (FG) which is derived from a group of peptides of interest (like the TiO2 exclusive peptides) and evaluates their significance against a background database (BG). A significant motif will score a high frequency of matches on the FG and is specific in scoring a much lower frequency on the BG at the same time.

Markus-Hermann Koch conducted two motif-x runs for motifs of length 7 requiring a phosphorylated S to be at the center of each motif; one for the TiO2-exclusive peptides and one for the pS-MIP-exclusive peptides. Such motifs are long enough to

Materials and Methods______be meaningful and short enough to ensure that not too many phosphorylated S are discarded due to the fact that they are too close to their peptide’s end. For each of the two exclusive sets we constructed an FG by extracting from all pertinent peptides all possible substrings of length 7 containing a phosphorylated S at their center. Analogously we generated a BG database using all identified peptides. For the ‘occurrences’ parameter we stayed with the motif-x default of 20. However, we chose a significance of 5e-4 rather than the 1e-6 default, the latter of which caused motif-x to yield no motifs at all. This, as warned by motif-x, may lead to a higher order of false positive motifs as more background-common residue-position pairs are accepted into the set of reported motifs. In fact the results reveals relatively low-fold increase values. However, in this case the elements of the BG are tailored to precise motif-length and hence there is less room for a given motif to fit into a BG element at random in the first place.

Results______

3. Results

Method establishment was carried out using two kinds MIP material-- phosphotyrosine imprinted polymer pY-MIP and phosphoserine imprinted polymer pS-MIP. In the MIP SPE development stage, the pY-MIP and pS-MIP were tested using standard peptide mixture to achieve the optimum conditions for sample loading, washing and eluting in SPE experiment the detection system (MALDI) was optimized regarding the matrices. Both pY-MIP and pS-MIP were synthesized and characterized by Dr. Sudhirkumar Shinde at TU-Dortmund (Helling at al. 2011 & Chen at al. 2015).

After the best SPE conditions were decided, the application result of pY-MIP and pS- MIP was presented using peptide mixture, peptides spiked in biological sample and the efficiency of the plastic antibodies was compared to the existing approaches respectively. The efficiency of pY-MIP was compared against the benchmarked method TiO2 and immunoprecipitations using anti-pY antibodies (Chen et al. manuscript submitted). The pS-MIP could exhibit high potential for further real sample application and was validated with different cell types and clinically relevant samples such as CSF and mouse brain digest (Chen et al. 2015, manuscript under consideration).

With the novel method of bottom-up proteomics strategy and sufficient statistic tests, we investigated the proteome change of the stressed SH-SY5Y cells with expression of wild type or G399S mutant HtrA2/Omi in combination with mouse brain model study to try to understand the link between HtrA2/Omi and PD. The identified differential proteins via LC-MS/MS measurement and statistic test were presented in several groups regarding the aspects of WT HtrA2/Omi over-expressing effect (cells and mouse brains), G399S mutant HtrA2/Omi over-expressing effect (cells and mouse brains), protein transfection /over-expression effect in general (cells and mouse brains), age effect (mouse brains) and stress effect (cells). These groups of differential proteins provide the first hint in number and different ways of regulation. Detailed scrutinization for network and pathway analyses will be discussed later with the help of IPA (ingenuity pathway analysis) tool.

Results______

3.1 Efficiency comparison of pY-MIP, TiO2 and anti-pY antibodies for tyrosine phosphorylated peptides

In the early stage, pY-MIP SPE experimental set-up was probed focusing on finding the best SPE conditions and detection system (MALDI). After extensive test experiment, SPE procedure was decided and the enrichment efficiency of pY-MIP,

TiO2 and anti-pY antibodies was compared using a standard peptide mixture. Further comparison conducted were three test methods using a standard peptide mixture spiked into mouse brain digest. Immunoprecipitation using anti-pY antibodies was investigated on sample/antibody ratio and different peptide sample amount. Three methodology work flows are schematically presented in Figure 3-1.

Figure 3-1: Schematic workflows of three phosphopeptide enrichment methods applied. The elution fractions from three methods were measured and analysed on MALDI.In the method development stage, flow through, washing and eluting fractions were measured on MALDI:

3.1.1 Method development and optimization

MALDI readout--matrices

Initially, the MIPs were extensively tested with the simple standard peptide mixture. The first goal was to apply the pY- and pS-MIPs for fractionating a mixture of nonphospho-, mono and multiply phosphorylated peptides listed in Table 3-1 into three fractions – a phosphotyrosine peptide fraction, a phosphoserine fraction and a fraction lacking phosphorylated peptides. 45

Results______

Table 3-1: Tyrosine and serine containing standard peptides to test the phosphoselectivity of the pY-MIP and pS-MIP materials.

Peptide Sequence Abbreviation m/z (MH1+) Producer Chinapeptide, AVPSPPPApSP-amide T-SpS 998,609 Shanghai VILGpSPAHR GpS 1029.5241 ISAS, Dortmund DRVYIHPF Y 1046,5418 Calbiochem DRVpSIHPF pS 1050,4768 LifeTein, USA GADDSYYTAR YY 1118,475 LifeTein, USA DRVpYIHPF pY 1126,5081 Calbiochem AVPSPPPApSPR SpS 1155,5558 ISAS, Dortmund GADDSYpYTAR YpY 1198,4412 LifeTein,USA GADDSpYpYTAR pYpY 1278,4076 LifeTein USA WWGSGPSGSGGSGGGK 4S 1420,624 IMP,Vienne WWGSGPpSGSGGpSGGGK 2S2pS 1580,5567 IMP,Vienne TRDIYETDYYRK 3Y 1622,7809 AnaSpec, Germany TRDIpYETDpYpYRK 3pY 1862,6799 AnaSpec, Germany

The peptide sample was pooled in the concentration of 10 pmol/µl of each peptide in water with 0.1%TFA.The absolute quantification of each peptide was determined via amino acid analysis (AAA) on an ACQUITY-UPLC with AccQ Tag Ultra-UPLC column calibrated with Pierce Amino Acid Standard.

With 10 peptides, the MALDI read-out system was briefly cheked for peptide detection. Commonly used MALDI matrices available in house were 2,5- dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (CHCA). After MALDI measurement, each peptide intensity was normalized on the reference peptide Y. Here we have dried many different measurements and the peptide Y was always well ionized. Figure 3-2 shows how differently each peptide was ionized in one spot measurement by using different MALDI matrices (DHB and CHCA).

Figure 3-2: MALDI matrix test. The same concentration of each peptide with 1pmol/µl was applied and the intensity for each peptide was normalized on that of the peptide Y. Expected was all peptide with equal intensity as the reference Y. 46

Results______

Ideally, equal intensed peaks from all 10 peptides were expected. However, as we know that the ionization efficiency is sequence specific in MALDI, the difference of peptide intensity measured in MALDI is inevitable. Using CHCA, only 5 peptides could be identified and phosphopeptides 2S2pS, 3pY, pS, 2pY and YpY were absent, while DHB matrix could provid ionization for most of the peptides. Therefore, the DHB matrix in MALDI detection was chosen.

Sample washing condition test After standard peptide mixture concentration determined and MALDI matrix chosen, the SPE experimental conditions were probed for further application. The test series were performed with washing conditions changed and the initially suggested sample loading (99.9% MeCN acidified with 0.1% TFA) and elution (MeOH 90%, 4.9%H2O and 0.1%TFA) remained the same. Results are shown in Figure 3-3.

Figure 3-3: MALDI result from SPE washing condition test. The same concentration of each peptide with 1pmol/µl was applied and the intensity of loading, washing and eluting fractions for each peptide were summarized as 100%. Yellow indicates the relative intensity in flow through, red for washing and peptide intensity of elution fraction is indicated with purple color. With reduced water content of 5% in washing condition, all four tyrosine phosphorylated peptides are enriched and identified. However, there are unspecific bindings such as 4S and 2P2pS in purple present.

Results______

Sample loading condition test

SPE loading condition was firstly recommended with 0.1% TFA acidified MeCN. But as we see from the washing condition test in Figure 3-3, the unspecific bindings of 4S and 2S2pS need be moved. Further test in sample loading was conducted to try to remove the unspecific bindings. The fine-tuning of low content of water starting from 20%, 10% to 5% in loading condition tests are presented in Figure 3-4. In the end, 94.9% MeCN 0.1% TFA and 5% water was used for sample loading. As we see here in Figure 3-4, with 10% or 20% of water content, most peptides were found in flow through because the hydrogen binding between phosphotyrosine and the pY-MIP was not strong under high water content, hence we find pY, 2pY and YpY only in flow through.

Figure 3-4: MALDI result from SPE loading condition test in duplicates. The same concentration of each peptide with 1pmol/µl was applied and the intensity of loading, washing and eluting fractions for each peptide were summarized as 100%. Yellow indicates the relative intensity in flow through, red for washing (the same as sample loading respectively) and peptide intensity of elution fraction is indicated with purple color. With reduced water

Results______content 5%, three out of four tyrosine phosphorylated peptides are enriched and identified. There was still minor unspecific binding such as the unphosphorylated peptide 4S. These tests were performed using syringe by hand. Later experiments were further optimized by introducing the table centrifuge for liquid removal with programmed time span and an additional elution step of 50% MeOH, 0.1% TFA in water, therefore we could see 3pY in experiments thereafter.

3.1.2 Comparison results of PETRA and EDMA pY-MIPs

Solid phase extraction (SPE) was performed using micro columns in form of disposable pipette tips packed with imprinted polymer based stationary phases. For the comparative study, a mixture of 13 peptides (Table 3-1) was used, designed deliberately with and without phosphorylations, with mono and multiple phosphorylations at either serine or tyrosine. The purpose of using the peptide mixture is to address and understand the behavior of the two MIPs in terms of side chain selectivity (pY vs. pS side chains) and degree of phosphorylation. After loading of the mixture the resulting flow through fractions were compared with elution fractions with respect to the presence of tyrosine-, serine- and non-phosphorylated peptides.

Figure 3-5: MALDI-TOF/TOF-MS analysis of phosphorylated peptides when peptide mixture was applied for solid phase extraction (1 pmol of each peptide); (A) flow through from P1 pY MIP, showing 4 non-phosphorylated peptides and 3pY; (B) elution from P1, showing 8 out of 9 phosphorylated peptides were enriched without distingushing 49

Results______tyrosinephosphorylation from serinephosphorylation; (C) flow through from P2 pY-MIP, showing not only 4 non-phosphorylated peptides but also 4 serinephosphorylated peptides went through; (D) elution from P2, all 4 tyrosinephosphorylated peptides enriched.

P2 pY-MIP prepared using the hydrophilic cross-linker pentaerythritol triacrylate (PETRA) was first compared with pY-MIP P1 (Figure 3-5) made using the more hydrophobic cross-linker ethylene glycol dimethacrylate (EDMA) (Helling et al. 2011).

In Figure 3-6 we compare the structure and see that on PETRA (left) there is a hydroxide group, making the cross-linker more hydrophilic than EDMA.

Figure 3-6: Structure of the cross-linkers pentaerythritol triacrylate (PETRA) on the left and ethylene glycol dimethacrylate (EDMA) on the right side. The hydrophilicity of PETRA is higher than EDMA due to its hydroxide group.

As indicated in Figure 3-5, although P1 enriched monophosphorylated and multiphosphorylated peptides, no discrimination between serine and tyrosine phosphorylated peptides was observed. This contrasted with the behaviour of P2 pY- MIP. The flow through fractions contained in this case most of the non- phosphorylated and serine-phosphorylated peptides while all tyrosine- phosphorylated peptides could be detected in the elution fraction as shown in Figure 3-5. Based on these findings, we continued to compare the performance of the P2 pY-MIP SPE with two established phosphoenrichment techniques based on TiO2 and anti phosphotyrosine antibodies.

3.1.3 Comparison results of pY-MIP, TiO2 and anti-pY antibodies (standard peptides)

1. Result of TiO2 SPE phosphopeptide enrichment

TiO2–based affinity enrichment of the model peptide mixture using a well-established protocol was studied as shown in (Material and Method 2.2.7 TiO2 SPE). TiO2 beads reportedly bind all phosphorylated peptides without discrimination of phosphorylation of serine, tyrosine and threonine. As shown in Figure 3-7, the performance of TiO2 for

Results______phosphopeptide enrichment is illustrated with increasing sample amount and only when peptide amount applied is high (100 pmol), 2Y2pY was able to be detected.

Figure 3-7: MALDI-TOF spectra of elution fractions from TiO2 beads, when (A):1 pmol each peptide applied 8 out of 9 phosphopeptides were found without pYpY; (B): 10 pmol each peptide applied, showing the same identification as when 1 pmol each peptide applied, without the enrichment of pYpY; (C):100 pmol each peptide used for solid phase extraction, with the identification of all 9 phosphorylated peptides.

At sample load of 1 pmol and 10 pmol each peptide, the doubly phosphorylated peptide pYpY was not able to be detected via TiO2 SPE. Notably however, the doubly tyrosine-phosphorylated peptide pYpY could only be detected at higher sample loads (100 pmol) (Figure 3-7C) presumably due to the strong affinity and slow displacement of pYpY from TiO2 columns. This contrasted with the pY-MIP SPE where pYpY was detectable at 1pmol sample loads (Figure 3-5D) and showed that MIP SPE allows a more sensitive detection of tyrosine-phosphorylated peptides. In brief, SPE using P2 pY-MIP is much more sensitive than TiO2 when sample load is as low as 1pmol.

2. Result of phosphopeptide enrichment via three anti-pY antibodies

The performance of three different anti-phosphorytosine antibodies were assessed and compared with the TiO2 and pY-MIP further. Three generic anti phosphotyrosine antibodies 4G10 (Merck), pY100 (Cell Signalling) and pY20 (Biozol) widely used in

Results______the field of phosphotyrosine research were used to carry out immunoprecipitation (IP) with the 13 peptide mixture in a manner of increasing sample amount (1pmol, 5pmol, 10 pmol each peptide). Only the triply tyrosine phosphorylated peptide (3pY) was enriched by all pY-antibodies. Resuls were shown in Figure 3-8.

1).

2).

3). Figure 3-8: MALDI-TOF spectra of immunoprecipitation (IP) elution fractions with 3 µg pY-antibodies 1). 4G10; 2). PY20; 3). PY100 when (A):1 pmol each peptide applied; (B):5 1. 52

Results______pmol each peptide applied; (C):10 pmol each peptide used for IP. At 1 and 5 pmol peptide sample load, all three antibodies could only enrich the triply tyrosinephosphorylated peptide 3pY.When the sample load was increased to 10 pmol, IP could identify pY and 3pY using 4G10 antibody.

Results show that when low peptide amount was applied, all three antibodies were able to detect only one tyrosine-phosphorylated peptide 3pY, while the P2 pY-MIP was able to enrich all four tyrosine phosphorylated peptides (pY, YpY, pYpY, 3pY) at small sample amounts corresponding to 1 pmol of each peptide (Figure 3-5D).

3. Deeper understanding of the conventional anti-pY antibodies

To better comprehend the selectivity and sensitivity of the antibodies, an excessive sample of 200 pmol of each peptide was loaded. Tyrosine phosphorylated peptides could now be detected by MALDI-MS with all antibodies. Hence, all tyrosine phosphorylated peptides (pY, YpY, 2pY, 3pY) were selectively enriched via the pY antibody PY100 at high amounts of 200 pmol each (Figure 3-9C).

Figure 3-9: MALDI-TOF spectrum of immunoprecipitation (IP) elution fraction for peptide mixture using (A): 3 µg 4G10; (B): 3 µg PY20; (C): 3 µg PY100, when 200 pmol each peptide used for IP. PY100 antibody was able to enrich all four tyrosine-phosphorylated peptides, whereas 4G10 and PY20 have unspecific bindings listed in Table 3-2.

Results______

Table 3-2: Results of different antibodies tested at high sample amount 200 pmol each peptide

pTyr-antibody clone Enriched pTyr.peptides Unspecific bindings 4G10 pY, YpY, pYpY, 3pY 4S, 2S2pS PY20 pY, pYpY, 3pY Y, 3Y, 4S,2S2pS PY100 pY, YpY, pYpY, 3pY -

From the results above, we could see that all antibodies worked well and they could all enrich four tyrosine phosphorylated peptides when high sample load was applied. However, when the sample load was as low as 1 pmol, three tyrosine phosphorylated peptides were not identified via IP.

In contrast to antibodies, all tyrosine-phosphorylated peptides were enriched by P2 pY MIP with minimal concentration of 1 pmol (Figure 3-10).

Figure 3-10: MALDI spectra of peptide fractions after enrichment of the peptide mixture (1 pmol of each peptide) (A) elution from P2 pY-MIP, all tyrosinephosphorylated peptides were enriched and unspecific bindings were observed as T-SpS, SpS, 4S and

2S2pS; (B) elution from TiO2 Column, 8 out of 9 phosphopeptides enriched, 2S2pS not found; (C) elution fraction after immuniprecipitation from phosphotyrosine antibody 4G10 (3 µg, Merck); (D) elution fraction after immuniprecipitation from phosphotyrosine antibody PY20 (3 µg, Biozol); (E) elution fraction after immuniprecipitation from phosphotyrosine antibody PY100 (3 µg). Three antibodies could find only 3pY when loaded with 1pmol each peptide.

Results______

4. Results of IP with combination of anti-pY antibodies

A combination of 4G10 and PY100 is recommended (Tinti, M. et al. 2010) for the identification or the affinity purification of a larger number of tyrosine phosphorylated peptides for high sample input application. Our study with standard peptide mixture complemented that this combination of antibodies can also lead to an increase in unspecific bindings (Figure 3-11).

Figure 3-11: MALDI-TOF spectrum of immunoprecipitation elution fraction using 2 µg pY-antibody PY100 and 2 µg 4G10, when peptide mixture having 200 pmol each peptide used for IP. Unspecific bindings 4S, 2S2pS are due to the cross-reactions of the antibody 4G10.

In Figure 3-11, peptides as 4S, 2S2pS were also unspecifically bound, due to the cross-reactions of the anti-pY antibody 4G10. Here we could see that conventional antibodies were not able to purify the targeted antigen all the time. Out of three antibodies, only PY100 enriched four tyrosinephosphorylated perfectly without any unspecific bindings in this peptide mixture. Nevertheless, the sample or antibody amount is crucial in IP, non-sufficient sample load (1, 5 10 pmol) in this work resulted in only partial identification of 3pY.

3.1.4 Comparison results of pY-MIP, TiO2 and anti-pY antibodies (spiking experiment)

Encouraged by the above results, the SPE performance of P2 pY-MIP was further investigated for a complex sample and was again benchmarked against TiO2 and

Results______antibodies. The peptide mixture (1 pmol each peptide; ca. 1 ng) was spiked in a digested mouse brain lysate. The MALDI-MS approach was capable of detecting the target peptides easily.

1. Results of pY-MIPperformance

In the spiking experiment result, P2 pY-MIP was able to enrich all four tyrosine phosphorylated peptides out of the complex sample when sample load was as low as 500 fmol. Lower level of spiking was not tested due to the MALDI detection limit.

Figure 3-12: MALDI-TOF spectra of elution fractions from P2 pY-MIP, when (A):0.5 pmol of each peptide spiked into 3.36 µg digested mouse brain lysate; (B):1 pmol of each peptide spiked into 3.36 µg digested mouse brain lysate. In both cases, all four tyrosine phosphorylated peptides were enriched.

All four tyrosine phosphorylated peptides were enriched by P2 pY-MIP. Unspecific bindings 4S and 2S2pS were observed as shown in Figure 3-12

2. Results of TiO2 SPE performance

The same level of spiking was tested for TiO2 SPE. Nevertheless, when the amount of spiked peptides was decreased to 0.5 pmol, a loss of 2S2pS, pYpY and YpY was observed for the TiO2 method (Figure 3-13), whereas pY-MIP was still able to enrich all tyrosine-phosphorylated peptides as shown in Figure 3-12 56

Results______

Figure 3-13: MALDI-TOF spectra of elution fractions from TiO2 beads, when (A): 0.5 pmol of each peptide spiked into 3.36 µg digested mouse brain lysate; (B):1 pmol of each peptide spiked into 3.36 µg digested mouse brain lysate and used for enrichment experiment. The peptide pYpY was missing in both experiments. The other doubly phosphorylated peptides 2S2pS was enriched only when sample load is higher as 1pmol.

As expected, peptides with multiple phosphorylations were not eluted from the TiO2 SPE column easily. With increased amount of spiked peptide standard at 1 pmol, doubly phosphorylated peptide 2S2pS was observed in Figure 3-13B. The peptide pYpY was not able to be enriched at both spiking levels.

3. Results of IP performance using 3 anti-pY antibodies

In IP experiments using three different anti-pY antibodies, we tested three different sample load of 1 pmol, 5 pmol and 10 pmol each peptide spiked in the trypsinized mouse brain sample. Observed was that the three antibodies behaved similar in comparison to the test with only peptide standard mixture as demonstrated in Figure 3-8. At 1 and 5 pmol level of spiking, only the phosphotyrosine peptide 3pY was able to be enriched via IP in all three experiments. Increased to 10pmol, PY20 and PY100 were also able to enrich the tyrosine phosphorylated peptide pY.

Results______

1).

2).

3).

Figure 3-14: MALDI-TOF spectra of immunoprecipitation (IP) elution fractions with 3 µg pY-antibodies 1). 4G10; 2). PY20; 3). PY100 when (A):1 pmol each peptide spiked into 3.36 mouse brain digest; (B):5 pmol each peptide spiked into 3.36 µg mouse brain digest; (C):10 pmol each peptide spiked into 3.36 µg mouse brain digest. At 1 and 5 pmol peptide sample load, all three antibodies could only enrich the triply tyrosine-phosphorylated peptide 3pY.

Results______

When the sample load was increased to 10 pmol, IP could identify pY and 3pY using PY20 and PY100 antibodies.

This result shows that P2 pY-MIP worked favorably in complex samples for both mono- and multiply tyrosine phosphorylated peptides using standard peptide mixture or peptides spiked in complex mouse brain digest. In addition, the method relying on P2 pY-MIP displayed the highest sensitivity of all methods as reflected in the enrichment of all four tyrosine phosphorylated peptides from complex mouse brain lysate (Figure 3-12). However, when we further went on for real sample application loading 10 µg SCX fractionated peptides, pY-MIP was not able to enrich tyrosine- phosphorylated peptides from the complex cell digestion (see discussion P2 pY-MIP efficiency).

In contrast, antibodies and TiO2 failed at lower sample loads (Figure 3-13, 3-14).

Real sample application using 10 µg with TiO2 or anti-pY antibodies were not performed but this is for sure not possible. However, when sample amount is as much as milligram range, both methods worked (Palma et al 2013). We can conclude here that IP and TiO2 are very useful methods as long as sample amount is sufficient. The plastic antibody P2 pY-MIP could work well in standard peptide mixture application and spiking experiment. Real sample application is not possible in this work.

3.2 Results of pS-MIP phosphopeptide enrichment from biological samples

The pS-MIP material was synthesized by collaboration partner Dr. Sudhirkumar Shinde and the template rebinding test was performed in an offline HPLC to show the imprinting effect and compared together with P2 pY-MIP template rebinding test as shown in Figure 3-16. The binding capacity for both pY-MIP and pS-MIP were determined as ca. 1 nmol/mg, meaning that 1 mg polymer could bind to 1nmol target template.

3.2.1 Template rebinding test (research conducted by Dr. Shinde in TU- Dortmund)

The pS-MIP imprinting effects were first assessed by chromatography using the crushed polymer monoliths as a stationary phase. Thus, the pS-MIP template Fmoc

Results______amino acid derivative was injected onto the columns in an acetonitrile rich mobile phase acidified with TFA. The UV chromatogram of the HPLC binding test is shown below in Figure 3-15.

Figure 3-15: HPLC binding test of the phosphoserine derivative Fmoc-pSer-OH for MIP and NIP polymers in acidic buffered mobile phase (MeCN:water:TFA=95:5:0.1). Injection Volume: 5 µl, flow rate: 0.5 ml/min, DAD at 254 nm.

In Figure 3-15 we can see that when NIP was applied as the stationary phase, the Fmoc-pSer-OEt was eluted very early in the first ten minutes (blue), while it retained much more strongly on pS-MIP column since we detected nothing using pS-MIP as the LC column eluted with the given mobile phase (red). The P2 pY-MIP template rebinding test was performed in the same manner. Both MIPs selectively retained pS and pY Fmoc derivates with preference for the complementary amino acid side chains whereas the non-imprinted polymer displayed weak retentivity for all analytes (Figure 3-16).

Figure 3-16: retention factor (k) of Fmoc protected amino acids injected onto pS- and pY- MIP and NIP columns in different mobile phases. (A) (MeCN:water:TFA=95:5:0.1) (B) (MeCN:water:TFA = 50:50:0.1). Injection Volume: 5 µl, flow rate: 0.5 ml/min, detection absorbance at 254 nm.

This effect depends strongly on the aqueous content in the mobile phase. Whereas the pS-MIP showed optimum discrimination between Fmoc-pS and Fmoc-pY in a mobile phase containing 5% water, the P2 pY-MIP operated best in more aqueous mobile phases. However, when further P2 pY-MIP SPE was performed, we observed

Results______that high water content could not retain tyrosine-phosphorylated peptides (Figure 3-3 and 3-4), which is contradictive to the template rebinding test condition (MeCN:water:TFA = 50:49.9:0.1) using only Fmoc-pTyr-OH single amino acid derivate. The final SPE condition for P2 pY-MIP was decided based on its performance using standard peptide mixture. We suspect that the behaviour of P2 pY-MIP binding to single amino acid derivate Fmoc-pTyr-OH is different from binding to the tyrosine-phosphorylated peptides.

3.2.2 Results of pS-MIP SPE sample loading and elution condition test

From the template rebinding test, the suggested condition for sample loading was 95% MeCN, 4.9% water and 0.1% TFA for pS-MIP. The SPE loading condition for pS-MIP was tested with reduced water content starting from 20% water as indicated in Figure 3-17.

Figure 3-17: MALDI result from pS-MIP SPE loading condition test in duplicates for 10% and 5% water content and single experiment in 20% water content. The same concentration of each peptide with 1pmol/µl was applied and the intensity of loading, washing and eluting fractions for each peptide were summarized as 100%. Yellow indicates the

Results______relative intensity in flow through, red for washing (the same as sample loading respectively) and peptide intensity of elution fraction is indicated with purple color. With reduced water content 5%, serine-phosphorylated peptides SpS and GpS could be identified in one trial with slight improvement than loading with higher water content. These SPE were performed by using syringe to remove the liquid by hand. The improved results later were achieved by introducing table centrifuge to program the experimental time for each step and by an additional elution step in Figure 3-18.

With decreased water content of sample loading, we expect that all serine phosphorylated peptides (2S2pS, pS, SpS and GpS) could be identified in elution fraction. However, the results indicated unselective bindings of tyrosine phosphorylated peptides pYpY and YpY. Moreover, the enrichment of serine- phosphorylated peptides were as good as expected. We suspected that the release of the target serine-phosphorylated peptides were not thorough, therefore, we added more elution steps to probe for better elution performance.

Figure 3-18: MALDI result from pS-MIP SPE sequential elution condition test in duplicates. The same concentration of each peptide with 1pmol/µl was applied and the intensity of loading, washing and eluting fractions for each peptide were summarized as 100%. Green indicates the relative intensity in flow through with the condition 95% MeCN, 4.9% water and 0.1% TFA; pink for washing (the same as sample loading respectively, with almost nothing identified); yellow for elution 1 90% MeOH, 9.9% water and 0.1% TFA; red for elution 2 50% MeOH, 49.9% water and 0.1% TFA; blue for aqueous buffer at pH= 7.8; black

Results______for basic buffer at pH = 10.8. When eluting with higher water content of 49.9% a second time, release of T-SpS and SpS was improved (red).

The results above show that loading condition with 4.9% water, 95% MeCN, 0.1 TFA was chosen and the addition of eluting step with 49.9% water could slightly improve the release of serine-phosphorylated peptides. However, the pS-MIP SPE results also suggest that the unselective bindings are present, as we can see that all tyrosine-phosphorylated peptides are found in the SPE test. These experiments at the method development stage were all performed by using syringe manually. Later when we further optimized the method using table centrifuge to remove the liquid, we could better control the time span of sample loading, washing, elution steps regarding the SPE. Moreover, an additional elution step using 50% MeOH, 0.1%TFA and water was applied, which improved the elution efficiency (red columns in Figure 3-18) than eluting with only 1 step (yellow columns in Figure 3-18 and Figure 3-17). The laborious SPE using hand for liquid removal was replaced by 20 minutes low spin centrifugation, which dramatically improved the SPE robustness and reproducibility.

3.2.3 Further probing for phosphopeptide recognition using pS-MIP

The orthogonality of the receptor was further demonstrated in cross comparisons and experiments involving a standard peptide mixture, sequential elution and spiked mouse brain extracts. The versatility of the MIP receptors was then demonstrated for four different biological samples (trypsinized HEK 293T and SH-SY5Y cell lines, mouse brain and human cerebrospinal fluid (CSF)), paying special attention to the minimum required sample amounts, analytical throughput and amino acid sequence bias.

Based on the mobile phase compositions identified as optimal for the amino acid solutes, further pS-MIP SPE experiments for the capture of serine phosphorylated peptides using standard peptide mixture were performed.

The first goal was to apply the pY- and pS-MIPs for fractionating a mixture of mono and multiply phosphorylated peptides listed in Table 1 into three fractions – a tyrosine phosphorylated peptide fraction, a serine-phosphorylated peptide fraction and a fraction lacking phosphorylated peptides. 63

Results______

Figure 3-19: MIPs SPE work flow of sequential elution of the standard peptide mixture and MALDI-TOF/TOF-MS confirmation. A mixture of 12 peptides (1 pmol each) was loaded in loading solution on P2 pY-MIP column first. The flow-through was collected and loaded on the pS-MIP column. Bound peptides from pY-MIP (c) and pS-MIP (b) were eluted and measured. The flow through fraction from pS column (a) was also collected for MALDI- TOF/TOF-MS analysis.

The resulting MALDI-TOF/TOF-MS spectra of the flow through and elution fractions are shown in the Figure 3-19. Hence the pS-MIP enriched 3 out of 4 of the serine- phosphorylated peptides, whereas the pY-MIP in contrast captured 3 out of the 4 tyrosine-phosphorylated peptides, too. Both MIPs demonstrate a small degree of unspecific binding to the opposite phosphopeptides, which is consistent with the template rebinding test in Figure 3-16. These experiments provided first hint for site- selectivities of the polymer and encouraged us to study the phosphopeptides binding capabilities in more complex biological matrices.

3.2.4 Results of pS-MIP SPE taggeting the single peptide spiked in mouse brain matrix

To further consolidate the site selectivity of the pS-MIP we performed mouse brain protein lysate spike in experiments. This experimental design may further prove if the plastic antibody pS-MIP could be applied in "real sample analogue" at first place. Hereto, a phosphopeptide belonging to the N-terminus of the mature serine protease HtrA2/Omi containing a phosphoserine motive (SpS: AVPSPPPApSPR)

Results______

(concentration 5 fmol, 10 fmol, 50 fmol, 100 fmol, 0.5 pmol, 1 pmol and 2 pmol) was used.

To facilitate the initial detection, a high level spiking (2 pmol, 1pmol and 0.5 pmol) of the SpS peptide in a digested mouse brain protein lysate (1.68 µg) was performed and the samples were subjected to pS-MIP enrichment. SPE flow through and elution fractions were measured by MALDI-TOF/TOF-MS as shown in Figure 3-20, 3-21, 3- 22 and 3-23.

Figure 3-20: MALDI-TOF/TOF-MS spectra of pS-MIP treated SPE fractions of 2 pmol SpS peptide spiked into 1.68 µg digested mouse brain lysate. (a) elution fraction contained only SpS. (b) flow through fraction consisting comlex peptide ions.

In Figure 3-20 we see that the spiked target peptide SpS also appeared in the flow- through fraction, implying that the spiked peptide level should be reduced. So we continued the experiment with low level of spiked peptide then measured the fractions from SPE on MALDI (Figure 3-21 and 3-22).

Results______

Figure 3-21: MALDI-TOF/TOF-MS spectra of pS-MIP treated SPE fractions of 1 pmol SpS peptide spiked into 1.68 µg digested mouse brain lysate. (a) elution fraction contained only SpS. (b) flow through fraction consisting comlex peptide ions.

Figure 3-22: MALDI-TOF/TOF-MS spectra of pS-MIP treated SPE fractions of 0.5 pmol SpS peptide spiked into 1.68 µg digested mouse brain lysate. The SPE flow through fraction (a) contained a complex mixture of peptide ions. Contrary, displayed SPE elution fractions of 0.5 pmol (b) spiked peptide clearly demonstrates the SpS peptide selectivity of pS-MIP.

Down to 0.5 pmol spiked peptide in SPE eluates was able to be detected demonstrating clear SpS peptide enrichment (Figure 3-22). Subsequent

Results______fragmentation analysis of the SpS precursor ion (m/z 1155.556) confirmed peptide identity as shown below.

Figure 3-23: MALDI-TOF/TOF-MS/MS of pS-MIP enriched peptide ion m/z=1155.556 confirmed identity of the SpS peptide assisted by theoretical fragment interpretation and by reference examinations using MS/MS fragmentation of the pure peptide. (a) MS/MS fragmentation of the precursor ion in standard pure peptide (b) MS/MS fragmentation of the precursor ion in the elution fraction of 2 pmol spiking experiment.

Elution from lower level spike-in (5 fmol, 10 fmol, 50 fmol, 100 fmol) were measured further on a more sensitive nano LC-ESI-MS/MS system (Q ExactiveTM Thermo Scientific) and MS/MS spectra shown in Figure 3-24, 3-25, 3-26 and 3-27. The unambiguous assignment of this peptide by data base searches was hampered by limited availability of sequence explaining fragment ions, due to the prominent cleavage at three of the four prolines within the amino acid sequence and neutral loss of H3PO4. Therefore, the MS fragments assignment was only possible by de- novo interpretation using the peptide specific fragmentation fingerprint provided by ProteinProspector v 5.12.1 online. The employed nano LC-ESI-MS/MS analysis was able to identify down to 5 fmol of the spiked peptide in the elution fraction.

Results______

Figure 3-24: NanoLC-ESI-MS/MS spectrum of pS-MIP elution fraction from 100 fmol SpS peptide spiked to 1.68 µg mouse brain lysate. Spiking : lysate = 6.8 : 10000 (w/w)

Figure 3-25: NanoLC-ESI-MS/MS spectrum of pS-MIP elution fraction from 50 fmol SpS peptide spiked to 1.68 µg mouse brain lysate. Spiking :lysate = 3.4 : 10000 (w/w).

Results______

Figure 3-26: NanoLC-ESI-MS/MS spectrum of pS-MIP elution fraction from 10 fmol SpS peptide spiked to 1.68 µg mouse brain lysate. Spiking : lysate = 6.8 : 100000 (w/w)

Figure 3-27: NanoLC-ESI-MS/MS identification of 5 fmol pS-MIP enriched SpS peptide spiked into a complex mouse brain protein digest. (a) amino acid sequence explaining fragment ions are limited to peptide typical prominent proline breakages. By manual spectrum interpretation the peptide AVPSPPPApSPR could unequivocally be assigned. (b) extracted ion chromatogram of the spiked peptide SpS.

These results from nanoLC-ESI-MS/MS identification suggest promising sensitivity and selectivity in targeting the spiked peptide AVPSPPPApSPR with serine-

Results______phosphorylation site, which implied further application in targeting the endogenous phosphopeptides directy in biological sample was possible.

3.2.5 Results of pS-MIP SPE application in human cell samples and comparison with TiO2 SPE

In the next step, the work was focused in the potential of pS-MIP for a global phosphopeptide enrichment of serine phosphorylated peptides from a real sample— trypsinized complex protein lysate. HEK 293T cells were firstly used. After cell lysis, proteins were tryptically digested into peptides. In order to reduce sample complexity after the tryptic digestion, and to enhance the phosphopeptide recovery for the SPE, the digest was pre-fractionated by SCX chromatography. The workflow and planned experiments are shown in Figure 3-28.

Figure 3-28: Work flow of phosphoproteomic analysis using SCX prefractionation and pS-MIP/TiO2 SPE. HEK 293T cells were harvested in the cell culture, lysed to obtain the 70

Results______protein lysate. The proteins were then digested with trypsin. The protein digest was fractionated with SCX and the fraction from the retention time 4-40 min was collected and applied to pS-MIP or TiO2 SPE.

The experiment was performed firstly without SCX, using 10 µg cell digest directly on the pS-MIP and TiO2. The results suggested further improvement. As we have a well developed fractionation offline method SCX, it was combined prior to the pS-MIP SPE. In total, seven different fractions were analyzed in technical replicates n=3 for all 10 µg sample load analysis and n=2 for TiO2 approach with 100 µg sample load as shown in Figure 3-29.

Figure 3-29: Phosphopeptides identified after nano LC-ESI-MS/MS analysis of HEK 293T cell lysate digest. The content of tryptic phosphopeptides was determined after measurement of the unfractionated sample (I), SCX prefractionation (II), pS-MIP (III) or TiO2

SPE (IV) as well as after SCX fractionation in combination with pS-MIP (V) or TiO2 SPE (VI).

SPE sample loads of 10 µg were used for the pS-MIP and TiO2 methods (n = 3) and 100 µg

(*) for an additional TiO2 SPE (VII) (n = 2). The mean values of identified phosphopeptides (column diagram) revealed maximal phosphopeptide recovery for combined SCX/pS-MIP purified samples. An overview of phosphopeptides identified in all replicates (Venn diagram) pointed out an overlap of 17 phosphopeptides detected for all analyses as well as huge differences in selectivity of the different approaches demonstrated by e.g. 398 SCX+pS-MIP and 182 in TiO2* specifically enriched peptides.

We analysed the unfractionated lysate, the TiO2 and pS-MIP treated samples, SCX prefractionated peptides as well as TiO2 and pS-MIP in combination with SCX. The number of identified phosphorylated peptides was counted via Proteome Discoverer V1.4 analysis, including a false discovery rate approach and the phosphoRS 3.1 tool 71

Results______for the correct assignment of phosphorylations sites (Chapter 2.2.10). In the unfractionated sample (I), 17 phosphopeptides were found. After pS-MIP SPE (III) was performed on 10 µg digested HEK 293T cell lysate, this number increased to 237. After reduction of the sample complexity by SCX pre-fractionation of the complex peptide samples, the number of identified phosphopeptides roughly doubled to 39 (II) and in combination with the pS-MIP enrichment, the number of identified phosphopeptides reached 924 (V). Technical replication (n = 3) of these analysis clearly shows acceptable repeatibility with 610 phosphopeptides identified in all three measurements. Our phosphopeptide extraction yield using pS-MIP combined with

SCX (V) exceeded the yield achieved by TiO2 beads using the same and also ten- fold higher sample loads of 100 µg digested proteins with maximal positive identifications of 711 phosphopeptides (VII).

To assess the effectiveness of the pS-MIP approach in different cell types, samples were also analyzed using a tryptic digest of the human neuroblastoma cell line SH-

SY5Y. In this case, 1,403 phosphopeptides from 100 µg of digest via TiO2 SPE alone were identified. By using pS-MIPs alone with 10 µg of peptides from the SH-SY5Y cell digest, 648 phosphopeptides were found; by adding SCX pre-fractionation, a total of 1,271 phosphopeptides were observed, a greater number than that obtained using HEK 293T cells. These data show that general phosphopeptide availability strongly depends on sample type.

All of these results demonstrated significant performance improvement with the plastic antibody, which remained upon more stringent data interpretation by neglecting unrepeatable phosphopeptide identifications as illustrated in the Venn diagram in Figure 3-28. With respect to the five relevant enrichment approaches, the results revealed capture phase and sample load-dependent differences in phosphopeptide specificities. A total of 476 phosphopeptides were found exclusively by pS-MIP based analyses (III and V), while 274 TiO2-specific peptides from 3 groups of analyses (IV, VI, VII) were found. Based on GPMAW lite analysis to determine the amino acids distribution from both groups of exclusively enriched phosphopeptides together with the untreated lysate sample in Figure 3-29, TiO2 preferred phosphopeptides with more of the acidic amino acid residues aspartic acid (D) and glutamic acid (E). Encouraged by this observation the data sets for spefic motifs using the motif-x online tool (Schartz et al. 2005 and Chou et al. 2011) was analyzed

Results______by Markus Hermann Koch and found the motif xxxSPxx to be specific for pS-MIP, and the motifs xxxSxxE and xxxSExx to be specific for TiO2 (Figure 3-30, Table 3-5 and Table 3-6).

Numerous overlapping peptides identified with the other approaches were also detected, as well as 17 peptides that were present in all of the analyses. These 17 peptides were all phosphorylated at serine and were likely present at a higher abundance in the sample based on the numbers of spectra matched to each peptide (Table 3-3).

Table 3-3: Repeatably nanoLC-ESI-MS/MS identified phosphopeptides present in all pS-MIP and previous TiO2 SPE experiments.

Sequence Accession PS Modification Ion [M+H]+1 M score§ SSSPAPADIAQTVQEDLR Q13283 69 S3(Phosp) 94 1964,89702 SASSDTSEELNSQDSPPK O14745 47 S3(Phosp) 113 1958,78569 EGEEPTVYSDEEEPKDESAR O00264 45 S9(Phosp) 49 2375,9395 HTGPNSPDTANDGFVR P31943;P5579 44 S6(Phosp) 81 1764,73381 5 FASDDEHDEHDENGATGPVK P05455 39 S3(Phosp) 86 2249,8604 ESEDKPEIEDVGSDEEEEKK P07900 36 S13(Phosp) 55 2400,98223 YGLQDSDEEEEEHPSK P52948 32 S6(Phosp) 69 1971,74504 KPVTVSPTTPTSPTEGEAS Q9Y6G9 32 S12(Phosp) 40 1965,90679 FEEESKEPVADEEEEDSDDDVEPITEF P54105 32 S17(Phosp) 107 3394,35385 R ESEDKPEIEDVGSDEEEEK P07900 31 S13(Phosp) 48 2272,8897 NKPGPNIESGNEDDDASFK O60841 30 S9(Phosp) 47 2113,87139 GNSRPGTPSAEGGSTSSTLR P35269 25 S3(Phosp); 70 2078,85624 T7(Phosp) VFDDESDEKEDEEYADEK O43719 23 S6(Phosp) 91 2271,83306 FNDSEGDDTEETEDYR Q9NYF8 21 S4(Phosp) 112 2001,68633 SPVSTRPLPSASQK Q8ND56 21 S1(Phosp) 46 1534,76201 DHSPTPSVFNSDEER Q6UN15 19 S3(Phosp) 61 1796,71306 SPVPSAFSDQSR Q9UQ35 14 S1(Phosp) 50 1357,57854 PSM: Peptide spectra match; §: maximal ion score of the Mascot search algorithm

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Figure 3-30: Characterization of exclusively identified phosphopeptides by TiO2 and pS-MIP based methods. (a) amino acids distribution of the trypsinized peptides identified in the lysate, 274 phosphopeptides exclusively identified by TiO2, and 476 phosphopeptides exclusively identified by pS-MIP based analysis. (b) Peptide motif from exclusively identified phosphopeptides via pS-MIP. (c) and (d) Peptide motif from exclusively identified phosphopeptides via TiO2. The peptides used for analysis were those identified in all replicates. (b,c,d) graphical representations of the motifs were generated by motif-x for all length 7-subsequences with center phosphorylated S as found in peptides identified by either enrichment method. Motif-x significance was at 0.0005, occurrences at 20.

3.2.6 Results of pS-MIP SPE application in clinical relevant samples

Given the compatibility of the pS-MIP approach with low sample loads, attention was focused on the analysis of scarce biological and clinically relevant samples, using the SCX/pS-MIP enrichment approach to analyze 10 µg of trypsinized human CSF and mouse brain samples. In non-depleted CSF lysates, identified were 29 and 25 phosphopeptides from in-gel digested and in-solution digested samples, respectively. In addition, 18 phosphopeptides from depleted CSF (14 highly abundant proteins were depleted). Collectively, 47 phosphopeptides were detected from 24 proteins (Table 3-4 and Attachment 1), among which the work report for the first time 8 phosphoproteins with a total of 19 phosphopeptides in human CSF. The 16

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overlapping phosphoproteins confirmed former results published (Bahl et al 2008)

obtained by TiO2-based phosphopeptide enrichment of 200 µg filtered CSF protein digest. In the mouse brain samples, a total of 673 phosphopeptides were identified, underlining the versatility of the SCX/pS-MIP approach for the analysis of complex biological and clinical samples.

Table 3-4: nanoLC-ESI-MS/MS-identified phosphorylated proteins present in human CSF

treated with SCX/pS-MIP and compared with the previous TiO2 research

Number Protein name Protein Group Accessions Found by TiO2 1 Golgi intergral membrane protein 4 O00461 no 2 Apolipoprotein L1 O14791 yes 3 Extracellular matrix protein 2 O94769 yes 4 Cystein C P01034 yes 5 Ig kappa chain C region P01834 no 6 Apolipoprotein E P02649 yes 7 Serum Albumin P02768 yes 8 Secretogranin-1 P05060 yes 9 Amyloid beta A4 P05067 yes 10 Osteopontin P10451 yes 11 Chromogranin-A P10645 yes 12 Clusterin P10909 no 13 Coagulation Factor P12259 no 14 Secretogranin-2 P13521 yes 15 Versican core protein P13611 yes 16 Cadherin-2 P19022 yes 17 Inter-alpha-trypsin inhibitor heavy chain H2 P19823 yes 18 Actin,cytoplasmic P60709 no 19 Nucleobindin-1 Q02818 no 20 SPARC-like protein 1 Q14515 yes 21 Rho GTPase-activating protein 22 Q7Z5H3 no 22 Golgi intergral membrane protein 1 Q8NBJ4 yes 23 Secretogranin-3 Q8WXD2 yes 24 Receptor-type tyrosine-protein phosphatase N2 Q92932 no

The above results demonstrate the high potential of the pS-MIP as a new tool for the enrichment of serine phosphorylated peptides. This was clearly demonstrated by the application in complex HEK 293T cell lysate digest detected by nano LC-ESI- MS/MS, and further application in SH-SY5Y cell digest as well as clinical relevant samples such as CSF and trypsinized mouse brain sample.

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3.3 Analysis of HtrA2/Omi model studies with novel methods in relation to PD

3.3.1 Results from global proteomic analysis of transgenic mouse brain samples

1. LC-MS/MS identified proteins with different ways of regulation

195 proteins were identified regulated when 3 groups of samples were analyzed by statistic tests "R" program (6 months and 12 months sample types), where Anova test was performed to decide which protein has significant regulation and Tuckey Post Hoc test was carried out later to pinpoint the significance between two groups. The filters were applied here with the ANOVA smaller than 0.05. No filters of fold change were applied. However, we define the proteins as confidently identified when they are present at least 5 out of 6 samples per group, and 4 out of 5 samples for G399S mutant effect (6M and 12M).

Among three groups of samples with the same age 6M and 12M, we made several comparisons respectively based on simple combination principle in mathematics as demonstrated in Figure 3-31. Proteins with all types of regulations are discussed later (Chapter 4.2.2)

1).

2).

3).

Figure 3-31: Significant regulations for proteins candidates identified between 2 groups resulted in protein regulations. 1). G399S mutant HtrA2/Omi over-expression effect: significance between NT and G399S and between WT and G399S but no significant difference between NT and WT. 2). WT HtrA2/Omi over-expression effect: significance between NT and WT and between WT and G399S, but no significance between NT and

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G399S. 3). Protein over-expression effect: proteins found regulated due to over-expression of any protein: significance beween NT and WT and between NT and G399S but no significance between WT and G399S.

2. G399S mutant HtrA2/Omi over-expression effect leading to 18 differential proteins in 6-month-old mouse brains, and 12 differential proteins in 12-month- old mouse brains.

Based on the comparison as illustrated in Figure 3-31-1), we identified 18 regulated proteins (Table 3-5 and Figure 3-32) and 12 proteins (Table 3-6 and Figure 3-33) due to the overexpression of G399S mutant HtrA2/Omi protein in mouse brains at 6 months and 12 months old respectively.

Table 3-5: G399S HtrA2/Omi overexpression differential proteins in 6 monthes mouse brains. Gene NT 6M/NT Omi- Omi- (Uniprot) 6M WT OE G339S 6M/ 6M/ NT 6M NT 6M Potassium/sodium hyperpolarization-activated cyclic Hcn2 1 1,02 0,25 nucleotide-gated channel 2 Zinc finger protein 354B Znf354b 1 1,03 0,39 Syntaxin-12 Stx12 1 1,39 0,44 Gap junction alpha-1 protein Gja1 1 0,86 0,58 Cytochrome b-c1 complex subunit 8 Uqcrq 1 1,02 0,74 Reticulon-1 Rtn1 1 1,03 0,78 Cytosolic non-specific dipeptidase Cndp2 1 0,98 1,20 Enoyl-CoA hydratase, mitochondrial Echs1 1 0,96 1,21 60S acidic ribosomal protein P0 Rplp0 1 1,07 1,44 Nucleoside diphosphate kinase B Nme2 1 1,11 1,44 Parkinson disease 7 domain-containing protein 1 Pddc1 1 1,03 1,59 Argininosuccinate synthase Ass1 1 1,14 1,84 Neuroligin-3 Nlgn3 1 1,01 1,93 Serine/threonine-protein phosphatase PP1-gamma catalytic Ppp1cc 1 1,21 1,97 subunit Monofunctional C1-tetrahydrofolate synthase, Mthfd1l 1 1,39 1,98 mitochondrial Rho guanine nucleotide exchange factor 7 Arhgef7 1 0,51 3,30 Serine/threonine-protein phosphatase 6 regulatory subunit Ppp6r3 1 1,02 7,60 3 Splicing factor 3B subunit 3 Sf3b3 1 6,14 15,17

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Figure 3-32: G399S mutant HtrA2/Omi over-expression effect regulated 18 proteins in moue brain at 6 monthes old. The spectral counting result enabled the comparison of the normalized protein level for each protein candidate. The spectra counts (averaged on the biological replicates) ratio between Omi-G399S 6M and non transgenic 6M and between Omi-WT 6M and non-transgenic 6M were plotted for all 18 proteins.

Table 3-6: G399S HtrA2/Omi overexpression differential proteins in 12 monthes mouse brains.

Gene NT Omi- Omi- (Uniprot) 12M/NT12M WT G339S OE 12M/ 12M/ NT NT 12M 12M Serine/threonine-protein Ern2 1 1,19 0,22 kinase/endoribonuclease IRE2 LIM zinc-binding domain-containing Nebulette Nebl 1 1,03 0,43 Diacylglycerol kinase zeta Dgkz 1 0,99 0,61 Mitochondrial import inner membrane Timm50 1 1,06 0,65 translocase subunit TIM50 Ubiquitin-conjugating enzyme E2 L3 Ube2l3 1 0,98 1,34 Tubulin beta-6 chain Tubb6 1 1,02 1,42 6-phosphogluconolactonase Pgls 1 0,91 1,43 Uncharacterized protein C1orf168 homolog Uncharacterized protein 1 0,99 1,60 C1orf168 homolog Small glutamine-rich tetratricopeptide repeat- Sgtb 1 0,99 1,61 containing protein beta Aldose reductase-related protein 1 Akr1b7 1 1,05 3,78 Haloacid dehalogenase-like hydrolase domain- Hdhd3 1 0,99 5,03 containing protein 3 Kinesin-like protein KIFC3 Kifc3 1 2,03 6,30

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Figure 3-33: G399S mutant HtrA2/Omi over-expression effect regulated 12 proteins in mouse brain at 12 monthes old. The spectral counting result enabled the comparison of the normalized protein level for each protein candidate. The spectra counts (averaged on the biological replicates) ratio between Omi-G399S 6M and non transgenic 6M and between Omi-WT 6M and non-transgenic 6M were plotted for all 12 proteins.

3. WT HtrA2/Omi effect leading to 8 proteins differential in 6-month-old mouse brains, and 8 differential proteins in 12-month-old mouse brains

Comparison performed as Figure 3-31-2), we pinpointed the WT HtrA2/Omi over- expression regulated 8 proteins in mouse brains as shown in Table 3-7 and Figure 3- 34) and 8 proteins (Table 3-8 and Figure 3-35) due to the WT HtrA2/Omi protein effect in the mouse brain proteome at 6 months and 12 months old respectively.

Table 3-7: WT HtrA2/Omi overexpression regulated proteins in 6 monthes mouse brains.

Gene NT Omi-WT OE Omi-G339S (Unipro 6M/NT6M 6M/ 6M/ t) NT 6M NT 6M WD repeat-containing protein 44 Wdr44 1 7,56 0,00 Purine nucleoside phosphorylase Pnp 1 1,88 1,08 Transmembrane channel-like protein 7 Tmc7 1 1,75 1,01 DNA-(apurinic or apyrimidinic site) lyase Apex1 1 1,43 1,01 Citrate synthase, mitochondrial Cs 1 0,87 0,97 Eukaryotic translation initiation factor 3 subunit Eif3k 1 0,22 1,17 K Ubiquitin domain-containing protein UBFD1 Ubfd1 1 0,20 1,17 Signal-induced proliferation-associated 1-like Sipa1l1 1 0,17 0,78 protein 1 79

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Figure 3-34: WT HtrA2/Omi over-expression effect regulated 8 proteins in moue brain at 6 monthes old. The spectral counting result enabled the comparison of the normalized protein level for each protein candidate. The spectra counts (averaged on the biological replicates) ratio between Omi-G399S 6M and non transgenic 6M and between Omi-WT 6M and non-transgenic 6M were plotted for all 8 proteins.

Table 3-8: WT HtrA2/Omi overexpression differential proteins in 12 monthes mouse brains.

Gene NT 12M/NT Omi-WT OE Omi-G339S (Uniprot 12M 12M/ 12M/ ) NT12M NT 12M MAP7 domain-containing protein 1 Map7d1 1 3,45 0,60 Protein disulfide-isomerase A6 Ppfia3 1 1,75 1,01 Dystrobrevin alpha Dtna 1 1,62 0,75 Heterogeneous nuclear Syncrip 1 1,45 0,97 ribonucleoprotein Q Unconventional myosin-VI Myo6 1 1,36 1,06 Protein disulfide-isomerase A6 Pdia6 1 1,20 0,87 Beta-actin-like protein 2 Actbl2 1 0,62 1,10 Gap junction gamma-3 protein Gjc3 1 0,34 1,03

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Figure 3-35: WT HtrA2/Omi over-expression effect regulated 8 proteins in moue brain at 12 monthes old. The spectral counting result enabled the comparison of the normalized protein level for each protein candidate. The spectra counts (averaged on the biological replicates) ratio between Omi-G399S 6M and non transgenic 6M and between Omi-WT 6M and non-transgenic 6M were plotted for all 8 proteins.

4. Protein over-expression leading to 17 proteins regulated in mouse brains at 6 months old and 2 at 12 months old.

Based on the comparison as illustrated in Figure 3-31-3), we identified 17 regulated proteins (Table 3-9 and Figure 3-36) and 2 proteins (Table 3-10 and Figure 3-37) due to protein overexpression effect in general at 6 months and 12 months old respectively.

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Table 3-9 Protein overexpression regulated proteins in 6 monthes mouse brains.

Gene NT 6M/NT Omi-WT OE Omi-G339S (Unipro 6M 6M/ 6M/ t) NT 6M NT 6M Ubiquitin carboxyl-terminal hydrolase isozyme L5 Uchl5 Oxysterol-binding protein-related protein 11 Osbpl1 1 3,02 2,90 1 Integrin alpha-V Itgav 1 2,85 2,67 Fumarylacetoacetate hydrolase domain-containing Fahd2 1 1,58 1,93 protein 2A Coatomer subunit delta Arcn1 1 1,54 1,65 Eukaryotic translation initiation factor 3 subunit A Eif3a 1 1,52 1,78 Sepiapterin reductase Spr 1 1,35 1,31 Hypoxia up-regulated protein 1 Hyou1 1 1,34 1,40 Neural cell adhesion molecule L1 L1cam 1 1,28 1,36 Lactoylglutathione lyase Glo1 1 1,19 1,24 DmX-like protein 2 Dmxl2 1 1,18 1,20 Stress-induced-phosphoprotein 1 Stip1 1 1,13 1,15 Fatty acid synthase Fasn 1 1,12 1,17 ATP synthase subunit b, mitochondrial Atp5f1 1 0,91 0,88 Protein QIL1 Qil1 1 0,82 0,82 Proline dehydrogenase 1, mitochondrial Prodh 1 0,48 0,56 Nicalin Ncln 1 0,29 0,33

Figure 3-36: Over-expression effect regulated 17 proteins in moue brain at 6 monthes old. The spectral counting result enabled the comparison of the normalized protein level for

Results______each protein candidate. The spectra counts (averaged on the biological replicates) ratio between Omi-G399S 6M and non transgenic 6M and between Omi-WT 6M and non- transgenic 6M were plotted for all 17 proteins.

Table 3-10: Protein overexpression regulated proteins in 12 monthes mouse brains.

Gene NT 12M/NT 12M Omi-WT OE 12M/ Omi-G339S 12M/ (Uniprot) NT 12M NT 12M DYN3_MOUSE Dnm3 1 1,18 1,34 KCAB2_MOUSE Kcnab2 1 0,84 0,67

Figure 3-37: Over-expression effect regulated 2 proteins in moue brain at 12 monthes old. The spectral counting result enabled the comparison of the normalized protein level for each protein candidate. The spectra counts (averaged on the biological replicates) ratio between Omi-G399S 6M and non transgenic 6M and between Omi-WT 6M and non- transgenic 6M were plotted for all 2 proteins.

Interestingly, we could see that the overexpression effect was quite dominant when the mice were 6 months old, while it is reduced to 2 proteins when the mice getting older. The age effect might be an important factor regarding the protein transfection effect. It seems that the proteome change at early stage was a combination of protein transfection effect and protein (HtrA2/Omi or its mutant) effect. We therefore analyzed age effect for different mouse types and try to find some common proteins differential both in age and transgenic mice.

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5. Age effect regulated proteins in 3 types of mouse brain samples.

We identified age effect regulated proteins in 3 types of mouse brain samples. The protein lists are shown in Table 3-11, 3-12 and 3-13 and Figure 3-38, 3-39 and 3-40.

Table 3-11: Age effect regulated proteins in non-transgenic mouse brains.

Gene NT (Uniprot) NT 12M/ 6M/NT6 NT 6M M Myelin-associated oligodendrocyte basic protein Mobp 1 1,54 Hyaluronan and proteoglycan link protein 1 Hapln1 1 1,38 NEDD8-conjugating enzyme Ubc12 Ube2m 1 0,16 Dihydropyrimidinase-related protein 1 Crmp1 1 0,80 Signal-induced proliferation-associated 1-like protein 1 Sipa1l1 1 0,00

Figure 3-38: Age effect regulated 5 proteins in non-transgenic mouse brains. The spectral counting result enabled the comparison of the normalized protein level for each protein candidate. The spectra counts (averaged on the biological replicates) ratio between NT 12 M and 6 M were plotted for 5 proteins.

Table 3-12: Age effect regulated proteins in WT HtrA2/Omi overexpressing mouse brains.

Gene Omi-WT Omi-WT OE (Uniprot) 6M/Omi- 12M/ WT 6M Omi-WT OE 6M Very long-chain specific acyl-CoA Acadvl 1 1,63 dehydrogenase, mitochondrial Poly(rC)-binding protein 3 Pcbp3 1 1,91 Gap junction gamma-3 protein Gjc3 1 0,34

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Figure 3-39: Age effect regulated 3 proteins in WT HtrA2/Omi overexpressing mouse brains. The spectral counting result enabled the comparison of the normalized protein level for each protein candidate. The spectra counts (averaged on the biological replicates) ratio between WT 12 M and 6 M were plotted for 3 proteins.

Table 3-13: Age effect regulated proteins in G399S HtrA2/Omi overexpressing mouse brains.

Gene Omi- Omi- (Uniprot) G399S G339S 6M/Omi- 12M/ G399S Omi- 6M G339S 6M Tubulointerstitial nephritis antigen-like Tinagl1 1 8,708 Beta-chimaerin Chn2 1 2,643 Beta-actin-like protein 2 Actbl2 1 2,273 Alpha-1-antitrypsin 1-4 Serpina1d 1 1,829 Tubulin beta-6 chain Tubb6 1 1,588 Ubiquitin-conjugating enzyme E2 L3 Ube2l3 1 1,421 Cytochrome b-c1 complex subunit 8 Uqcrq 1 1,420 Myelin basic protein Mbp 1 1,351 Thioredoxin-dependent peroxide reductase, mitochondrial Prdx3 1 1,310 Cell division control protein 42 homolog Cdc42 1 1,309 Nucleoside diphosphate kinase B Nme2 1 0,670 Coatomer subunit delta Arcn1 1 0,585 Diacylglycerol kinase zeta Dgkz 1 0,577 Rho-associated protein kinase 2 Rock2 1 0,527 116 kDa U5 small nuclear ribonucleoprotein component Eftud2 1 0,280 NEDD8-activating enzyme E1 catalytic subunit Uba3 1 0,264 FUN14 domain-containing protein 1 Fundc1 1 0,242 Serine/threonine-protein kinase/endoribonuclease IRE2 Ern2 1 0,193

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Figure 3-40: Age effect regulated 18 proteins in G399S HtrA2/Omi overexpressing mouse brains. The spectral counting result enabled the comparison of the normalized protein level for each protein candidate. The spectra counts (averaged on the biological replicates) ratios between G399S 12 M and 6 M were plotted for 18 proteins.

When age effect regulated proteins of G399S type of mouse brains were compared to the G399S over-expression effect (both 6 and 12 months old mouse brains), we found two proteins Ubiquitin-conjugating enzyme E2 L3 (UBE2L3) and Serine/threonine-protein kinase/endoribonuclease IRE2 (ERN2) were overlapped, implying that both proteins were influenced by both G399S mutant HtrA2/Omi effect and age effect. Protein validation was carried out via Western blotting for the ubiquitin-conjugating enzyme E2 L3 protein candidate found overlapping in both age and G399S mutant HtrA2/Omi effect.

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6. Validation of protein candidate ubiquitin-conjugating enzyme E2L3 (UBE2L3) identified by LC-MS/MS

In the spectral counting analysis, we found that the transgenic G399S mouse brain at the age of 12 months old could show significant high regulation of the protein UBE2L3 compared to the NT 12 or WT 12 as indicated in Figure 3-41. This protein is considered as one of the G399S mutant effect regulated proteins

Figure 3-41: Spectral counting results of the ubiquitin-conjugating enzyme UBE2L3 level in 3 mouse brain groups. For each sample type, the biological replicates n = 6.The significance is between NT 12 and G399S12 and also between WT 12 and G399S 12 indicated with **.

For validation we tried western blot using total protein label for quantification and the SMA label at 80 kDa as the protein loading control. The blot is shown in Figure 3-42 1).

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1). Western blot of the ubiquitin-conjugating enzyme UBE2L3 (18 kDa).

2). Scanned total proteins with labeling in gels before blotting (as the loading control)

Figure 3-42: Western blot of the ubiquitin-conjugating enzyme UBE2L3 (18 kDa) 1).For each sample type, the biological replicates n = 6 with the TG G399S 12 months n=5. The marked signals are the three groups to be compared. 10 µg protein lysate was loaded for each lane and 80 kDa was used as the protein loading control. 2). Gel before blotting with total labeled protein as part of the normalization. 10 µg protein each lane was loaded. The reference signal between two gels is hardly seen here.

In the blot, we could see the signals of UBE2L3 in the NT 12 type samples are weaker. The quantification of the signal intensity was conducted and the quantification result is shown in Figure 3-43.

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Figure 3-43: Western blot quantification of the ubiquitin-conjugating enzyme UBE2L3 intensity and normalized on the reference protein SMA 80 kDa (internal standard added) and the total protein amount (total protein labeling). For each sample type, the biological replicates n = 6 with the TG G399S 12 months n=5.

Here we validated the UBE2L3 via Western blot. The result partly agrees with the LC- MS/MS spectral counting result. In Figure 3-41, we see that the protein amount is significantly different between NT_12 : G399S_12 and between WT_Omi_12 and G399S Omi_12. The missing significance between WT Omi_12 and G399S Omi_12 in Western blot validation could be due to outliers such as the protein band Lane 19 and Lane 32 in Figure 3-42-1. The statistic significance was performed using Anova and Tuckey post hoc tests with p value cut-off of 0.05.

3.3.2 Results from global proteomics study of human HtrA2/Omi WT and mutation G399S transfected neuroblastoma SH-SY5Y cell lines

1. Stress induced SH-SY5Y cell apoptosis

Staurosporine is the inhibitor of protein kinases through the prevention of ATP binding to the kinase. It binds to many kinases with high affinity and low selectivity (Karaman et al. 2008) so that the kinases fail to bind the ATP. The differential analysis can be performed to investigate the stress effect when apoptosis pathway is initiated. Biologically, anti-cleaved caspase-3 was applied as the indicator (Chapter 1.1.4). The apoptosis pathway was initiated in all samples as shown in Figure 3-44

Results______for the vector control, WT and G399S overexpressing SH-SY5Y cells. The cleaved caspase-3 intensity on Western blot was quantified using Image Studio Lite 3.1 as described in the user tutorial guide with reference protein band ß-actin.

1).

2).

3).

Figure 3-44: Western blot detection of the activated caspase 3 (5A1E, Cell Signaling Technology) indicated apoptotic signaling after staurosporine treatment (anti-actin for 90

Results______loading control) for 1). vector SH-SY5Y; 2). WT HtrA2/Omi stably transfected; 3). SH-SY5Y and G-mutant HtrA2/Omi stably transfected SH-SY5Y cells. The significant difference between the ß-actin normalized signal intensity of cleaved caspase-3 was indicated with*.

The abundance of cleaved caspase-3 normalized to the expression of ß-actin yielded in significant increase after 6 h of exposure to staurosporine for all cells and the significance of cleaved caspase-3 signal between cells with and without stressor. Based on this result, 6 groups of 30 sample types (VC_0h, VC_6h, WT_0h, WT_6h, G399S_0h G399S_6h, n=5) were analyzed for proteomics study.

2. Staurospoine induced cell stress effect on the phophorylation state of HtrA2/Omi at serine 400

It was observed that HtrA2/Omi is phosphorylated by Cyclin-dependent kinase-5 (Cdk5) in a 38-dependent manner (Fitzgerald et al. 2012). As the protein kinase inhibitor, staurosporine can inhibit the phosphorylation at S400 of HtrA2/Omi. The phosphorylation state of serine 400 HtrA2/Omi in three different cell types was also checked with the help of the anti-pSer400-HtrA2/Omi antibody produced in Tübingen. Only the WT cell line could show a phosphorylation state change after 1 h of cell stress. Figure 3-45 reveals the reduction of S400 phosphorylation after 1h of stress.

1) WT type of cells

2). G399S type of cells

Figure 3-45: Western blot detection of serine 400 phosphorylated HtrA2/Omi in WT transfected cells. WT cells show the most intensive signal, non pS400 HtrA2/Omi signal was observed in G399S type cells.

The absence of S400 phosphorylation of HtrA2/Omi in G399S cells may indicate toxicity due to the G399S mutant effect. Inducing the kinase inhibitor staurosporine

Results______further contributed to the dysfunction of the serine protease HtrA2/Omi via the regulation of HtrA2/Omi phosphorylation by the kinase Cyclin-dependent kinase-5 Cdk5 in a p38-dependent manner (Fitzgerald et al. 2012).

3. LC-MS/MS identified proteins with different ways of regulation in SH-SY5Y cell samples

From the literature we know that that HtrA2 knock down mice leads to mitochondrial uncoupling accompanied by altered breathing pattern and ATP depletion and vulnerability to chemical ischaemia (Plun-Favreau et al. 2012). And G399S mutant HtrA2/Omi over-expression results in reduced proteolytic activity of HtrA2/Omi and more sensitive against induced cell stress than WT HtrA2/Omi overexpressing cells (Strauss et al. 2015) using biochemical methods. We hypothesize that neuroblastoma cells SH-SY5Y with overexpressing of WT HtrA2/Omi and G399S mutant should also have toxic effect on mitochondrial dysfunction and cell death, apoptosis pathways representing the neurodegeneration, which will be reflected by protein level change in proteomics study. More than 1300 proteins were identified as being influenced by inducing the Staurosporine after 6 hours after statistic tests. Anova and Tuckey post hock statistic tests were performed to find the difference of 6h or 0h conditions within 3 cell types VC : WT : G399S of all 6 sample groups. Student T test was carried out to find the stress effect between 6h and 0h for each cell line.

To make sure that the identified protein candidates were not due to "one-hit-wonder", further filtering strategies include: 1). Minimum peptide spectra found from one sample contain at least 4 spectra counts; 2). The regulated proteins should be identified in all experiments and no less than 4 sample groups. In this case, there were altogether 469 regulated proteins found, within which 81 (the protein list is in the Attachment 2) were involved in apoptosis or cell death biological process after Uniprot oncology analysis.

To compare with the mouse brain study, we analyzed G399S mutant HtrA2/Omi effect, WT HtrA2/Omi effect and protein over-expression effect and stress effect on the proteome change of the neuron cells.

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4. G399S mutant HtrA2/Omi effect resulting in 95 proteins differential (protein list in the Attachment 3)

Figure 3-46: G399S mutant HtrA2/Omi over-expression effect regulated 95 proteins in SH-SY5Y cells. The spectral counting result enabled the comparison of the normalized protein level for each protein candidate. The spectra counts (averaged on the biological replicates) ratio between Omi-G399S transfected and vector controls and between Omi-WT transfected and vector controls were plotted for all 95 proteins.

5. WT HtrA2/Omi effect resulting in 72 proteins differential (protein list in the Attachment 4)

Figure 3-47: WT HtrA2/Omi over-expression effect regulated 72 proteins in SH-SY5Y cells. The spectral counting result enabled the comparison of the normalized protein level for 93

Results______each protein candidate. The spectra counts (averaged on the biological replicates) ratio ratio between Omi-WT transfected and vector controls and between Omi-G399S transfected and vector controls were plotted for all 72 proteins.

6. Transfection effect resulting in 15 proteins differential (protein list in the Attachment 5).

Figure 3-48: Protein over-expression effect regulated 15 proteins in SH-SY5Y cells. The spectral counting result enabled the comparison of the normalized protein level for each protein candidate. The spectra counts (averaged on the biological replicates) ratio between Omi-WT transfected and vector controls and between Omi-G399S transfected and vector controls were plotted for all 15 proteins.

7. Stress effect for V, WT and G399S SH-SY5Y cell lines

We identified proteins with different regulations as presented in Table 3-14

Table 3-14: numbers of proteins regulated after induced cell stress No. of proteins Total Up Down involved in apoptosis and cell death VC 143 102 41 32 WT 132 74 58 22 G399S 249 151 98 47

For vector control SH-SY5Y, 143 proteins were found differential with down regulation of 41 proteins and up-regulation of 102 proteins after 6 hours of staurosporine stressor are observed, within which 32 were involved in apoptosis process as shown in the Attachment 6.

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For WT HtrA2/Omi overexpressing SH-SY5Y, a down regulation was observed for 25 proteins and up-regulation of 37 proteins, within which 22 were involved in apoptosis process as shown in the Attachment 7.

For G399S HtrA2/Omi overexpressing SH-SY5Y, a down regulation was observed for 58 proteins and up-regulation of 151 proteins, within which 47 were involved in apoptosis process as shown in the Attachment 8.

Alone from the number of regulated proteins (Table 3-18) regarding the stress effect, G399S mutant HtrA2/Omi transfected SH-SY5 cells has much severe change in the proteome and the highest numbers of proteins which are involved in apoptosis and cell death analyzed by Uniprot oncology analysis (Chapter 2.2.10). The pathway and network analysis will be discussed later.

8. Validation of protein candidate DNA replication licensing factor MCM4 identified by LC-MS/MS

In the spectral counting analysis, we found that the DNA replication licensing factor 4 MCM4 is regulated in many ways as indicated in Figure 3-49. The protein level was higher in vector and WT cells after stress, while G399S mutant transfected cells showed the opposite. WT HtrA2/Omi overexpression seems to have an effect on the MCM4 level demonstrated by the significant down regulation (VC 0h and WT 0h).

Figure 3-49: Spectral counting results of the DNA replication licensing factor MCM4. For each sample type, the biological replicates n = 5.The significance between two groups is indicated with **.

Results______

For results validation we performed Western blot using total protein labeling and adding internal standard for normalized quantification of the Western blot. The Western blot is shown in Figure 3-50. Suspiciously, the internal standard SMA at 80 kDa appeared less in the 5 WT samples: W 01-W 05 marked in Figure 3-63 (1) than those in the vector control in. To double-check the sample load, the original SDS- page for the V, G and WT type of cells (corresponding to the left half of the western blot in Figure 3-50-1) was shown here in Figure 3-50-2).

1). Western blot of the DNA replication licensing factor MCM4 (96.6 kDa) and added internal standard (-80 kDa).

2). Scanned total proteins with labeling in gels before blotting (as the loading control)

Results______

Figure 3-50: Western blot of the DNA replication licensing factor MCM4 (96.6 kDa) for cell lysate. For each sample type, the biological replicates n = 5. The marked signals are the 2 groups with significance. 10 µg protein lysate was loaded for each lane and 80 kDa was used as the protein loading control. 2). Gel before blotting with total labeled protein as part of the normalization. 5 µg protein lysate was loaded for each lane and the total intensity was taken for the normalization.

By checking the SDS page with eyes first then with precise quantification using the software, we are sure of the sample loading amount. Normalized intensity of the MCM4 signal was quantified and the result is shown in Figure 3-51.

Figure 3-51: Western blot quantification of DNA replication licensing factor 4 MCM4 intensity and normalized on the reference protein SMA 80 kDa (internal standard added) and the total protein amount (total protein labeling). For each sample type, the biological replicates n=5.

Here we validated the MCM4 via Western blot. The down-regulation of MCM4 in cells transfected with WT HtrA2/Omi agrees with that in LC-MS/MS spectral counting as shown in Figure 3-49. The relevant significant protein level change lies between the vector control (VC_0h) and WT HtrA2/Omi transfected cells (WT_0h). The statistic significance was tested by Anova and Tuckey post hoc test with a p value cut-off at 0.05.

Discussion______

4. Discussion

4.1 Development of novel imprinted polymer based phosphoenrichment techniques

4.1.1 Efficiency of pY-MIP, TiO2, and anti-pY antibodies enrichment of phosphopeptides

Enrichment efficiency of pY-MIP, TiO2 and anti-pY antibodies were tested using standard peptide mixture. Further comparison included also three methods test using standard peptide mixture spiked into mouse brain digest. Immunoprecipitation using anti-pY antibodies was investigated on sample/antibody ratio and different peptide sample amount.

MALDI for phosphopeptides identification

In the early stage, pY-MIP SPE experimental setup was probed focusing on the SPE conditions and detection system (MALDI). In Result Figure 3-2, DHB MALDI matrix provided improved ionization of the 10 peptides than CHCA. The reason could lie in the ionization process of different peptide, since it is common the phosphopeptide molecules are not as well ionized as other peptides. In comparison to DHB, all phosphopeptides were poorly or not able to be measured when CHCA matrix was applied, whereas the phosphopeptides intensity was improved when DHB was introduced. DHB is known as a “cool” matrix for MALDI which results in low energy ionization during the mass analysis. However, it generated quite heterogeneous and large crystalline spots (Kjellström & Jensen 2004). CHCA provides more homogeneous matrix and analyte deposits during MALDI target preparation, but the “hot” matrix can lead to significant decomposition of phosphopeptide ions during mass analysis, as observed here in this study that only non phosphopeptides could be detected. Therefore, when phosphopeptides were analyzed on MALDI, only DHB matrix was applied.

During early stage MIP SPE development, the detection limit of MALDI-TOF/TOF-MS was also tested. A confident detection after MIP SPE was possible when sample loading amount was not lower than 500 fmol. Standard peptide detection limit could go 10 times lower. Depending on different questions and studies, the detection systems should be chosen appropriately.

Discussion______

Using standard peptide mixture, pY-MIP could display a high selectivity and sensitivity (Results Figure 3-9). Four phosphopeptides were all enriched via pY-MIP, whereas TiO2 missed the doubly phosphorylated pYpY and antibodies were only able to enrich the triply phosphopeptides 3pY. Increased sample complexity on pY-MIP was tested by spiking the peptide mixture into the mouse brain digest (Figure 3-11). Results encourage further complex sample direct test. Application on HEK 293T cell digest was performed during this work with 3 replicates as shown in Table 4-1.

Table 4-1: result from biological sample test using SCX/pY-MIP No. of identified No. of identified Enrichment efficiency phosphorylation total peptide % phosphorylation 232 2878 0,080611536 443 4384 0,10104927 186 1302 0,142857143

Unfortunately, pY-MIP was not able to enrich the tyrosine phosphorylated peptides. The reason of the failed pY-MIP application on real sample test could be due to low sample amount applied (10 µg). The natural abundance of phosphotyrosine is less than 1% within all phosphorylations. Zoumaro-Djayoon et al. (2012) digested 5 mg protein then further purified with with PY99 anti-phosphotyrosine antibody for immuno-affinity purification of all tyrosine phosphorylated peptides then measured with 6 h LC gradiant, leading to a significant number of more than 1000 tyrosinephosphorylated peptides identification. pY-MIP SPE was able to enrich not more than 5 tyrosinephosphorylated peptides using 10 µg of HEK 293T cell digest. Compared to the 5 mg, the sample amount applied here is too low. Therefore, large- scale sample load is the preresuisite to successful enrichment of the extremely low abundant phosphotyrosine peptides from biological samples.

In this work, the pY-MIP application was illustrated with a comparison study using three approaches: anti-pY antibodies, TiO2 and pY-MIP. We purchased three lgG anti-pY antibodies (100 µg/each, ca. 400 Euro each). With a large-scale sample load (mg) for more than 10 experiments for each antibody, the whole study could have been incredibly expensive. On the other hand, when 5 mg lysate digest would have required 400 mg pY-MIP (10 µ digest/800 µg pY-MIP) for one single experiment. The

Discussion______total amount of pY-MIP available is not more than 50 mg. That's the reason why large-scaled test was not performed during the comparison study. During the IP study, approproiate peptide/antibody ratio was calculated. lgG antibody has the size of 150 kDa. In the standard peptide mixture IP, 3 µg (20 pmol) of anti-pY antibody was used. The calculation was based on the applied standard peptide mixture amount 13 pmol (1 pmol each peptide), among which only 4 pmol was tyrosine phosphorylated ones. Despite the fact that excessive antibody amount is used (20 pmol antibody, binding theoretically 40 pmol pY peptides) compared to the sample load, IP was still not able to capture all 4 pmol phosphotyrosine peptides pY, pYpY, YpY and 3 pY. This could most possibly lie in 3 different reasons: 1). There are too many washing steps in IP (Figure 3-1), contributing to big sample loss in the beginning. 2). Large reaction container (1.5 ml Eppendorf tube) of IP makes it harder for the 20 pmol low concentrated antibody to find its 4 pmol target within 16 h of incubation time. In principle, all reaction could be understood as a matter of kinetic. The antibody binding to antigen kinetic should be dependent on only concentration and temperature. The 1.5 mL Eppendorf tube is a common choice for performing IP. The solvent or buffer volumn (1ml) was always adapted to the reactor, leading to a low concentration of both antibodies and peptide sample. The temperature is set at 4°C 3). The use of detergent for specific binding is the crucial obstacle in further MS analysis.

The1.5 ml Eppendorf tube and the commercial magnetic stand were all set for IP, the only parameter can be improved here is the concentration of antibody or sample. In the protocol communication regarding phosphotyrosine peptide profiling IP with Prof. Dr. Forest White from MIT USA, I leanred that they used 30 µg 3 types of anti- pYantibodies against 500 µg of trypsinized lysate, which is a excessive amout of antibodies. The success of an enrichment protocol could be completed by the result with excessive load of 200 pmol each peptide and 3 pmol of the anti-pY antibodies as shown in Figure 3-8 in our work too. Summerized from both cases (MIT protocol and our peptide overloading trial), results indicated that either increasing sample amount or antibody amount could significantly improve the immunoprecipitation outcome for extremely low abundant antigen enrichment such as tyrosine-phosphorylated peptides.

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4.1.2 Efficiency of phosphopeptide enrichment using pS-MIP

In the spiking results measured on MALDI (Figure 3-21 and 3-22), the spectra of loading and elution show clear specific enrichment of the spiked peptide.

The results demonstrate the high potential of pS-MIP as a new tool for the enrichment of serine-phosphorylated peptides in small-scale biological samples. This potential was primarily demonstrated by the positive identification of a serine- phosphorylated peptide in mouse brain lysate at spike-in levels as low as those in complex mammalian samples detectable by nanoLC-ESI- MS/MS.

Without the addition of synthetic peptides, using tryptic digests of HEK 293T cell lysates and combining SCX fractionation with pS-MIP enrichment resulted in the identification of 924 phosphopeptides in only 10 µg of sample, 610 of which were found in all 3 replicates, suggesting a high reproducibility of the data obtained by this method.

It is to be noted that higher numbers of phosphopeptides were found in large-scale phosphoproteomic studies using multi-dimentional approaches based on electrostatic repulsion-hydrophilic interaction chromatography (ERLIC), hydrophilic interaction liquid chromatography (HILIC) or SCX prefractionation followed by TiO2 or IMAC affinity-based chromatography (Palma et al. 2013, Villen & Steven 2008, Zarei et al. 2011). These experiments were, however, performed on other cell types with considerably higher sample sizes, e.g., nearly 1,000-fold larger amounts of protein digest (4 to 15 mg). A study on human mesenchymal stem cell lysate (120 µg of tryptic digest) identified 350 phosphopeptides using the TiO2 technique, whereas sequential elution from IMAC captured 716 phosphopeptides (Thingholm et al. 2006). We demonstrated here that the total number of identified phosphopeptides differed significantly from sample type to sample type. For instance, TiO2 alone was able to enrich 330 phosphopeptides from 10 µg of HEK 293T cell lysate and 1,351 phosphopeptides from 100 µg of SH-SY5Y cell lysate digests. The presented HEK 293T cell experiments showed that pS-MIP enrichment combined with SCX resulted in a comparable number of phosphopeptides (924) as TiO2 (711) but with 10-fold less peptide sample. Moreover, the TiO2 approach showed bias towards the acidic amino acids D and E and motif-x analysis revealed a potential casein kinase motif SxxE and

SE, indeed, this acidic residue preference of TiO2 was observed in previous research

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(Bahl et al. 2008), whereas pS-MIP showed no obvious preference for acidic amino acid residues, but a distinct SP-motif reflecting a major regulatory phosphorylation motif of Pro-directed protein kinases, encompassing numerous kinase classes of CDKs, MAPKs, JNKs or GSK-3, involved in diverse cellular processes (Lu et al. 2002).

MIP was able to enrich phosphopeptides out of human cell digest. We also tested the pS-MIP material in clinical relevant samples such as CSF or mouse brain digest. In the CSF sample test, we were able to enrich 47 phosphopeptides (attachment) belonging to the 24 identified phosphoprotein by SCX+pS-MIP method. Previous research on human CSF phosphoproteome mapping showed that TiO2 was able to identify 44 phosphoproteins (Bahl et al. 2008), however, our method could complementarily identify 8 more phosphoproteins in human CSF phosphoproteome. Discovery of Five phosphorylation sites not annotated in the UniProt database, namely at Y-297 and S-298 in the Rho GTPase-activating protein 22 and at Y-84 in the immunoglobulin kappa chain C region, at T-304 in actin, cytoplasmic and at S- 360 in Golgi integral membrane protein 1 are reported here for the first time. Identification of of amyloid-ß precursor protein a4 phosphorylated at S-441 via SCX/pS-MIP enables further investigation disease case study as amyloid-ß A4 binds to the FE65, a cytosolic adapter protein, whose interactome is of central interest in Alzheimer disease research (Nensa et al. 2013) and the phosphorylation of Amyloid- ß a4 may play a critical role. This phosphosite S-441 was found by all three different CSF sample preparation approach (i) in solution digestion, (ii) in gel short separation and removal of albumin, (iii) depletion of 14 hot abundant proteins). As we do not see any significant difference between (i) and (ii), further research should follow the most simplified protocol (i) to reduce sample preparation procedure. CSF using depletion protocol provided the worst result and showed high sample loss, which indicated that protein depletion might also remove low abundant interesting proteins in the CSF, therefore is not recommended.

TiO2 based capture of phosphopeptides is a well-enginieered technique for routine laboratory applications, pS-MIP can fill a gap when sensitivity is demanded by the study design as it addresses the restrictions faced when sample availability is low. This is often typical for clinical samples, such as human brain tissue for the analysis of subfractions (e.g. neuromelanin granules Plum et al. 2013) or samples generated

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Discussion______by laser microdissection of tissue (Kley et al. 2013). Especially intriguing is the revelation of a large number of phosphopeptides which were exclusively enriched by the plastic antibody.

Besides the successful applications demonstrated in this work, one negative point concerning pS-MIP application in biological samples should be kept in minds: the selectivity of the plastic antibody. Despite so many advantages like small sample load, fast and robust, low bias towards amino acids, one major drawback for pS-MIP is the phosphopeptide selectivity. The analysis results are shown in Table 4-2.

Table 4-2 Phosphoenrichment Selectivity of pS-MIP and compared to TiO2

No. of identified No. of identified Enrichment efficiency analysis phosphorylation total peptide % phosphorylation group 1 lysate 1 16 10013 0% lysate 2 17 9003 0% lysate 3 20 8825 0% group 2 SCX 1 42 14123 0% SCX 2 39 13982 0% SCX 3 36 12949 0% group 3 pSMIP/SCX 1 982 6036 16% pSMIP/SCX 2 893 7989 11% pSMIP/SCX 3 897 7146 13% group 4 pYMIP/SCX 1 232 2878 8% pYMIP/SCX2 443 4384 10% pYMIP/SCX 3 186 1302 14% group 5 TiO2 1* 743 845 88% TiO2 2* 680 694 98%

group 6 TiO2 /SCX2 145 892 16% TiO2 /SCX3 146 246 59% TiO2 /SCX1 241 276 87% TiO2/SCX 4 228 359 64% TiO2 /SCX5 203 406 50% group 7 TiO2 1 383 392 98% TiO2 2 267 569 47% TiO2 3 340 541 63% group 8 pSMIP 1 193 7759 2% 1 mg MIP pSMIP 2 221 7372 3% pSMIP 3 205 7585 3%

group 9 pSMIP 4 241 6023 4% 800 µg MIP pSMIP 5 223 5814 4% pSMIP 6 249 5846 4%

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From the list, we could see that combined with SCX, the pS-MIP could reach the highest phosphoenrichment efficiency of 16%. By removing the low number PSM (peptide spectral matching) <= 2, the phosphoenrichment efficiency can reach up to 40%. The reason for low selectivity behind could be hydrophobic solvent conditions throughout SPE process, contributing much to the unspecific bindings as the interaction was theoretically only dependent on the hydrogen bond between the phosphate in the analyte and the urea monomer. Nevertheless, the interaction behavior was not possible to be well characterized due to the format of our bulk polymer. In other study however, the hydrogen binding in a different polymer format nano-particle pY-MIP between the phosphate in the template and urea monomer carrying a nitrobenzoxadiazole fluorophore was able to be confirmed via NMR titration (Wan et al. 2013).

In presence of hydrophobic organic solvents such as acetronitrile or methanol, the hydrogen bonding interaction is not the only one as hydrophobic amino acids residues (alanine, isoleucine, leucine, phenyalanine, valine and proline) could easily bind to the MIP when the mobile phase is highly hydrophobic organic solvents. The hydrophobic interaction might also positively contribute to the discovery of phosphopeptide motif xxxSPxx as the amino acid proline is hydrophobic and is likely to interact with the pS-MIP polymer in the presence of organic solvents.

Compared to pS-MIP, TiO2 show high selectivity with the phosphoenrichment efficiency as high as 98% due to its high affinity in binding to the phosphopeptides in a highly acidified condition (loading condition: 80% MeCN, 1%TFA pH<0). The elution condition in TiO2 SPE was pure aqueous basic (pH 11.3) condition with the absence of hydrophobic solvent. It should be noted that the reason that TiO2 fails in the enrichment of those hydrophobic proline-rich peptides can lie in the pure aqueous elution condition without any organic solvent composition, which is not preferred by those hydrophobic phosphopeptides, therefore may remain on the TiO2 beads. Another theory is that the loading solution 80% MeCN, 1%TFA pH<0 was favored by hydrophobic phosphopeptides in competition with the binding activity of TiO2 to the phosphate groups, therefore, they were not bound to TiO2 in the first place. The determination of the sequence bias feature of TiO2 regarding its phosphopeptide enrichment is a new discovery after more than a decade of research in this field. Hence, neither of the two methods is perfect —either we choose to use pS-MIP and

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Discussion______benefit from its low sample-load, time saving and non-bias merit but with low selectivity, or we choose the high sample-load, sequence-biased high phosphor- selective method using TiO2, knowing that we are loosing those proline-rich hydrophobic phosphopeptides. A combination of both methods may lead to an improved cellular phosphoproteome profiling for sure.

4.2 Global proteomics study for mouse and cell models

4.2.1 Advantages and disadvantages in protein validation using Western blot

7 proteins were tried for validation (1). ADSS2-adenylosuccinate synthetase 2; (2). UBE2L3-unibiquitin-conjugating enzyme E2 L3; (3). ARHG7-rho guanine nucleotide exchange factor 7; (4) ADSL-adnylosuccinate lyase; (5) AMPD2 adenosine monophosphate deaminase 2; (6) MCM4-DNA replication licensing factor; (7) PHB1- Prohibitin 1. Only two proteins- UBE2L3 (from mouse study) and MCM4 (cell study) were able to be validated using Western blot and the results partly agree with the LC- MS/MS data. To obtain the significance between different sample groups in Western blot data evaluation, we performed also Anova and Tucky Post Hoc statistic tests. We didn't get significant difference for UBE2L3 beween sample groups of WT Omi_12 and G399S Omi_12 after statistic tests (p>0.05) as showing significant in spectra counting result, the Tuckey Post Hoc p for Western blott between these two groups is 0.065, indicating a confidence interval of 93,5% instead of normal accepted conventional 95% confidence interval. Whether or not this should be taken into account

We have encountered many obstacles such as low selectivity of the antibodies or similar protein molecular weight requiring different blots. Moreover, it is an expensive method with each antibody costs ca. 300 Euro/100µg. Nevertheless, it is still convenient in case the antibody works well and sample amount is sufficient. Our latest modern Western blot system enables us to normalize the protein signal intensity on both the added internal standard protein band and the total protein amount per lane, where we use the suitable molecular weight of protein as the internal standard freely, as well as take the loading control of total protein into consideration. 105

Discussion______

4.2.2 Discussion of different effect cases based on the proteomics data in mouse model study

The large scale of proteomics data was tested first on statistics Anova and tukey post hoc. First Anova test delivered the results that if the protein is significantly regulated between 3 groups (NT, WT and G399S) of samples at the same age. The tukey post hoc test helps to localize where the significant is by making comparisons in pair-- NT: WT, NT: WT and WT: G399S. For the differentiao proteins (which passed the Anova test with p value < 0.05) we did tockey post hoc test.

After tuckey post hoc test, we got 3 numbers resembling the Anova p value for each protein and can divide the proteins which have different ways of regulations into 7 groups theoretically.

1. *NT:WT, *NT:G399S, non-significant between WT:G399S 2. *NT:WT, *WT:G399S, non-significant between NT:G399S 3. *NT:G399S, *WT:G399S, non-significant between NT:WT 4. *NT:WT, non-significant between NT:G399S and WT:G399S 5. *WT:G399S, non-significant between NT:WT and NT:G399S 6. *NT:G399S, non-significant between NT:WT and WT:G399S 7. *NT:WT, *WT:G399S, *NT:G399S

The first three cases 1-3 were discussed in the result part (Chapter 3.3.1). Case 1 indicates the over-expression effect regardless of any protein that is over-expressed in the mouse brains. Case 2 represents WT HtrA2/Omi effect. Case 3 is G399S mutant HtrA2/Omi effect.

Differential proteins that are regulated as in the Case 4, 5 and 6 are not able to be specified. Taking Case 4 as the example, the significance is between only NT and WT HtrA2/Omi as shown in Figure 4-1. Case 5 and Case 6 can be understood analogue to Case 4.

Proteins that are significantly regulated between every group as in Case 7 in our study don't exist.

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Figure 4-1: Protein regulation Case 4. Protein expression is regulated between only NT and WT HtrA2/Omi groups. Each single expression value is plotted as in one blue point.

4.2.3 Discussion on pathway analysis for regulated proteins in mouse brain study

It was shown that mice omitting WT HtrA2 expression display a neurodegenerative phenotype like PD and one important pathway involved to PD was observed as mitochondrial dysfunction (Jones et al. 2003; Martins et al. 2004). Further study showed that the neurodegeneration and early death in mnd2 mice is rescued by a neuron targeted human HtrA2 gene, which explains the neuroprotective role of HtrA2 (Kang et al. 2013). Recent research reported that the mutation of G at position 399 replaced by S serine in HtrA2/Omi was identified in sporadic PD patients in Germany (Strauss et al. 2005).

The WT HtrA2/Omi and G399S HtrA2/Omi were over-expressed in all areas of the mouse brains. The transgenic WT HtrA2/Omi mice could show decrease in learning ability and movement disorder from collaboration partner's animal behavioral test in Tübingen, leading to the conclusion that HtrA2/Omi protein might be a double edged sword—either loss or elevated , could lead to PD phenotype mice. The G399S

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Discussion______overexpressing mice did not show clear phenotype behaviour but both transgenic mouse brains were observed with cell death (Poonam dissertation).

Based on the previous researches, we expected a negative role of overexpression of both G399S and WT HtrA2/Omi in toxic gain of mitochondrial dysfunction and cell death and wanted to understand the mechanism in a proteomics perspective.

As presented in results, we found out that there are 30 proteins regulated due to the G399S HtrA2/Omi effect (Chapter 3.3.1 Figure 3-32 and 3-33). For a more comprehensive analysis, we applied IPA (ingenuity pathway analysis, report saved in IPA software) to try to get an overview by networking the regulated 30 proteins based on related researches implemented into IPA. The cellular cross-talks between different signalling pathways within numbers of proteins are too complicated for us to solve. IPA pathway analysis enables us to get an idea for possible pathways that involved on a large-scale proteomics dataset.

1. Networks and canonical pathways identified by proteins having G399S HtrA2/Omi over-expression effect

From IPA analysis, we could find out the top network of differential proteins in G399S HtrA2/Omi over-expression with the highest score 28 is the neurological disease, psychological disorders, hereaditary disorder and this network with identified differential proteins is illustrated as in Figure 4-2.

1). Top networks (9 identified but)

2). Top canonical pathways

Figure 4-2: IPA analysis of G399S HtrA2/Omi regulated differential proteins. 1). Top network identified is the neurological disease, psychological disorder, hereditary disorder with 13 interesting protein candidates. 2). Top canonical pathways identified by IPA.

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We found a top network associated with neurological disease, while the well explained canonical pathways were found more involved in metabolic pathways, and may indirectly related to cell death.

2. Networks and canonical pathways identified by proteins having WT HtrA2/Omi over-expression and protein over-expression effect

On the contrary, the top network identified for the 16 regulated proteins identified in WT HtrA2/Omi overexpressing mouse brains (Figure 3-34 and 3-35) was not directly related to neurodegenerative disorders and the identified canonical pathways are listed in Figure 4-3-1.

1). Top canonical pathways WT HtrA2/Omi effect

2). Top canonical pathways protein overexpression effect

Figure 4-3: IPA analysis of WT HtrA2/Omi regulated differential proteins and protein- overexpression regulated differential proteins. 1).Top canonical pathways identified for WT effect with 16 molecules 2). Top canonical pathways identified for over-expression effectis with 19 protein candidates.

Less confidence in network score can lie in the fact of less regulated protein number identified in WT HtrA2/Omi overexpressing mouse brains and in the protein transfection (over-expression) effect. Possibly, the number of differential proteins already reveals that to what extent the proteome of the whole mouse brain was influenced by the effect of WT or G399S HtrA2/Omi. As we observe that 30 proteins regulated in G399S mutant mouse brains while only 16 were found in the WT HtrA2/Omi over-expressing mouse brains and 11 in protein over-expression effect regulated mouse brains in general.

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Discussion______

The differential proteins identified due to protein over-expression effect are associated to the top network of nutritional disease, psychological disorders, endocrine system disorders, and the identified canonical pathways are related to biosynthesis of fatty acid or proline degradation (Figure 4-3-2). All the three analyses: G399S effect, WT effect and over-expression effect cannot find direct link to cell death or mitochondrial dysfunction based on the canonical pathway analyses by IPA.

Intriguingly, we could see from Figure 3-36 and 3-37 that the protein over-expression effect declined much more when the mice were older (12 months old) with only 2 differential protein identified than the early stage of 6 months old with 17 differential proteins. The proteome of mouse brains must have developed with age and the protein over-expression effect seems to fade with time. The G399S HtrA2/Omi and WT HtrA2/Omi effect can be more enhanced when we analyse the regulated proteins using a developing perspective with the age factor taken into account.

3. Networks and canonical pathways identified by proteins having age effect in NT, WT and G399S HtrA2/Omi over-expressing mouse brains

Another IPA analysis is performed for the regulated proteins showing age effect in non-transgenic, WT and G399S HtrA2/Omi overexpressing mouse brain types respectively. Only one network with confident score can be identified in the G399S HtrA2/Omi overexpressing mouse brain type as shown in Figure 4-4 and the top canonical pathway is exactly mitochondrial dysfunction. The top canonical pathway identified for other analysis was also listed together with the top network below.

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1).Proteins regulated in G399S HtrA2/Omi over-expressed mouse brains with consideration of age factor (age effect)

2). Proteins regulated in WT HtrA2/Omi overexpressed mouse brains with consideration of age factor (age effect)

3). Proteins regulated in NT mouse brains with consideration of age factor (age effect)

Figure 4-4: IPA analysis of age effect regulated differential proteins 1). Top network and top canonical pathways for G399S mutant HtrA2/Omi overexpressing mouse brains in age effect. 2). Top network of age effect regulated proteins in WT HtrA2/Omi overexpressing mouse brains. No canonical pathway was identified 3). Top network and top canonical pathways of age effect regulated proteins in non-transgenic mouse brains. Top network identified is only confident in the G399S HtrA2/Omi over-expressing mice with high score. No significant networks or canonical pathways were found analyzing

From the IPA analysis above we can conclude that proteins regulated due to G399S HtrA2/Omi overexpressing in mice indicate high potential cause of mitochondrial dysfunction and cell death. The exact protein molecules involved in the mitochondrial dysfunction pathway are PRDX3 and UQCRQ.

The G399S HtrA2/Omi effect is more obvious than the WT HtrA2/Omi effect based on our pathway analysis by IPA with the consideration of age factor. This conclusion agrees with the previous study that cells overexpressing the G399S mutant HtrA2/Omi were more sensitive and vulnerable to stress-induced cell death than the WT HtrA2/Omi (Strauss et al. 2005). However, it is contradictive to the animal

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Discussion______behavioral study (as mice overexpressing G399S HtrA2/Omi in the brain didn't show phenotype as the mice overexpressing WT HtrA2/Omi did).

Nevertheless, the age factor should play an important role. At the early age (6M), protein over-expression effect seemed to be quite dominant (17 over-expression effect regulated proteins identified) and only later at 12 monthes old we saw that the over-expression effect faded (with only 2 protein regulated). We assumed that the G399S mutant HtrA2/Omi effect or WT HtrA2/Omi effect was then enhanced at later stage of the mice.

The effect of G399S mutant HtrA2/Omi overexpressing was then further evaluated via a similar cell model study. The proteomics study based on single type of neuron cells would reveal more information regarding the cellular cross-talks of proteins (Chaper 4.2.3).

Adding to pathway analysis, extra manual literature research was performed for the validated protein candidate UBE2L3, which belongs to G399S mutant HtrA2/Omi effect and also showed age effect (Figure 3-46 and 3-53).

4. Short review of the validated protein in mouse brain proteomics study

Ubiquitination and the ubiquitin-conjugating enzyme E2L3 (UBE2L3)

Ubiquitination is a post-translational modification where the small protein ubiquitin attaches to a substrate protein then further degrade the tagged protein. The ubiquitination can change proteins in many aspects: it is able to signal for protein degradation via the proteasome, change their location in the cell and affect their activity, and promote or prevent protein interactions (Glickman & Ciechanover, 2002; Schnell & Hicke, 2003).) Ubiquitination plays an important role in aggregate formation and proteins intended for proteasomal degradation and Lewy bodies typically contain ubiquitinated α-synuclein (Tofaris et al. 2002). Ubiquitination and the ubiquitin- proteasome system plays a central and complex role also in regulating apoptosis by binding to key cell death proteins such as the executioners of apoptosis-caspases (Bader & Steller 2009). An non-classic K63-linked ubiquitination is considered to be related to neurodegeneration (Lim et al. 2010).

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Figure 4-5: Ubiquitination system (Perrett et al. 2011)

The ubiquitin- activating enzyme E1 is to activate the small protein ubiquitination by using the ATP for the complex formation. Ubiquitin is then transferred to an ubiquitin- conjugating enzyme E2, who further binds to an E3 ubiquitin-protein ligase, making the binding of protein substrate to the ubiquitins. The ubiquitin-protein ligase E3 makes it possible for one or more ubiquitin molecules to bind the substrate (S).

The protein candidate found in the differential analysis is an E2 ubiquitin-conjugating enzyme, playing an essential role of the post-translational protein ubiquitination pathway, facilitating the transfer of activated ubiquitin to bind the substrate proteins (Moynihan et al. 1996). The identified protein with the gene UBE2L3 was named as UbcM4 is the mouse homologue of the human UbcH7 protein involved in the in vitro ubiquitination of several proteins including the tumor suppressor protein p53 with 100% identical amino acid sequence as the human UbcH7 enzyme (Harbers et al. 1996). Disruption of the gene encoding UbcM4 has no effect on proliferation or in vitro differentiation of mouse embryonic stem cells (Pringa et al. 2000). Within all the 12 complex multidomain enzyme RBR (RING-BetweenRING-RING) E3 ligases, 9 interact with the E2 enzyme UbcH7 (Spratt et al. 2014).

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In the latest report, ubiquitin was identified as the genuine substrate for PINK1 and PINK1 phosphorylates ubiquitin at Ser65. The phosphomimetic ubiquitin accelerates the removal of the conjugate “UbcH7 + ubiquitin” in presence of parkin (E3 ubiquitin- protein ligase) in vitro, indicating that phosphorylated ubiquitin is the activator of parkin (Koyano et al. 2014). Our results show that the ubiquitin-conjugating enzyme E2 UbcH7 (UBE2L3) are up-regulated by G399S mutant overexpression but not the WT HtrA2/Omi, which implies that the G399S mutation elevated E2 level is a result of cellular response to the disturbed mitochondrial function, which may further suggest an increased UBE2L3 involved protein ubiquitination level resulting in high protein degradation rate contributing to cell death and neurodegeneration.

4.2.4 Discussion on pathway analysis for regulated proteins in cell model study

We established a cell model study with induced stress using the neuroblastoma cell lines SH-SY5Y with the vector control DNA WT HtrA2/Omi or G399S HtrA2/Omi stably transfected cells to try to further understand the molecular cellular pathways of differential proteins then compare them to the mouse model study. IPA analysis was performed regarding the G399S mutant effect, WT HtrA2/Omi effect, protein transfection effect in regulating proteins for three SH-SY5Y cell lines (detailed reports saved in IPA software) as briefly shown in Figure 4-5.

1) Networks and canonical pathways identified in regulated proteins from G399S HtrA2/Omi over-expressed cells

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2). Networks and canonical pathways identified in regulated proteins from WT HtrA2/Omi over-expressed cells.

3). Networks and canonical pathways identified in regulated proteins from vector control cells.

Figure 4-6: IPA analysis of regulated differential proteins 1). Top network and top canonical pathways for G399S mutant HtrA2/Omi overexpressing SH-SY5Y cells with confidence scores. Mitochontrial dysfunction and oxidative phosphorylation are related to PD and identified as top canonical pathways. 2). Top network for WT HtrA2/Omi overexpressing SH-SY5Y cells. Top networks found are also related to cell development. 3). Top network (with low score 5) and top canonical pathways for vector control SH-SY5Y cells indicating no direct relation to neurodegeneration and cell death.

Compared to the mouse brain study, both model over-expressing G399S HtrA2/Omi were identified as related to mitochondrial dysfunction and cell death based on the canonical pathway analysis from IPA. Interestingly, combining to the animal 115

Discussion______behavioural study in Tübingen, although the G399S HtrA2/Omi over-expressing mice did not show clear phenotype behaviour, possibly due to less protein over-expression level (Poonam dissertation), we still obtain strong indication from the proteomics result of both models that more signal pathways related to mitochondrial dysfunction and nervous system function development were activated from the G399S HtrA2/Omi over-expressed mouse brains and transfected SH-SY5Y cells.

The WT HtrA2/Omi over-expressing mice showed phenotype symptoms in animal behavioural experiment but according to our proteomics study, we cannot verify it through mouse brain study as we identified too few differential proteins (Figure 3-34 and 3-35). We assume that the heterogeneity of the cell types in the mouse brains might have overshadowed the WT HtrA2/Omi effect. However, with the help of the WT HtrA2/Omi transfected SH-SY5Y cell line, we can clearly see that the proteome of the SH-SY5Y cells was also altered to the direction of cellular development, cell function and maintenance (Figure 4-6-2). Hence, the combination of both model studies can provide much more valuable information in the proteomics perspective.

4.2.5 Discussion on pathway analysis for common proteins in mouse brain and cell model studies

We investigated further the overlapping differential proteins identified from both mouse brains and neuroblastoma cell lines. In total, an overlapp of 118 proteins were found as regulated in both model studies in Attachment Table 3. We used IPA tool again to see what pathways could be involved when these overlapping proteins are differential. Astonishingly, we found highly confident top network in neurological disease, hereditary disorder and cell cycle as shown in Figure 4-7.

1). Top networks

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2). Top canonical pathways

Figure 4-7: IPA analysis of regulated overlapping proteins in both model studies. 1). Top networks identified from overlapping regulated proteins in both studies. 2). Top canonical pathways identified from overlapping regulated proteins in both studies

With the identification of network function as "Neurological Disease, Hereditary Disorder, Cell Cycle" and top canonical pathways, we again demonstrate the benefit of the combination of two model studies in the proteomics study linking the proteome change to neurodegeneration diseases. Intriguingly, the top canonical pathways (Figure 4-7) identified should draw attention in the PD related research beyond direct cell death, apoptosis and mitochondrial dysfunction related proteins.

In the literature, we found out that EIF2 activity is regulated by a mechanism related to both guanine nucleotide exchange and phosphorylation (Hershey 1989). An increase in phosphorylated (inactive) EIF2 was identified in patients suffering from Alzheimer's, Parkinson's and Huntington's disease (Chang et al. 2007) suggesting the EIF2 signalling related to neurodegenerative diseases. Mutations of PARK2 and PINK1 genes, which encoded in the cytosolic E3 uniquitin-protein ligase Parkin and the mitochondrial serine/threonine kinase PINK1, case early-onset PD (Bertolin et al. 2015). Our validated differential protein uniquitin ligase UBE2L3 in the mouse brain also suggests the close correlation between PD and altered protein ubiquitin pathways. TCA cycle in the mitochondria is known as citric acid cycle or Krebs cycle for the generation of energy through the oxidation of carbonhydrates, fats and proteins into chemical energy in the form of adenosine triphosphate ATP (Krebs et al. 1987). A deficits in the TCA cycle enzyme was identified in the brains of PD patients (Gibson et al. 2003) implying the energy metabolism malfunction of the TCA cycle contributes highly to the mitochondrial dysfunction which is related to PD.

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4.2.6 Discussion on down regulation of DNA replication licensing factor 4 MCM4

In the work from collaboration partner from Tübingen, IP was performed on M17—a neuroblastic cell line resembling our SH-SY5Y cell with staurosporine for the discovery of potential interaction partners of HtrA2/Omi. The result is shown in Figure 4-8

10 9 8 7 6 5 4 3 2 1 0 HtrA2-IP HtrA2-IP HtrA2-IP bov IgG-IP bov IgG-IP bov IgG-IP untreated STS 17-AAG untreated STS 17-AAG Figure 4-8: Normalized HtrA2/Omi-IP result (measured on LC-MS/MS) using staurosporine treated M17 neuroblastic cells (results from collarboration partner in Tübingen). The PSM (peptide spectra matching) of MCM4 was normalized on the PSM of HtrA2/Omi protein. The first column is the MCM4 level in the untreated M17 cell lysate, the second column is the MCM4 level with staurosporine treatment, the third one is MCM4 level in the M17 cells treated with tanespimycin (HSP90 inhibitor). The antibody used here is anti- HtrA2/Omi antibody. As a control, the bolvine lgG control IP experiment was also performed and no MCM4 was identified (column 4-6).

Here in Figure 4-8, we can see that the conclusion that MCM4 is one of the substrate of HtrA2/Omi. In this work, the SH-SY5Y cell stress study found that the wild type HtrA2/Omi over-expression in the neuron cells can lead to down-regulation of the MCM4. This gives a hint that over-expression of not only the G399S mutant but also WT HtrA2/Omi can lead to cellular dysfunction, in particular in the nucleus. The protein DNA replication licening factor MCM4 is the putative replicative helicase essential for DNA replication in eukaryotic cells (Ishimi Y. 1997). It is localized in the nucleus and its helicase activity could be inhibited by phosphorylation with cyclin A/Cdk2 (Ishimi Y. 2000). MCM4 mutation leads to adrenal failure, short stature, and deficiency of natural killer cell in the humans (Hughes. et al. 2012). In our study, MCM4 was found down-regulated in LC-MS/MS (and validated) in WT HtrA2/Omi transfected SH-SY5Y cells compared to the vector control cells (Figure 3-62 and 3-

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64). However, the Wester blot validation could not show down regulation after stress for the vector control cells.

As an interaction partner of HtrA2/Omi in the nucleus, the MCM4 activity can be inhibited by phosphorylation with cyclin A/Cdk2 (Ishmi et al 2000). It has been suggested that phosphorylation of HtrA2 might modulate its proteolytic activity (Plun- Favreau et al. 2007) and the cyclin-dependent kinase-5 (Cdk5) is responsible for phosphorylating HtrA2/Omi at S400 (Fritzgerald et al. 2012). We could show that phosphosylated S400 HtrA2/Omi is localized in the nucleus and hypothesize that the decrease of S400 phosphorylation in WT type cells could contribute to the inhibition of MCM4 helicase activity, failing in protein DNA replication, contributing to the cellular proteome change.

4.2.7 Discussion on down-regulation of three key enzymes in purine nucleotide pathway

1. PURA2—adenylosuccinate synthetase isozyme 2

The protein adenylosuccinate synthetase isozyme 2 PURA2 was unable to be validated by Western blot. However, as we see the protein regulation in both cell and mouse brain proteomics studies showed exactly the same trend, it can be considered as validated via two sample types and important for further investigation.

In the mouse brain study, the protein adenylosuccinate synthetase isozyme 2 (PURA2) was identified as differential between non-transgenic and G399S transgenic mouse brain samples. Interestingly, similar regulation was found here also in human neuroblastoma cells after cellular stress (Figure 4-9).

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Figure 4-9: Spectral counting relatively quantified protein PURA2 amount in 6 different cell sample groups (left) and 3 groups of 6 monthes old mouse brains. The 12 monthes mouse brains have the same tendency. Significant differences are marked with *.

The relative quantification of PURA2 (adenylosuccinate synthetase isozyme 2) in both studies showed similar down regulation in the presence of G399S mutant transgenic mouse brains or transfected cell line compared to the non-transgenic mouse and vector controls respectively. Interestingly, the same regulation pattern in mouse only matches that in the staurosporine stressed cells. This can explain that the cell stress experimental design agrees with transgenic mouse model study.

Additionaly, the protein PURA2 in WT HtrA2/Omi over-expressing mouse brain was also found significantly down-regulated compared to NT mouse brains, whereas in the cell lines, WT HtrA2/Omi transfection did not show significant effect compared to both the VC cells and G399S HtrA2/Omi transfected cells.

2. AMPD2--AMP deaminase 2

Another enzyme AMP deaminase 2 (AMPD2) was also found as differential in both proteomics studies as shown in Figure 4-3 considered as two LC-MS/MS spectral counting results validated via two different sample types. The regulation however, is not exactly the same from both sample types as shown in Figure 4-10.

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0,70 0,30 ** 0,60 0,25 0,50 ** * *** 0,20 0,40 0,15 0,30

0,20 0,10

0,10 0,05 0,00 VC VC WT WT G399S G399S 0,00 0h 6h 0h 6h 0h 6h Non transg. 12M Omi-WT OE 12M Omi-G339S 12M AMP deaminase 2

Figure 4-10: Spectral counting relatively quantified protein AMPD2 amount in 6 different cell sample groups (left) and 3 groups of 12 monthes old mouse brains. Significant differences are marked with *.

The AMP deaminase 2 was significantly regulated by the induced stress in the cell model study, while in the mouse brain study, AMP2 in G399S HtrA2/Omi transgenic mouse brains was more influenced and down regulated compared to the WT transgenic mouse brains.

3. ADSL--adenylosuccinate lyase

A third enzyme found regulated in cell stress study was adenylosuccinate lyase as shown in Figure 4-11, this is however, no identified as regulated in the mouse brain study.

Figure 4-11: Spectral counting relatively quantified protein ADSL amount in 6 different cell sample groups. Significant differences are marked with *.

As shown in Figure 4-11, the adenylosuccinate lyase was significantly regulated by the induced cell stress for vector control and G399S trnasfected SH-SY5Y cells.

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Inducing stress resulted in significant decrease in adenylosuccinate lysase in the entire cell proteome.

Inspired by the two enzymes identified, attention was drawn to the purine nucleotide pathways as shown below in Figure 4-5. Out of three key enzymes (marked in red), two were found both regulated in both cell and mouse model studies.

Figure 4-12: Purine nucleotides cycle The purine nucleotide cycle serves an important function within exercising muscle and the disruption of the purine nucleotide cycle was a potential explanation for muscle dysfunction in Adenosine monophosphate deaminase deficiency type 1 myoadenylate deminase (MADD) deficiency (Rechard et al. 1980). AMP deaminase plays a critical role in energy metabolism. Catalyzes the deamination of AMP to IMP and plays an important role in the purine nucleotide cycle.

Disruption of the purine nucleotide cycle by inhibition of adenylosuccinate lyasev (ADSL) results in skeletal muscle dysfunction (Swain et al. 1984). In the mouse brain study, one eynzympe PURA2 was found significantly decreased in G399S mutant transgenic mouse brains compared to the non-transgenic mouse brains. In cell stress differential analysis, all three key enzymes as adenylosuccinate synthetase (PURA2),

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Discussion______adenylosuccinate lyase (ADSL, not found regulated in mouse brain study) and AMP deaminase 2 (AMPD2) were found deficient when compared to the non stressed SH- SY5Y cell lines respectively, which may imply a dysfunction in metabolism, resulting in movement disorder symptoms related PD. Tremor is one the PD synptoms. Essential tremor is one of the most frequent movement disorders for human beings and is considered as linked to substantial disability. Some persons suffering tremor further develop PD. In a study carried out through six generations of Turkish relatives with both tremor and PD, both HtrA2/Omi and G399S mutant were identified (Gulsuner et al. 2014). Relating the WT transgenic mice observed with movement disorders in animal behavioural experiment, and that G399S mutant over-expressing mice were observed cell death in the mouse brains (results from Tübingen), we hypothesize that 1). G399S mutant over-expressing may cause muscular disorder by down regulating 2 key enzymes (adenylosuccinate synthetase isozyme 2 and AMP2) in purine nucleotide metabolisms in the transgenic mouse brains study. 2). Staurosporine exerted toxic effect on AMP2 down-regulation in cellular proteome, contributing to dysfunction of metabolisms. 3). Staurosporine induced stress has similar toxic effect as G399S overexpression in the cells down-regulating PURA2 and ADSL enzyme in purine nucleotide cycle.

Last but not the least, level of all three enzymes in purine nucleotide metabolism was disrupted in the G399S transfected cells. Based upon all the facts the observation and results from cell and mouse brain differential studies, we speculate that the dysfunction of purine nucleotide cycle, which is the key in cellular energy metabolism and considered involved in TCA cycle (Aragon et al. 1980), can be linked to movement disorder, one of the important PD symdromes.

4.2.8 Discussion on the role of phosphor-S400 HtrA2/Omi

It was observed that HtrA2/Omi is phosphorylated by Cyclin-dependent kinase-5 (Cdk5) in a p38-dependent manner and the phosphorylation is involved in maintaining mitochondrial function under stressed conditions, compensating cells protection against cellular stress (Fitzgerald et al. 2012). They could show that the phosphorylation of S400 at HtrA2/Omi by Cdk5 can be induced significantly only when p38 signalling pathway was activated. The mitogen-activated protein (MAP)

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Discussion______kinases are involved in cellular behaviours in response to extracellular stimuli, of which the group of p38 MAP kinases work as key mediator in signal transduction, linking p38 to inflammation, cell cycle, cell death, development, cell differentiation and so on (Zarubin & Han 2005).

The phosphorylation by Cdk5 could not take place when the S400 peptide of HtrA2/Omi is mutated to an alanine (Fitzgerald et al 2012). Our Western blot result added that the phosphorylated S400 HtrA2/Omi is absent when the S400 peptide is next to a second mutation S (glycine replaced by serine, G399S mutation), which is highly conserved in sporadic PD patients (Yun et al. 2008, Strauss et al. 2005). This cell model result in house agrees well with the mouse brain study with reduced pS400 in G399S HtrA2/Omi transgenic overexpression in the cortex than in the WT HtrA2/Omi transgenic overexpression (Fitzgerald et al 2012).

Phosphorylation at S400 was shown to increase the serine protease activity in vitro (Plun-Favreau et al. 2007). In our western blot results where (Figure 3-48), apoptosis pathway initiated (clearly shown by clear signal of the cleaved caspase-3), implied that the protein kinase inhibitor staurosporine inhibited the phosphorylation at S400 of HtrA2/Omi. Combined with fact that the Cdk5 is responsible for phosphorylation of S400 at HtrA2/Omi (Fitzgerald et al. 2012), Cdk5 may also be able to down-regulate the pro-apoptotic function of HtrA2/Omi, contributing to cell death.

In previous in house study, Phospho-S400 HtrA2/Omi was found in nucleus in G399S and WT HtrA2/Omi transfected SH-SY5Y cells (in house work from Laura Abel und Piotr Chartowski). Our identified and validated potential HtrA2/Omi interacting partner MCM4 is found located in the nucleus. This may suggest that phosphoserine 400 HtrA2/Omi might be involved in caspase activation or apoptosis in the nucleus.

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5. Conclusion

The method development for phosphopeptide enrichment using the etipope imprinted polymer pY-MIP and pS-MIP was conducted and performance of both MIPs were evaluated--

The application results of pY-MIP was illustrated in a study and compared with TiO2 and immunoprecipitation approach using anti-pY antibodies using standard peptides spiked into trypsinized mouse brain matrix. Successful pY-MIP application without any spiking was not possible to show in this work, due to high amount of both sample and pY-MIP requirement.

Biological sample application and validation on pS-MIP was demonstrated using 4 different sample types (HEK 293T and SH-SY5Y cells, human CSF and mouse brain).

Results from cells digest were compared with the benchmarked TiO2 approach and were proved superior regarding big sample load and phosphopeptide sequence bias problems for TiO2. The engineering of method development was started with system setup with MALDI-readout optimization and MIP SPE contion using standard peptide mixture, followed by a semi-complex sample--to target the spiked phosphopeptide in typsinized mouse brain. It was successfully applied to real biological samples.

The meaning of HtrA2/Omi was investigated in the form of differential analyses on the total dynamic proteome of human neuroblastoma SH-SY5Y cell lines (i) vector control; (ii) wild type human HtrA2/Omi overespressing (WT); (iii) G399S mutant HtrA2/Omi overexpressing (G399S) with staurosporine induced cell stress condition.

We found out that phosphosylated S400 HtrA2/Omi is localized in the nucleus, and potentially interacting with MCM4. It has been suggested that phosphorylation of HtrA2 might modulate its proteolytic activity (Plun-Favreau et al. 2007). The induced kinase inhibitor stressor in cells caused disappearing of phosphor-S400 HtrA2/Omi, leading to loss of the protective function of S400 HtrA2/Omi.

After induced stressor, the phosphorylation S400 disappeared and MCM4 level increased. We speculate that the S400 phosphorylation might also contribute to the inhibition of MCM4 helicase activity, while MCM4 activity inhibited by phosphorylation with cyclin A/Cdk2 (Ishmi et al 2000) was proved.

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Three key enzymes in purine nucleotide metabolism were found down regulated with induced stress. The enzyme PURA2 in G399S HtrA2/Omi overexpressing cells was more vulnerable after 6 hours of cell stress, suggesting the G399S mutant HtrA2/Omi, which was also present in PD patients, is toxic to the purine nucleotide pathway and PURA2 is more sensitive to the mutant G399S HtrA2/Omi overexpression, reflecting a potentially disrupted energy metabolism in PD patients, leading to muscular disorder.

Mitochondrial dysfunction and cell apoptosis are considered as important pathways in relation to PD. Our research revealed the G399S mutated HtrA2/Omi over- expression effect regulated proteins are mostly involved in mitochondrial function pathway or cell death (IPA analysis reports) in both cell and mouse model studies. WT HtA2/Omi transfected cells have altered proteome change relating to cellular development and DNA replication or cancer but the WT HtrA2/Omi effect is not obvious in mouse brain study in proteomics perspective. Combining both models, the overlapped regulated proteins imply high potential networks and canonical pathways involved in neurological diseases and the identified canonical pathways such as TCA cycle, EIF2 signalling and protein ubiquitination pathways should be taken into consideration in down stream studies in the future.

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Attachment 1 NanoLC-ESI-MS/MS-identified phosphopeptides present in a human CSF sample treated with SCX+pS-MIP, compared with the previous research using TiO2 approach (Bahl et al. 2008).

Sequence found by TiO2 Protein name Protein Group Accessions Modifications

GREEHYEEEEEEEEDGAAVAEK no Golgi integral membrane protein 4 O00461 Y6(Phosp)

VTEPISAESGEQVER yes Apolipoprotein L1 O14791 T2(Phosp)

EALQSEEDEEVKEEDTEQKR yes Extracellular matrix protein 2 O94769 S5(Phosp)

EALQSEEDEEVKEEDTEQK yes O94769 S5(Phosp)

LVGGPMDASVEEEGVR yes Cystein C P01034 M6(Oxidation); S9(Phosp)

VYACEVTHQGLSSPVTK no Ig kappa chain C region P01834 Y2(Phosp)

AATVGSLAGQPLQER no Apolipoprotein E P02649 T3(Phosp)

KVPQVSTPTLVEVSR no Serum Albumin P02768 T7(Phosp)

ERADEPQWSLYPSDSQVSEEVK no Secretogranin-1 P05060 S13(Phosp)

GHPQEESEESNVSMASLGEK yes P05060 S7(Phosp); M14(Oxidation)

GEDSSEEKHLEEPGETQNAFLNER yes P05060 S5(Phosp)

GEDSSEEKHLEEPGETQNAFLNER yes P05060 S4(Phosp); S5(Phosp)

SAEFPDFYDSEEPVSTHQEAENEKDR yes P05060 S1(Phosp)

SQEESEEGEEDATSEVDKRR yes P05060 S1(Phosp)

SQEESEEGEEDATSEVDKR yes P05060 S5(Phosp)

VESLEQEAANER yes Amyloid beta A4 P05067 S3(Phosp)

KANDESNEHSDVIDSQELSK no Osteopontin P10451 S6(Phosp)

ANDESNEHSDVIDSQELSK yes P10451 S5(Phosp); S14(Phosp)

ANDESNEHSDVIDSQELSK yes P10451 S5(Phosp)

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Attachment______

ANDESNEHSDVIDSQELSKVSR yes P10451 S14(Phosp)

DSYETSQLDDQSAETHSHK yes P10451 S2(Phosp)

AIPVAQDLNAPSDWDSR yes P10451 S16(Phosp)

QNLLAPQNAVSSEETNDFKQETLPSK yes P10451 S11(Phosp)

QNLLAPQNAVSSEETNDFKQETLPSK yes P10451 S11(Phosp); S12(Phosp)

QNLLAPQNAVSSEETNDFK yes P10451 S12(Phosp)

EDSLEAGLPLQVR no Chromogranin-A P10645 S3(Phosp)

VTTVASHTSDSDVPSGVTEVVVK no Clusterin P10909 S11(Phosp)

EILSVDCSTNNPSQAK no P10909 S4(Phosp)

LEPEDEESDADYDYQNR no Coagulation Factor P12259 S8(Phosp)

QQAHKEESSPDYNPYQGVSVPLQQK no Secretogranin-2 P13521 S8(Phosp)

EESSPDYNPYQGVSVPLQQK no P13521 S4(Phosp)

IESQTQEEVRDSKENIEK yes P13521 S12(Phosp)

QEVNPVRQEIESETTSEEQIQEEK yes Versican core protein P13611 T15(Phosp)

ESAEVEEIVFPR yes Cadherin-2 P19022 S2(Phosp)

SLPGESEEMMEEVDQVTLYSYK no Inter-alpha-trypsin inhibitor heavy chain H2 P19823 S6(Phosp); M9(Oxidation); M10(Oxidation)

DLYANTVLSGGTTMYPGIADR no Actin, cytoplasmic P60709 T13(Phosp)

AQRLSQETEALGR no Nucleobindin-1 Q02818 S5(Phosp)

EHANSKQEEDNTQSDDILEESDQPTQVSK no SPARC-like protein 1 Q14515 S14(Phosp)

HSASDDYFIPSQAFLEAER no Q14515 S11(Phosp)

MQEDEFDQGNQEQEDNSNAEMEEENASNVNK yes Q14515 M1(Oxidation); S17(Phosp); M21(Oxidation)

DQGNQEQDPNISNGEEEEEKEPGEVGTHNDNQER yes Q14515 S12(Phosp)

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Attachment______

FLDEVQAYSNVNK no Rho GTPase-activating protein 22 Q7Z5H3 Y8(Phosp); S9(Phosp)

LRGEDDYNMDENEAESETDKQAALAGNDR no Golgi integral membrane protein 1 Q8NBJ4 M9(Oxidation); S16(Phosp)

ELSAERPLNEQIAEAEEDK yes Secretogranin-3 Q8WXD2 S3(Phosp)

ELSAERPLNEQIAEAEEDKIK yes Q8WXD2 S3(Phosp)

SEHPESSLSSEEETAGVENVK no Receptor-type tyrosine-protein phosphatase N2 Q92932 S10(Phosp)

Attachment 2 Protiens involved in the apoptosis process and cell death identified as differential in the cell stress study. Significant regulation ratio of each protein quantified between two sample types or conditions is listed in the table.

Accession Protein VC W G39 VC VC/G WT/G VC VC/G WT/G 0h T 9S /W 399S 399S /W 399S 399S /6 0h 0h/6 T 0h 0h T 6h 6h h /6 h 0h 6h h Q14204 Cytoplasmic dynein 1 heavy chain 1 (Cytoplasmic dynein heavy chain 1) (Dynein heavy chain, cytosolic) 1,4 2,1 2,0 0,6 1,8 Q13813 Spectrin alpha chain, non-erythrocytic 1 (Alpha-II spectrin) (Fodrin alpha chain) (Spectrin, non-erythroid alpha subunit) 1,3 1,2 3,4 2,9 3,0 Q15149 Plectin (PCN) (PLTN) (Hemidesmosomal protein 1) (HD1) (Plectin-1) 1,7 0,7 10,6 0,6 4,3 7,1 0,3 27,7 101,8 P78527 DNA-dependent protein kinase catalytic subunit (DNA-PK catalytic subunit) (DNA-PKcs) (EC 2.7.11.1) (DNPK1) (p460) 1,6 0,5 3,5 1,8 0,5 1,7 P08670 Vimentin 1,3 1,2 0,4 1,9 4,4 0,4 2,5 P02545 Prelamin-A/C [Cleaved into: Lamin-A/C (70 kDa lamin) (Renal carcinoma antigen NY-REN-32)] 0,3 0,6 2,5 4,3 0,6 P48681 Nestin 5,4 6,1 5,3 6,9 O43707 Alpha-actinin-4 (F-actin cross-linking protein) (Non-muscle alpha-actinin 4) 1,7 0,5 1,2 2,6 0,5 2,0 4,1 P20700 Lamin-B1 0,1 7,9 9,2 P43246 DNA mismatch repair protein Msh2 (hMSH2) (MutS protein homolog 2) 2,3 3,1 0,7 0,2 1,9 Q14203 Dynactin subunit 1 (150 kDa dynein-associated polypeptide) (DAP-150) (DP-150) (p135) (p150-glued) 2,8 2,5 0,7 0,3 1,6 P12814 Alpha-actinin-1 (Alpha-actinin cytoskeletal isoform) (F-actin cross-linking protein) (Non-muscle alpha-actinin-1) 1,8 0,5 2,0 0,4 1,7 4,0

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P52701 DNA mismatch repair protein Msh6 (hMSH6) (G/T mismatch-binding protein) (GTBP) (GTMBP) (MutS-alpha 160 kDa subunit) (p160) 2,3 0,4 2,5 O00567 Nucleolar protein 56 (Nucleolar protein 5A) 0,2 4,3 Q9Y2A7 Nck-associated protein 1 (NAP 1) (Membrane-associated protein HEM-2) (p125Nap1) 1,5 2,1 1,5 0,7 Q13616 Cullin-1 (CUL-1) 2,0 0,6 0,4 Q96ST3 Paired amphipathic helix protein Sin3a (Histone deacetylase complex subunit Sin3a) (Transcriptional corepressor Sin3a) 0,7 2,9 0,7 0,3 P41250 Glycine--tRNA ligase (EC 6.1.1.14) (Diadenosine tetraphosphate synthetase) (AP-4-A synthetase) (Glycyl-tRNA synthetase) (GlyRS) 0,7 0,6 0,6 1,9 0,5 O60341 Lysine-specific histone demethylase 1A (EC 1.-.-.-) (BRAF35-HDAC complex protein BHC110) (Flavin-containing amine oxidase 1,7 2,8 2,7 0,4 1,5 1,8 domain-containing protein 2) P08243 Asparagine synthetase [glutamine-hydrolyzing] (EC 6.3.5.4) (Cell cycle control protein TS11) (Glutamine-dependent asparagine 1,6 1,3 1,7 1,7 0,7 0,4 0,7 0,5 synthetase) P08758 Annexin A5 (Anchorin CII) (Annexin V) (Annexin-5) (Calphobindin I) (CBP-I) (Endonexin II) (Lipocortin V) (Placental anticoagulant 0,5 1,7 0,7 0,4 0,7 1,6 1,6 0,7 protein 4) (PP4) (Placental anticoagulant protein I) (PAP-I) (Thromboplastin inhibitor) (Vascular anticoagulant-alpha) (VAC-alpha) P49756 RNA-binding protein 25 (Arg/Glu/Asp-rich protein of 120 kDa) (RED120) (Protein S164) (RNA-binding motif protein 25) (RNA- 0,5 0,5 0,5 binding region-containing protein 7) P04792 Heat shock protein beta-1 (HspB1) (28 kDa heat shock protein) (Estrogen-regulated 24 kDa protein) (Heat shock 27 kDa protein) 1,4 0,6 0,6 1,4 2,4 0,7 (HSP 27) (Stress-responsive protein 27) (SRP27) P35222 Catenin beta-1 (Beta-catenin) 0,5 0,4 0,4 4,1 Q92974 Rho guanine nucleotide exchange factor 2 (Guanine nucleotide exchange factor H1) (GEF-H1) (Microtubule-regulated Rho-GEF) 2,7 1,9 1,6 (Proliferating cell nucleolar antigen p40) P49588 Alanine--tRNA ligase, cytoplasmic (EC 6.1.1.7) (Alanyl-tRNA synthetase) (AlaRS) (Renal carcinoma antigen NY-REN-42) 1,9 1,6 3,0 1,7 0,6 1,7 P62258 14-3-3 protein epsilon (14-3-3E) 1,5 0,7 1,3 0,7 2,1 0,7 0,3 O00232 26S proteasome non-ATPase regulatory subunit 12 (26S proteasome regulatory subunit RPN5) (26S proteasome regulatory subunit 2,2 2,0 2,8 p55) P09172 Dopamine beta-hydroxylase (EC 1.14.17.1) (Dopamine beta-monooxygenase) [Cleaved into: Soluble dopamine beta-hydroxylase] 0,4 5,3 1,6 0,3 P35221 Catenin alpha-1 (Alpha E-catenin) (Cadherin-associated protein) (Renal carcinoma antigen NY-REN-13) 0,3 0,3 2,7 1,6 1,8 O60216 Double-strand-break repair protein rad21 homolog (hHR21) (Nuclear matrix protein 1) (NXP-1) (SCC1 homolog) 2,4 0,7 0,3 2,0 0,5 Q9UKV3 Apoptotic chromatin condensation inducer in the nucleus (Acinus) 0,1 7,2 8,1 1,4 P54577 Tyrosine--tRNA ligase, cytoplasmic (EC 6.1.1.1) (Tyrosyl-tRNA synthetase) (TyrRS) [Cleaved into: Tyrosine--tRNA ligase, cytoplasmic, 1,4 0,6 2,8 0,4 N-terminally processed] P19367 Hexokinase-1 (EC 2.7.1.1) (Brain form hexokinase) (Hexokinase type I) (HK I) 2,0 P35232 Prohibitin 0,2 2,6 2,8 0,6 0,7 Q9H2P0 Activity-dependent neuroprotector homeobox protein (Activity-dependent neuroprotective protein) 0,5 2,3 P18583 Protein SON (Bax antagonist selected in saccharomyces 1) (BASS1) (Negative regulatory element-binding protein) (NRE-binding 0,2 5,1 6,9 protein) (Protein DBP-5) (SON3) O00429 Dynamin-1-like protein (EC 3.6.5.5) (Dnm1p/Vps1p-like protein) (DVLP) (Dynamin family member proline-rich carboxyl-terminal 0,6 2,9 2,0 0,5 3,9 domain less) (Dymple) (Dynamin-like protein) (Dynamin-like protein 4) (Dynamin-like protein IV) (HdynIV) (Dynamin-related protein 1)

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P25787 Proteasome subunit alpha type-2 (EC 3.4.25.1) (Macropain subunit C3) (Multicatalytic endopeptidase complex subunit C3) 0,3 0,3 0,5 (Proteasome component C3) P21796 Voltage-dependent anion-selective channel protein 1 (VDAC-1) (hVDAC1) (Outer mitochondrial membrane protein porin 1) 1,2 0,3 2,7 2,8 (Plasmalemmal porin) (Porin 31HL) (Porin 31HM) Q86UE4 Protein LYRIC (3D3/LYRIC) (Astrocyte elevated gene-1 protein) (AEG-1) (Lysine-rich CEACAM1 co-isolated protein) (Metadherin) 0,5 0,5 0,6 (Metastasis adhesion protein) Q9Y3T9 Nucleolar complex protein 2 homolog (Protein NOC2 homolog) (NOC2-like protein) (Novel INHAT repressor) 2,7 2,7 P61764 Syntaxin-binding protein 1 (MUNC18-1) (N-Sec1) (Protein unc-18 homolog 1) (Unc18-1) (Protein unc-18 homolog A) (Unc-18A) 6,6 0,1 0,5 (p67) O95831 Apoptosis-inducing factor 1, mitochondrial (EC 1.1.1.-) (Programmed cell death protein 8) 0,5 0,6 2,0 Q07812 Apoptosis regulator BAX (Bcl-2-like protein 4) (Bcl2-L-4) 0,6 0,4 0,5 P09972 Fructose-bisphosphate aldolase C (EC 4.1.2.13) (Brain-type aldolase) 1,5 1,7 2,1 0,6 Q9UBB4 Ataxin-10 (Brain protein E46 homolog) (Spinocerebellar ataxia type 10 protein) 2,9 4,6 0,3 Q13177 Serine/threonine-protein kinase PAK 2 (EC 2.7.11.1) (Gamma-PAK) (PAK65) (S6/H4 kinase) (p21-activated kinase 2) (PAK-2) (p58) 0,3 0,6 [Cleaved into: PAK-2p27 (p27); PAK-2p34 (p34) (C-t-PAK2)] P05556 Integrin beta-1 (Fibronectin receptor subunit beta) (Glycoprotein IIa) (GPIIA) (VLA-4 subunit beta) (CD antigen CD29) 0,6 0,0 0,6 #### ##### 4,0 4,7 #### ##### # Q9UNN5 FAS-associated factor 1 (hFAF1) (UBX domain-containing protein 12) (UBX domain-containing protein 3A) 2,9 0,4 0,3 P45880 Voltage-dependent anion-selective channel protein 2 (VDAC-2) (hVDAC2) (Outer mitochondrial membrane protein porin 2) 1,7 0,8 0,4 2,7 2,4 0,6 0,6 P98175 RNA-binding protein 10 (G patch domain-containing protein 9) (RNA-binding motif protein 10) (RNA-binding protein S1-1) (S1-1) 0,6 0,5 0,4 P49720 Proteasome subunit beta type-3 (EC 3.4.25.1) (Proteasome chain 13) (Proteasome component C10-II) (Proteasome theta chain) 0,5 0,7 0,4 Q92734 Protein TFG (TRK-fused gene protein) 2,2 0,6 0,4 Q99700 Ataxin-2 (Spinocerebellar ataxia type 2 protein) (Trinucleotide repeat-containing gene 13 protein) 0,6 0,4 0,3 0,4 P06400 Retinoblastoma-associated protein (p105-Rb) (pRb) (Rb) (pp110) 6,2 0,4 4,6 Q8IX12 Cell division cycle and apoptosis regulator protein 1 (Cell cycle and apoptosis regulatory protein 1) (CARP-1) (Death inducer with 0,5 0,3 3,2 0,6 0,2 3,5 SAP domain) O43464 Serine protease HTRA2, mitochondrial (EC 3.4.21.108) (High temperature requirement protein A2) (HtrA2) (Omi stress-regulated 0,1 0,1 0,1 0,1 0,5 endoprotease) (Serine protease 25) (Serine proteinase OMI) O95292 Vesicle-associated membrane protein-associated protein B/C (VAMP-B/VAMP-C) (VAMP-associated protein B/C) (VAP-B/VAP-C) 0,7 0,3 2,2 0,5 0,4 0,8 Q05639 Elongation factor 1-alpha 2 (EF-1-alpha-2) (Eukaryotic elongation factor 1 A-2) (eEF1A-2) (Statin-S1) 0,5 0,5 0,4 2,1 0,5 P35611 Alpha-adducin (Erythrocyte adducin subunit alpha) 2,3 1,5 Q01433 AMP deaminase 2 (EC 3.5.4.6) (AMP deaminase isoform L) 2,3 1,8 3,4 Q9Y6C9 Mitochondrial carrier homolog 2 (Met-induced mitochondrial protein) 0,1 14,6 16,9 0,2 0,3 Q06323 Proteasome activator complex subunit 1 (11S regulator complex subunit alpha) (REG-alpha) (Activator of multicatalytic protease 0,3 0,3 0,5 subunit 1) (Interferon gamma up-regulated I-5111 protein) (IGUP I-5111) (Proteasome activator 28 subunit alpha) (PA28a) (PA28alpha)

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Q96TA1 Niban-like protein 1 (Meg-3) (Melanoma invasion by ERK) (MINERVA) (Protein FAM129B) #### 0,6 3,1 0,4 #### ##### #### #### ##### # Q04760 Lactoylglutathione lyase (EC 4.4.1.5) (Aldoketomutase) (Glyoxalase I) (Glx I) (Ketone-aldehyde mutase) (Methylglyoxalase) (S-D- 0,4 5,7 0,1 lactoylglutathione methylglyoxal lyase) Q8IV08 Phospholipase D3 (PLD 3) (EC 3.1.4.4) (Choline phosphatase 3) (HindIII K4L homolog) (Hu-K4) (Phosphatidylcholine-hydrolyzing 3,5 0,3 0,3 3,5 phospholipase D3) P09651 Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) (Helix-destabilizing protein) (Single-strand RNA-binding protein) (hnRNP 1,8 0,4 3,8 2,9 core protein A1) [Cleaved into: Heterogeneous nuclear ribonucleoprotein A1, N-terminally processed] P42224 Signal transducer and activator of transcription 1-alpha/beta (Transcription factor ISGF-3 components p91/p84) 2,3 2,2 1,4 P78362 SRSF protein kinase 2 (EC 2.7.11.1) (SFRS protein kinase 2) (Serine/arginine-rich protein-specific kinase 2) (SR-protein-specific kinase 0,7 0,4 2) [Cleaved into: SRSF protein kinase 2 N-terminal; SRSF protein kinase 2 C-terminal] P09936 Ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCH-L1) (EC 3.4.19.12) (EC 6.-.-.-) (Neuron cytoplasmic protein 9.5) (PGP 9.5) 0,3 2,4 1,9 (PGP9.5) (Ubiquitin thioesterase L1) P12236 ADP/ATP translocase 3 (ADP,ATP carrier protein 3) (ADP,ATP carrier protein, isoform T2) (ANT 2) (Adenine nucleotide translocator 3) 0,4 (ANT 3) (Solute carrier family 25 member 6) [Cleaved into: ADP/ATP translocase 3, N-terminally processed] P05141 ADP/ATP translocase 2 (ADP,ATP carrier protein 2) (ADP,ATP carrier protein, fibroblast isoform) (Adenine nucleotide translocator 2) 0,1 5,9 6,3 0,7 (ANT 2) (Solute carrier family 25 member 5) [Cleaved into: ADP/ATP translocase 2, N-terminally processed] Q9Y3E5 Peptidyl-tRNA hydrolase 2, mitochondrial (PTH 2) (EC 3.1.1.29) (Bcl-2 inhibitor of transcription 1) 0,2 4,6 4,6 Q6Y7W6 PERQ amino acid-rich with GYF domain-containing protein 2 (GRB10-interacting GYF protein 2) (Trinucleotide repeat-containing 2,5 2,2 gene 15 protein) P37198 Nuclear pore glycoprotein p62 (62 kDa nucleoporin) (Nucleoporin Nup62) 2,7 Q8TB36 Ganglioside-induced differentiation-associated protein 1 (GDAP1) 0,5 0,2 0,5 P46379 Large proline-rich protein BAG6 (BAG family molecular chaperone regulator 6) (BCL2-associated athanogene 6) (BAG-6) (BAG6) 1,7 3,0 (HLA-B-associated transcript 3) (Protein G3) (Protein Scythe) Q86YP4 Transcriptional repressor p66-alpha (Hp66alpha) (GATA zinc finger domain-containing protein 2A) 0,5 0,4 0,6 Q8NBS9 Thioredoxin domain-containing protein 5 (Endoplasmic reticulum resident protein 46) (ER protein 46) (ERp46) (Thioredoxin-like 0,4 3,0 protein p46) O75340 Programmed cell death protein 6 (Apoptosis-linked gene 2 protein) (Probable calcium-binding protein ALG-2) 0,5

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Attachment 3 G399S mutant HtrA2/Omi effect resulted in 95 differential proteins. Significant regulation ratio of each protein quantified between two sample types is listed in the table.

Accession Protein VC VC/G WT/ /W 399S G399 T 0h S 0h 0h Q9Y6C9 Mitochondrial carrier homolog 2 (Met-induced mitochondrial protein) 14,6 16,9 P20700 Lamin-B1 7,9 9,2 Q9UKV3 Apoptotic chromatin condensation inducer in the nucleus (Acinus) 7,2 8,1 P18583 Protein SON (Bax antagonist selected in saccharomyces 1) (BASS1) (Negative regulatory element-binding protein) (NRE-binding protein) (Protein DBP-5) 5,1 6,9 (SON3) P05141 ADP/ATP translocase 2 (ADP,ATP carrier protein 2) (ADP,ATP carrier protein, fibroblast isoform) (Adenine nucleotide translocator 2) (ANT 2) (Solute carrier 5,9 6,3 family 25 member 5) [Cleaved into: ADP/ATP translocase 2, N-terminally processed] P48681 Nestin 5,4 6,1 Q9Y3E5 Peptidyl-tRNA hydrolase 2, mitochondrial (PTH 2) (EC 3.1.1.29) (Bcl-2 inhibitor of transcription 1) 4,6 4,6 P09651 Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) (Helix-destabilizing protein) (Single-strand RNA-binding protein) (hnRNP core protein A1) [Cleaved 3,8 2,9 into: Heterogeneous nuclear ribonucleoprotein A1, N-terminally processed] P35232 Prohibitin 2,6 2,8 P21796 Voltage-dependent anion-selective channel protein 1 (VDAC-1) (hVDAC1) (Outer mitochondrial membrane protein porin 1) (Plasmalemmal porin) (Porin 31HL) 2,7 2,8 (Porin 31HM) Q9Y3T9 Nucleolar complex protein 2 homolog (Protein NOC2 homolog) (NOC2-like protein) (Novel INHAT repressor) 2,7 2,7 P45880 Voltage-dependent anion-selective channel protein 2 (VDAC-2) (hVDAC2) (Outer mitochondrial membrane protein porin 2) 2,7 2,4 Q13177 Serine/threonine-protein kinase PAK 2 (EC 2.7.11.1) (Gamma-PAK) (PAK65) (S6/H4 kinase) (p21-activated kinase 2) (PAK-2) (p58) [Cleaved into: PAK-2p27 0,3 0,6 (p27); PAK-2p34 (p34) (C-t-PAK2)] P41250 Glycine--tRNA ligase (EC 6.1.1.14) (Diadenosine tetraphosphate synthetase) (AP-4-A synthetase) (Glycyl-tRNA synthetase) (GlyRS) 0,6 0,6 P25787 Proteasome subunit alpha type-2 (EC 3.4.25.1) (Macropain subunit C3) (Multicatalytic endopeptidase complex subunit C3) (Proteasome component C3) 0,3 0,5 P49756 RNA-binding protein 25 (Arg/Glu/Asp-rich protein of 120 kDa) (RED120) (Protein S164) (RNA-binding motif protein 25) (RNA-binding region-containing protein 0,5 0,5 7) Q13616 Cullin-1 (CUL-1) 0,6 0,4 Q05639 Elongation factor 1-alpha 2 (EF-1-alpha-2) (Eukaryotic elongation factor 1 A-2) (eEF1A-2) (Statin-S1) 0,5 0,4 Q99700 Ataxin-2 (Spinocerebellar ataxia type 2 protein) (Trinucleotide repeat-containing gene 13 protein) 0,4 0,3

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Q8IX12 Cell division cycle and apoptosis regulator protein 1 (Cell cycle and apoptosis regulatory protein 1) (CARP-1) (Death inducer with SAP domain) 0,6 0,2 Q9BVJ6 U3 small nucleolar RNA-associated protein 14 homolog A (Antigen NY-CO-16) (Serologically defined colon cancer antigen 16) #### #### #### #### ## ## Q16891 Mitochondrial inner membrane protein (Cell proliferation-inducing gene 4/52 protein) (Mitofilin) (p87/89) 44,9 49,0 Q02978 Mitochondrial 2-oxoglutarate/malate carrier protein (OGCP) (Solute carrier family 25 member 11) 39,1 44,4 O96008 Mitochondrial import receptor subunit TOM40 homolog (Protein Haymaker) (Translocase of outer membrane 40 kDa subunit homolog) (p38.5) 26,4 32,9 Q12788 Transducin beta-like protein 3 (WD repeat-containing protein SAZD) 22,4 29,3 Q13423 NAD(P) transhydrogenase, mitochondrial (EC 1.6.1.2) (Nicotinamide nucleotide transhydrogenase) (Pyridine nucleotide transhydrogenase) 24,3 27,9 Q03252 Lamin-B2 19,2 24,0 P53007 Tricarboxylate transport protein, mitochondrial (Citrate transport protein) (CTP) (Solute carrier family 25 member 1) (Tricarboxylate carrier protein) 20,5 18,7 Q8N766 ER membrane protein complex subunit 1 7,8 10,4 P22695 Cytochrome b-c1 complex subunit 2, mitochondrial (Complex III subunit 2) (Core protein II) (Ubiquinol-cytochrome-c reductase complex core protein 2) 6,0 7,3 P49454 Centromere protein F (CENP-F) (AH antigen) (Kinetochore protein CENPF) (Mitosin) 6,4 6,3 Q9Y3Y2 Chromatin target of PRMT1 protein (Friend of PRMT1 protein) (Small arginine- and glycine-rich protein) (SRAG) 4,3 5,2 Q8N2K0 Monoacylglycerol lipase ABHD12 (EC 3.1.1.23) (2-arachidonoylglycerol hydrolase) (Abhydrolase domain-containing protein 12) 4,7 5,1 P56182 Ribosomal RNA processing protein 1 homolog A (Novel nuclear protein 1) (NNP-1) (Nucleolar protein Nop52) (RRP1-like protein) 5,4 5,1 O60264 SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5 (SWI/SNF-related matrix-associated actin-dependent 4,8 4,8 regulator of chromatin A5) (EC 3.6.4.-) (Sucrose nonfermenting protein 2 homolog) (hSNF2H) P36542 ATP synthase subunit gamma, mitochondrial (F-ATPase gamma subunit) 5,3 4,5 P24539 ATP synthase F(0) complex subunit B1, mitochondrial (ATP synthase proton-transporting mitochondrial F(0) complex subunit B1) (ATP synthase subunit b) 3,5 4,4 (ATPase subunit b) Q9Y277 Voltage-dependent anion-selective channel protein 3 (VDAC-3) (hVDAC3) (Outer mitochondrial membrane protein porin 3) 3,9 4,3 Q9UJZ1 Stomatin-like protein 2, mitochondrial (SLP-2) (EPB72-like protein 2) (Paraprotein target 7) (Paratarg-7) 3,6 3,9 P35613 Basigin (5F7) (Collagenase stimulatory factor) (Extracellular matrix metalloproteinase inducer) (EMMPRIN) (Leukocyte activation antigen M6) (OK blood group 3,7 3,9 antigen) (Tumor cell-derived collagenase stimulatory factor) (TCSF) (CD antigen CD147) Q9UJS0 Calcium-binding mitochondrial carrier protein Aralar2 (Citrin) (Mitochondrial aspartate glutamate carrier 2) (Solute carrier family 25 member 13) 5,0 3,9 Q9UNX4 WD repeat-containing protein 3 3,1 3,8 Q969V3 Nicalin (Nicastrin-like protein) 2,7 3,5 Q9H307 Pinin (140 kDa nuclear and cell adhesion-related phosphoprotein) (Desmosome-associated protein) (Domain-rich serine protein) (DRS protein) (DRSP) 3,4 3,1 (Melanoma metastasis clone A protein) (Nuclear protein SDK3) (SR-like protein) P05023 Sodium/potassium-transporting ATPase subunit alpha-1 (Na(+)/K(+) ATPase alpha-1 subunit) (EC 3.6.3.9) (Sodium pump subunit alpha-1) 2,0 2,9

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O95864 Fatty acid desaturase 2 (EC 1.14.19.-) (Delta(6) fatty acid desaturase) (D6D) (Delta(6) desaturase) (Delta-6 desaturase) 2,9 2,8 Q14165 Malectin 2,6 2,7 Q9BYG3 MKI67 FHA domain-interacting nucleolar phosphoprotein (Nucleolar phosphoprotein Nopp34) (Nucleolar protein interacting with the FHA domain of pKI-67) 2,5 2,6 (hNIFK) P00403 Cytochrome c oxidase subunit 2 (Cytochrome c oxidase polypeptide II) 2,7 2,4 P25705 ATP synthase subunit alpha, mitochondrial 1,9 2,2 P42167 Lamina-associated polypeptide 2, isoforms beta/gamma (Thymopoietin, isoforms beta/gamma) (TP beta/gamma) (Thymopoietin-related peptide isoforms 2,1 2,1 beta/gamma) (TPRP isoforms beta/gamma) [Cleaved into: Thymopoietin (TP) (Splenin); Thymopentin (TP5)] Q9Y5M8 Signal recognition particle receptor subunit beta (SR-beta) (Protein APMCF1) 2,1 2,1 Q01813 ATP-dependent 6-phosphofructokinase, platelet type (ATP-PFK) (PFK-P) (EC 2.7.1.11) (6-phosphofructokinase type C) (Phosphofructo-1-kinase isozyme C) 1,5 2,1 (PFK-C) (Phosphohexokinase) O75369 Filamin-B (FLN-B) (ABP-278) (ABP-280 homolog) (Actin-binding-like protein) (Beta-filamin) (Filamin homolog 1) (Fh1) (Filamin-3) (Thyroid autoantigen) 2,4 2,1 (Truncated actin-binding protein) (Truncated ABP) Q99623 Prohibitin-2 (B-cell receptor-associated protein BAP37) (D-prohibitin) (Repressor of estrogen receptor activity) 1,7 1,9 Q9NZ01 Very-long-chain enoyl-CoA reductase (EC 1.3.1.93) (Synaptic glycoprotein SC2) (Trans-2,3-enoyl-CoA reductase) (TER) 1,8 1,8 Q9BTV4 Transmembrane protein 43 (Protein LUMA) 1,6 1,7 O95232 Luc7-like protein 3 (Cisplatin resistance-associated-overexpressed protein) (Luc7A) (Okadaic acid-inducible phosphoprotein OA48-18) (cAMP regulatory 1,7 1,7 element-associated protein 1) (CRE-associated protein 1) (CREAP-1) P62851 40S ribosomal protein S25 1,5 1,5 Q9Y490 Talin-1 1,2 1,4 Q6PI48 Aspartate--tRNA ligase, mitochondrial (EC 6.1.1.12) (Aspartyl-tRNA synthetase) (AspRS) 0,6 0,6 Q9Y606 tRNA pseudouridine synthase A, mitochondrial (EC 5.4.99.12) (tRNA pseudouridine(38-40) synthase) (tRNA pseudouridylate synthase I) (tRNA-uridine isomerase 0,6 0,6 I) Q92620 Pre-mRNA-splicing factor ATP-dependent RNA helicase PRP16 (EC 3.6.4.13) (ATP-dependent RNA helicase DHX38) (DEAH box protein 38) 0,5 0,6 Q92522 Histone H1x 0,6 0,6 O15347 High mobility group protein B3 (High mobility group protein 2a) (HMG-2a) (High mobility group protein 4) (HMG-4) 0,6 0,6 Q6P2E9 Enhancer of mRNA-decapping protein 4 (Autoantigen Ge-1) (Autoantigen RCD-8) (Human enhancer of decapping large subunit) (Hedls) 0,4 0,5 Q05519 Serine/arginine-rich splicing factor 11 (Arginine-rich 54 kDa nuclear protein) (p54) (Splicing factor, arginine/serine-rich 11) 0,4 0,5 P18615 Negative elongation factor E (NELF-E) (RNA-binding protein RD) 0,3 0,5 Q08J23 tRNA (cytosine(34)-C(5))-methyltransferase (EC 2.1.1.203) (Myc-induced SUN domain-containing protein) (Misu) (NOL1/NOP2/Sun domain family member 2) 0,5 0,5 (Substrate of AIM1/Aurora kinase B) (tRNA (cytosine-5-)-methyltransferase) (tRNA methyltransferase 4 homolog) (hTrm4) Q92747 Actin-related protein 2/3 complex subunit 1A (SOP2-like protein) 0,5 0,5 Q15435 Protein phosphatase 1 regulatory subunit 7 (Protein phosphatase 1 regulatory subunit 22) 0,7 0,5

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P42166 Lamina-associated polypeptide 2, isoform alpha (Thymopoietin isoform alpha) (TP alpha) (Thymopoietin-related peptide isoform alpha) (TPRP isoform alpha) 0,6 0,5 [Cleaved into: Thymopoietin (TP) (Splenin); Thymopentin (TP5)] O95819 Mitogen-activated protein kinase kinase kinase kinase 4 (EC 2.7.11.1) (HPK/GCK-like kinase HGK) (MAPK/ERK kinase kinase kinase 4) (MEK kinase kinase 4) 0,5 0,5 (MEKKK 4) (Nck-interacting kinase) Q15291 Retinoblastoma-binding protein 5 (RBBP-5) (Retinoblastoma-binding protein RBQ-3) 0,5 0,4 Q6PD62 RNA polymerase-associated protein CTR9 homolog (SH2 domain-binding protein 1) 0,5 0,4 P51858 Hepatoma-derived growth factor (HDGF) (High mobility group protein 1-like 2) (HMG-1L2) 0,5 0,4 P17174 Aspartate aminotransferase, cytoplasmic (cAspAT) (EC 2.6.1.1) (EC 2.6.1.3) (Cysteine aminotransferase, cytoplasmic) (Cysteine transaminase, cytoplasmic) 0,5 0,4 (cCAT) (Glutamate oxaloacetate transaminase 1) (Transaminase A) P62841 40S ribosomal protein S15 (RIG protein) 0,3 0,4 Q96CW1 AP-2 complex subunit mu (AP-2 mu chain) (Adapter-related protein complex 2 subunit mu) (Adaptin-mu2) (Adaptor protein complex AP-2 subunit mu) 0,3 0,4 (Clathrin assembly protein complex 2 mu medium chain) (Clathrin coat assembly protein AP50) (Clathrin coat-associated protein AP50) (HA2 50 kDa subunit) (Plasma membrane adaptor AP-2 50 kDa protein) Q14677 Clathrin interactor 1 (Clathrin-interacting protein localized in the trans-Golgi region) (Clint) (Enthoprotin) (Epsin-4) (Epsin-related protein) (EpsinR) 0,5 0,4 Q6P158 Putative ATP-dependent RNA helicase DHX57 (EC 3.6.4.13) (DEAH box protein 57) 0,5 0,4 P62899 60S ribosomal protein L31 0,5 0,4 Q8WWY3 U4/U6 small nuclear ribonucleoprotein Prp31 (Pre-mRNA-processing factor 31) (Serologically defined breast cancer antigen NY-BR-99) (U4/U6 snRNP 61 kDa 0,5 0,4 protein) (Protein 61K) (hPrp31) Q8TCS8 Polyribonucleotide nucleotidyltransferase 1, mitochondrial (EC 2.7.7.8) (3'-5' RNA exonuclease OLD35) (PNPase old-35) (Polynucleotide phosphorylase 1) 0,6 0,4 (PNPase 1) (Polynucleotide phosphorylase-like protein) O43847 Nardilysin (EC 3.4.24.61) (N-arginine dibasic convertase) (NRD convertase) (NRD-C) 0,6 0,4 Q9UPQ0 LIM and calponin homology domains-containing protein 1 0,5 0,4 P33316 Deoxyuridine 5'-triphosphate nucleotidohydrolase, mitochondrial (dUTPase) (EC 3.6.1.23) (dUTP pyrophosphatase) 0,5 0,3 P26038 Moesin (Membrane-organizing extension spike protein) 0,6 0,3 P53004 Biliverdin reductase A (BVR A) (EC 1.3.1.24) (Biliverdin-IX alpha-reductase) 0,6 0,3 Q8ND56 Protein LSM14 homolog A (Protein FAM61A) (Protein SCD6 homolog) (Putative alpha-synuclein-binding protein) (AlphaSNBP) (RNA-associated protein 55A) 0,4 0,3 (hRAP55) (hRAP55A) Q7Z2T5 TRMT1-like protein (EC 2.1.1.-) 0,3 0,3 Q92541 RNA polymerase-associated protein RTF1 homolog 0,5 0,3 Q6YP21 Kynurenine--oxoglutarate transaminase 3 (EC 2.6.1.7) (Cysteine-S-conjugate beta-lyase 2) (EC 4.4.1.13) (Kynurenine aminotransferase III) (KATIII) (Kynurenine-- 0,4 0,3 glyoxylate transaminase) (EC 2.6.1.63) (Kynurenine--oxoglutarate transaminase III) Q9Y520 Protein PRRC2C (BAT2 domain-containing protein 1) (HBV X-transactivated gene 2 protein) (HBV XAg-transactivated protein 2) (HLA-B-associated transcript 2- 0,2 0,2 like 2) (Proline-rich and coiled-coil-containing protein 2C) P11171 Protein 4.1 (P4.1) (4.1R) (Band 4.1) (EPB4.1) 0,5 0,2

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Attachment 4 WT HtrA2/Omi effect resulted in 72 differential proteins. Significant regulation ratio of each protein quantified between two sample types is listed in the table.

Accession Protein VC VC/ WT/ /W G39 G39 T 9S 9S 0h 0h 0h P35222 Catenin beta-1 (Beta-catenin) 0,4 4,1 Q96TA1 Niban-like protein 1 (Meg-3) (Melanoma invasion by ERK) (MINERVA) (Protein FAM129B) 0,6 3,1 P35221 Catenin alpha-1 (Alpha E-catenin) (Cadherin-associated protein) (Renal carcinoma antigen NY-REN-13) 0,3 2,7 P12814 Alpha-actinin-1 (Alpha-actinin cytoskeletal isoform) (F-actin cross-linking protein) (Non-muscle alpha-actinin-1) 0,5 2,0 P62258 14-3-3 protein epsilon (14-3-3E) 1,3 0,7 Q9Y2A7 Nck-associated protein 1 (NAP 1) (Membrane-associated protein HEM-2) (p125Nap1) 1,5 0,7 O00429 Dynamin-1-like protein (EC 3.6.5.5) (Dnm1p/Vps1p-like protein) (DVLP) (Dynamin family member proline-rich carboxyl-terminal domain less) (Dymple) 2,0 0,5 (Dynamin-like protein) (Dynamin-like protein 4) (Dynamin-like protein IV) (HdynIV) (Dynamin-related protein 1) P52701 DNA mismatch repair protein Msh6 (hMSH6) (G/T mismatch-binding protein) (GTBP) (GTMBP) (MutS-alpha 160 kDa subunit) (p160) 2,3 0,4 P54577 Tyrosine--tRNA ligase, cytoplasmic (EC 6.1.1.1) (Tyrosyl-tRNA synthetase) (TyrRS) [Cleaved into: Tyrosine--tRNA ligase, cytoplasmic, N-terminally processed] 2,8 0,4 O60341 Lysine-specific histone demethylase 1A (EC 1.-.-.-) (BRAF35-HDAC complex protein BHC110) (Flavin-containing amine oxidase domain-containing protein 2) 2,7 0,4 P61764 Syntaxin-binding protein 1 (MUNC18-1) (N-Sec1) (Protein unc-18 homolog 1) (Unc18-1) (Protein unc-18 homolog A) (Unc-18A) (p67) 6,6 0,1 P49006 MARCKS-related protein (MARCKS-like protein 1) (Macrophage myristoylated alanine-rich C kinase substrate) (Mac-MARCKS) (MacMARCKS) 0,5 8,6 O00159 Unconventional myosin-Ic (Myosin I beta) (MMI-beta) (MMIb) 0,3 7,7 Q9P2E9 Ribosome-binding protein 1 (180 kDa ribosome receptor homolog) (RRp) (ES/130-related protein) (Ribosome receptor protein) 0,2 6,2 Q9UIG0 Tyrosine-protein kinase BAZ1B (EC 2.7.10.2) (Bromodomain adjacent to zinc finger domain protein 1B) (Williams syndrome transcription factor) (Williams- 0,3 5,6 Beuren syndrome chromosomal region 10 protein) (Williams-Beuren syndrome chromosomal region 9 protein) (hWALp2) Q12797 Aspartyl/asparaginyl beta-hydroxylase (EC 1.14.11.16) (Aspartate beta-hydroxylase) (ASP beta-hydroxylase) (Peptide-aspartate beta-dioxygenase) 0,2 5,4 Q96G23 Ceramide synthase 2 (CerS2) (LAG1 longevity assurance homolog 2) (SP260) (Tumor metastasis-suppressor gene 1 protein) 0,4 5,0 Q9NR12 PDZ and LIM domain protein 7 (LIM mineralization protein) (LMP) (Protein enigma) 0,3 4,6 Q09666 Neuroblast differentiation-associated protein AHNAK (Desmoyokin) 0,3 4,4 P18754 Regulator of chromosome condensation (Cell cycle regulatory protein) (Chromosome condensation protein 1) 0,5 3,2 Q05682 Caldesmon (CDM) 0,5 2,9 O75475 PC4 and SFRS1-interacting protein (CLL-associated antigen KW-7) (Dense fine speckles 70 kDa protein) (DFS 70) (Lens epithelium-derived growth factor) 0,4 2,9 (Transcriptional coactivator p75/p52)

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P16401 Histone H1.5 (Histone H1a) (Histone H1b) (Histone H1s-3) 0,5 2,8 Q32MZ4 Leucine-rich repeat flightless-interacting protein 1 (LRR FLII-interacting protein 1) (GC-binding factor 2) (TAR RNA-interacting protein) 0,4 2,7 Q15942 Zyxin (Zyxin-2) 0,5 2,6 P08133 Annexin A6 (67 kDa calelectrin) (Annexin VI) (Annexin-6) (Calphobindin-II) (CPB-II) (Chromobindin-20) (Lipocortin VI) (Protein III) (p68) (p70) 0,3 2,6 Q96HC4 PDZ and LIM domain protein 5 (Enigma homolog) (Enigma-like PDZ and LIM domains protein) 0,3 2,5 P49257 Protein ERGIC-53 (ER-Golgi intermediate compartment 53 kDa protein) (Gp58) (Intracellular mannose-specific lectin MR60) (Lectin mannose-binding 1) 0,6 2,5 P13674 Prolyl 4-hydroxylase subunit alpha-1 (4-PH alpha-1) (EC 1.14.11.2) (Procollagen-proline,2-oxoglutarate-4-dioxygenase subunit alpha-1) 0,5 2,4 Q12792 Twinfilin-1 (Protein A6) (Protein tyrosine kinase 9) 0,4 2,4 Q9UHB6 LIM domain and actin-binding protein 1 (Epithelial protein lost in neoplasm) 0,6 2,3 P50454 Serpin H1 (47 kDa heat shock protein) (Arsenic-transactivated protein 3) (AsTP3) (Cell proliferation-inducing gene 14 protein) (Collagen-binding protein) 0,4 2,2 (Colligin) (Rheumatoid arthritis-related antigen RA-A47) Q9UMS6 Synaptopodin-2 (Genethonin-2) (Myopodin) 0,6 2,2 Q9Y678 Coatomer subunit gamma-1 (Gamma-1-coat protein) (Gamma-1-COP) 0,6 2,2 Q6ZXV5 Transmembrane and TPR repeat-containing protein 3 (Protein SMILE) 0,6 2,1 Q9Y4P3 Transducin beta-like protein 2 (WS beta-transducin repeats protein) (WS-betaTRP) (Williams-Beuren syndrome chromosomal region 13 protein) 0,5 2,0 O60701 UDP-glucose 6-dehydrogenase (UDP-Glc dehydrogenase) (UDP-GlcDH) (UDPGDH) (EC 1.1.1.22) 0,6 1,9 Q96CN7 Isochorismatase domain-containing protein 1 0,7 1,8 Q15738 Sterol-4-alpha-carboxylate 3-dehydrogenase, decarboxylating (EC 1.1.1.170) (Protein H105e3) 0,6 1,6 O14974 Protein phosphatase 1 regulatory subunit 12A (Myosin phosphatase-targeting subunit 1) (Myosin phosphatase target subunit 1) (Protein phosphatase 0,6 1,4 myosin-binding subunit) Q16352 Alpha-internexin (Alpha-Inx) (66 kDa neurofilament protein) (NF-66) (Neurofilament-66) (Neurofilament 5) 2,1 0,6 P78347 General transcription factor II-I (GTFII-I) (TFII-I) (Bruton tyrosine kinase-associated protein 135) (BAP-135) (BTK-associated protein 135) (SRF-Phox1- 1,5 0,6 interacting protein) (SPIN) (Williams-Beuren syndrome chromosomal region 6 protein) Q8IZL8 Proline-, glutamic acid- and leucine-rich protein 1 (Modulator of non-genomic activity of estrogen receptor) (Transcription factor HMX3) 2,0 0,6 O14531 Dihydropyrimidinase-related protein 4 (DRP-4) (Collapsin response mediator protein 3) (CRMP-3) (UNC33-like phosphoprotein 4) (ULIP-4) 1,7 0,6 Q07955 Serine/arginine-rich splicing factor 1 (Alternative-splicing factor 1) (ASF-1) (Splicing factor, arginine/serine-rich 1) (pre-mRNA-splicing factor SF2, P33 2,3 0,5 subunit) P33991 DNA replication licensing factor MCM4 (EC 3.6.4.12) (CDC21 homolog) (P1-CDC21) 1,9 0,5 O14744 Protein arginine N-methyltransferase 5 (EC 2.1.1.-) (72 kDa ICln-binding protein) (Histone-arginine N-methyltransferase PRMT5) (EC 2.1.1.125) (Jak-binding 1,5 0,5 protein 1) (Shk1 kinase-binding protein 1 homolog) (SKB1 homolog) (SKB1Hs) [Cleaved into: Protein arginine N-methyltransferase 5, N-terminally processed] Q8N7H5 RNA polymerase II-associated factor 1 homolog (hPAF1) (Pancreatic differentiation protein 2) 1,9 0,5

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Q10570 Cleavage and polyadenylation specificity factor subunit 1 (Cleavage and polyadenylation specificity factor 160 kDa subunit) (CPSF 160 kDa subunit) 1,6 0,5 P55010 Eukaryotic translation initiation factor 5 (eIF-5) 2,0 0,4 P13984 General transcription factor IIF subunit 2 (EC 3.6.4.12) (ATP-dependent helicase GTF2F2) (General transcription factor IIF 30 kDa subunit) (Transcription 2,4 0,4 initiation factor IIF subunit beta) (TFIIF-beta) (Transcription initiation factor RAP30) P29144 Tripeptidyl-peptidase 2 (TPP-2) (EC 3.4.14.10) (Tripeptidyl aminopeptidase) (Tripeptidyl-peptidase II) (TPP-II) 2,7 0,4 Q99426 Tubulin-folding cofactor B (Cytoskeleton-associated protein 1) (Cytoskeleton-associated protein CKAPI) (Tubulin-specific chaperone B) 2,1 0,4 Q92888 Rho guanine nucleotide exchange factor 1 (115 kDa guanine nucleotide exchange factor) (p115-RhoGEF) (p115RhoGEF) (Sub1.5) 2,1 0,4 Q9UNH7 Sorting nexin-6 (TRAF4-associated factor 2) [Cleaved into: Sorting nexin-6, N-terminally processed] 1,8 0,4 P14550 Alcohol dehydrogenase [NADP(+)] (EC 1.1.1.2) (Aldehyde reductase) (Aldo-keto reductase family 1 member A1) 2,1 0,4 P38606 V-type proton ATPase catalytic subunit A (V-ATPase subunit A) (EC 3.6.3.14) (V-ATPase 69 kDa subunit) (Vacuolar ATPase isoform VA68) (Vacuolar proton 2,0 0,4 pump subunit alpha) Q9BTV5 Fibronectin type III and SPRY domain-containing protein 1 (MID1-related protein 1) (Microtubule-associated protein GLFND) 2,1 0,4 Q14241 Transcription elongation factor B polypeptide 3 (Elongin 110 kDa subunit) (Elongin-A) (EloA) (RNA polymerase II transcription factor SIII subunit A1) (SIII 2,3 0,4 p110) Q9GZR7 ATP-dependent RNA helicase DDX24 (EC 3.6.4.13) (DEAD box protein 24) 2,2 0,3 Q52LJ0 Protein FAM98B 2,8 0,3 Q05193 Dynamin-1 (EC 3.6.5.5) 3,4 0,3 O14497 AT-rich interactive domain-containing protein 1A (ARID domain-containing protein 1A) (B120) (BRG1-associated factor 250) (BAF250) (BRG1-associated 3,3 0,3 factor 250a) (BAF250A) (Osa homolog 1) (hOSA1) (SWI-like protein) (SWI/SNF complex protein p270) (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin subfamily F member 1) (hELD) Q9UPN3 Microtubule-actin cross-linking factor 1, isoforms 1/2/3/5 (620 kDa actin-binding protein) (ABP620) (Actin cross-linking family protein 7) (Macrophin-1) 2,0 0,3 (Trabeculin-alpha) P35249 Replication factor C subunit 4 (Activator 1 37 kDa subunit) (A1 37 kDa subunit) (Activator 1 subunit 4) (Replication factor C 37 kDa subunit) (RF-C 37 kDa 2,4 0,3 subunit) (RFC37) Q00688 Peptidyl-prolyl cis-trans isomerase FKBP3 (PPIase FKBP3) (EC 5.2.1.8) (25 kDa FK506-binding protein) (25 kDa FKBP) (FKBP-25) (FK506-binding protein 3) 2,5 0,3 (FKBP-3) (Immunophilin FKBP25) (Rapamycin-selective 25 kDa immunophilin) (Rotamase) P08237 ATP-dependent 6-phosphofructokinase, muscle type (ATP-PFK) (PFK-M) (EC 2.7.1.11) (6-phosphofructokinase type A) (Phosphofructo-1-kinase isozyme A) 3,3 0,3 (PFK-A) (Phosphohexokinase) P61011 Signal recognition particle 54 kDa protein (SRP54) 3,2 0,3 Q9BV38 WD repeat-containing protein 18 2,8 0,3 O43237 Cytoplasmic dynein 1 light intermediate chain 2 (Dynein light intermediate chain 2, cytosolic) (LIC-2) (LIC53/55) 3,5 0,2 Q9H078 Caseinolytic peptidase B protein homolog (Suppressor of potassium transport defect 3) 6,3 0,2 Q969Z0 Protein TBRG4 (Cell cycle progression restoration protein 2) (Cell cycle progression protein 2) (FAST kinase domain-containing protein 4) (Transforming 7,7 0,1 growth factor beta regulator 4)

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Attachment 5 Transfection effect resulted in 15 proteins differential. Significant regulation ratio of each protein quantified between two sample types is listed in the table.

Accession Protein VC/ VC/G3 WT/G3 WT 99S 99S 0h 0h 0h P46379 Large proline-rich protein BAG6 (BAG family molecular chaperone regulator 6) (BCL2-associated athanogene 6) (BAG-6) (BAG6) (HLA-B-associated 1,7 3,0 transcript 3) (Protein G3) (Protein Scythe) Q12888 Tumor suppressor p53-binding protein 1 (53BP1) (p53-binding protein 1) (p53BP1) 2,0 2,3 P27708 CAD protein [Includes: Glutamine-dependent carbamoyl-phosphate synthase (EC 6.3.5.5); Aspartate carbamoyltransferase (EC 2.1.3.2); Dihydroorotase 2,3 2,0 (EC 3.5.2.3)] P30876 DNA-directed RNA polymerase II subunit RPB2 (EC 2.7.7.6) (DNA-directed RNA polymerase II 140 kDa polypeptide) (DNA-directed RNA polymerase II 2,0 1,7 subunit B) (RNA polymerase II subunit 2) (RNA polymerase II subunit B2) Q14739 Lamin-B receptor (Integral nuclear envelope inner membrane protein) (LMN2R) 1,5 1,7 Q8TD19 Serine/threonine-protein kinase Nek9 (EC 2.7.11.1) (Nercc1 kinase) (Never in mitosis A-related kinase 9) (NimA-related protein kinase 9) (NimA-related 2,4 1,7 kinase 8) (Nek8) P42704 Leucine-rich PPR motif-containing protein, mitochondrial (130 kDa leucine-rich protein) (LRP 130) (GP130) 1,8 1,6 Q9BYD1 39S ribosomal protein L13, mitochondrial (L13mt) (MRP-L13) 2,0 1,6 P21266 Glutathione S-transferase Mu 3 (EC 2.5.1.18) (GST class-mu 3) (GSTM3-3) (hGSTM3-3) 2,3 1,5 P52943 Cysteine-rich protein 2 (CRP-2) (Protein ESP1) 0,4 0,4 P08962 CD63 antigen (Granulophysin) (Lysosomal-associated membrane protein 3) (LAMP-3) (Melanoma-associated antigen ME491) (OMA81H) (Ocular 0,3 0,4 melanoma-associated antigen) (Tetraspanin-30) (Tspan-30) (CD antigen CD63) O15260 Surfeit locus protein 4 0,3 0,4 O15270 Serine palmitoyltransferase 2 (EC 2.3.1.50) (Long chain base biosynthesis protein 2) (LCB 2) (Long chain base biosynthesis protein 2a) (LCB2a) (Serine- 0,2 0,3 palmitoyl-CoA transferase 2) (SPT 2) Q8IV08 Phospholipase D3 (PLD 3) (EC 3.1.4.4) (Choline phosphatase 3) (HindIII K4L homolog) (Hu-K4) (Phosphatidylcholine-hydrolyzing phospholipase D3) 0,3 0,3 O43464 Serine protease HTRA2, mitochondrial (EC 3.4.21.108) (High temperature requirement protein A2) (HtrA2) (Omi stress-regulated endoprotease) 0,1 0,1 (Serine protease 25) (Serine proteinase OMI)

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Attachment 6 Cell stress induced 143 protein regulated for vector control cells. Significant regulation ratio of each protein quantified between two conditions (before and after stress) is listed in the table.

Accession Protein VC 0h/6h

Q5T4S7 E3 ubiquitin-protein ligase UBR4 (EC 6.3.2.-) (600 kDa retinoblastoma protein-associated factor) (N-recognin-4) (Retinoblastoma-associated factor of 600 kDa) 4,3 (RBAF600) (p600) (Zinc finger UBR1-type protein 1) Q9UFC0 Leucine-rich repeat and WD repeat-containing protein 1 (Centromere protein 33) (CENP-33) (Origin recognition complex-associated protein) (ORC-associated protein) 3,4 (ORCA) Q8TD19 Serine/threonine-protein kinase Nek9 (EC 2.7.11.1) (Nercc1 kinase) (Never in mitosis A-related kinase 9) (NimA-related protein kinase 9) (NimA-related kinase 8) 3,4 (Nek8) P46782 40S ribosomal protein S5 [Cleaved into: 40S ribosomal protein S5, N-terminally processed] 3,1 A5YKK6 CCR4-NOT transcription complex subunit 1 (CCR4-associated factor 1) (Negative regulator of transcription subunit 1 homolog) (NOT1H) (hNOT1) 2,7 P13984 General transcription factor IIF subunit 2 (EC 3.6.4.12) (ATP-dependent helicase GTF2F2) (General transcription factor IIF 30 kDa subunit) (Transcription initiation 2,6 factor IIF subunit beta) (TFIIF-beta) (Transcription initiation factor RAP30) Q9UPN3 Microtubule-actin cross-linking factor 1, isoforms 1/2/3/5 (620 kDa actin-binding protein) (ABP620) (Actin cross-linking family protein 7) (Macrophin-1) (Trabeculin- 2,5 alpha) O14974 Protein phosphatase 1 regulatory subunit 12A (Myosin phosphatase-targeting subunit 1) (Myosin phosphatase target subunit 1) (Protein phosphatase myosin-binding 2,4 subunit) Q9H4M9 EH domain-containing protein 1 (PAST homolog 1) (hPAST1) (Testilin) 2,4 P13674 Prolyl 4-hydroxylase subunit alpha-1 (4-PH alpha-1) (EC 1.14.11.2) (Procollagen-proline,2-oxoglutarate-4-dioxygenase subunit alpha-1) 2,3 P09493 Tropomyosin alpha-1 chain (Alpha-tropomyosin) (Tropomyosin-1) 2,3 Q05193 Dynamin-1 (EC 3.6.5.5) 2,2 O60524 Nuclear export mediator factor NEMF (Antigen NY-CO-1) (Serologically defined colon cancer antigen 1) 2,2 Q14739 Lamin-B receptor (Integral nuclear envelope inner membrane protein) (LMN2R) 2,1 Q14195 Dihydropyrimidinase-related protein 3 (DRP-3) (Collapsin response mediator protein 4) (CRMP-4) (Unc-33-like phosphoprotein 1) (ULIP-1) 2,1 Q92888 Rho guanine nucleotide exchange factor 1 (115 kDa guanine nucleotide exchange factor) (p115-RhoGEF) (p115RhoGEF) (Sub1.5) 2,1 P61201 COP9 signalosome complex subunit 2 (SGN2) (Signalosome subunit 2) (Alien homolog) (JAB1-containing signalosome subunit 2) (Thyroid receptor-interacting protein 2,1 15) (TR-interacting protein 15) (TRIP-15) Q96SB3 Neurabin-2 (Neurabin-II) (Protein phosphatase 1 regulatory subunit 9B) (Spinophilin) 2,0 A6NKG5 Retrotransposon-like protein 1 (Mammalian retrotransposon derived protein 1) (Paternally expressed gene 11 protein) (Retrotransposon-derived protein PEG11) 2,0 Q9UNH7 Sorting nexin-6 (TRAF4-associated factor 2) [Cleaved into: Sorting nexin-6, N-terminally processed] 2,0 P30566 Adenylosuccinate lyase (ASL) (EC 4.3.2.2) (Adenylosuccinase) (ASase) 1,9

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P83731 60S ribosomal protein L24 (60S ribosomal protein L30) 1,9 Q02952 A-kinase anchor protein 12 (AKAP-12) (A-kinase anchor protein 250 kDa) (AKAP 250) (Gravin) (Myasthenia gravis autoantigen) 1,9 P49792 E3 SUMO-protein ligase RanBP2 (EC 6.3.2.-) (358 kDa nucleoporin) (Nuclear pore complex protein Nup358) (Nucleoporin Nup358) (Ran-binding protein 2) (RanBP2) 1,9 (p270) P30876 DNA-directed RNA polymerase II subunit RPB2 (EC 2.7.7.6) (DNA-directed RNA polymerase II 140 kDa polypeptide) (DNA-directed RNA polymerase II subunit B) (RNA 1,8 polymerase II subunit 2) (RNA polymerase II subunit B2) Q9NUL3 Double-stranded RNA-binding protein Staufen homolog 2 1,8 Q07020 60S ribosomal protein L18 1,8 P11172 Uridine 5'-monophosphate synthase (UMP synthase) [Includes: Orotate phosphoribosyltransferase (OPRT) (OPRTase) (EC 2.4.2.10); Orotidine 5'- 1,8 phosphate decarboxylase (ODC) (EC 4.1.1.23) (OMPdecase)] P52888 Thimet oligopeptidase (EC 3.4.24.15) (Endopeptidase 24.15) (MP78) 1,7 Q14558 Phosphoribosyl pyrophosphate synthase-associated protein 1 (PRPP synthase-associated protein 1) (39 kDa phosphoribosypyrophosphate synthase- 1,6 associated protein) (PAP39) Q9UI10 Translation initiation factor eIF-2B subunit delta (eIF-2B GDP-GTP exchange factor subunit delta) 1,5 P29144 Tripeptidyl-peptidase 2 (TPP-2) (EC 3.4.14.10) (Tripeptidyl aminopeptidase) (Tripeptidyl-peptidase II) (TPP-II) 1,5 O15173 Membrane-associated progesterone receptor component 2 (Progesterone membrane-binding protein) (Steroid receptor protein DG6) 1,5 Q7Z6Z7 E3 ubiquitin-protein ligase HUWE1 (EC 6.3.2.-) (ARF-binding protein 1) (ARF-BP1) (HECT, UBA and WWE domain-containing protein 1) (Homologous to E6AP carboxyl 1,5 terminus homologous protein 9) (HectH9) (Large structure of UREB1) (LASU1) (Mcl-1 ubiquitin ligase E3) (Mule) (Upstream regulatory element-binding protein 1) (URE-B1) (URE-binding protein 1) O60271 C-Jun-amino-terminal kinase-interacting protein 4 (JIP-4) (JNK-interacting protein 4) (Cancer/testis antigen 89) (CT89) (Human lung cancer oncogene 6 protein) (HLC- 1,5 6) (JNK-associated leucine-zipper protein) (JLP) (Mitogen-activated protein kinase 8-interacting protein 4) (Proliferation-inducing protein 6) (Protein highly expressed in testis) (PHET) (Sperm surface protein) (Sperm-associated antigen 9) (Sperm-specific protein) (Sunday driver 1) Q92616 Translational activator GCN1 (HsGCN1) (GCN1-like protein 1) 1,5 P53992 Protein transport protein Sec24C (SEC24-related protein C) 1,5 P27708 CAD protein [Includes: Glutamine-dependent carbamoyl-phosphate synthase (EC 6.3.5.5); Aspartate carbamoyltransferase (EC 2.1.3.2); Dihydroorotase (EC 3.5.2.3)] 1,4 O75369 Filamin-B (FLN-B) (ABP-278) (ABP-280 homolog) (Actin-binding-like protein) (Beta-filamin) (Filamin homolog 1) (Fh1) (Filamin-3) (Thyroid autoantigen) (Truncated 1,4 actin-binding protein) (Truncated ABP) O60763 General vesicular transport factor p115 (Protein USO1 homolog) (Transcytosis-associated protein) (TAP) (Vesicle-docking protein) 1,4 P42704 Leucine-rich PPR motif-containing protein, mitochondrial (130 kDa leucine-rich protein) (LRP 130) (GP130) 1,3 P25325 3-mercaptopyruvate sulfurtransferase (MST) (EC 2.8.1.2) 0,9 Q07065 Cytoskeleton-associated protein 4 (63-kDa cytoskeleton-linking membrane protein) (Climp-63) (p63) 0,8 Q9Y4L1 Hypoxia up-regulated protein 1 (150 kDa oxygen-regulated protein) (ORP-150) (170 kDa glucose-regulated protein) (GRP-170) 0,8 O43660 Pleiotropic regulator 1 0,8

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P62805 Histone H4 0,8 Q92747 Actin-related protein 2/3 complex subunit 1A (SOP2-like protein) 0,7 Q9HCC0 Methylcrotonoyl-CoA carboxylase beta chain, mitochondrial (MCCase subunit beta) (EC 6.4.1.4) (3-methylcrotonyl-CoA carboxylase 2) (3-methylcrotonyl-CoA 0,7 carboxylase non-biotin-containing subunit) (3-methylcrotonyl-CoA:carbon dioxide ligase subunit beta) Q09666 Neuroblast differentiation-associated protein AHNAK (Desmoyokin) 0,7 Q9GZR7 ATP-dependent RNA helicase DDX24 (EC 3.6.4.13) (DEAD box protein 24) 0,7 Q96CN7 Isochorismatase domain-containing protein 1 0,7 Q8TEX9 Importin-4 (Imp4) (Importin-4b) (Imp4b) (Ran-binding protein 4) (RanBP4) 0,7 Q9UJZ1 Stomatin-like protein 2, mitochondrial (SLP-2) (EPB72-like protein 2) (Paraprotein target 7) (Paratarg-7) 0,7 P05023 Sodium/potassium-transporting ATPase subunit alpha-1 (Na(+)/K(+) ATPase alpha-1 subunit) (EC 3.6.3.9) (Sodium pump subunit alpha-1) 0,7 O60716 Catenin delta-1 (Cadherin-associated Src substrate) (CAS) (p120 catenin) (p120(ctn)) (p120(cas)) 0,7 P50454 Serpin H1 (47 kDa heat shock protein) (Arsenic-transactivated protein 3) (AsTP3) (Cell proliferation-inducing gene 14 protein) (Collagen-binding protein) (Colligin) 0,6 (Rheumatoid arthritis-related antigen RA-A47) Q9Y606 tRNA pseudouridine synthase A, mitochondrial (EC 5.4.99.12) (tRNA pseudouridine(38-40) synthase) (tRNA pseudouridylate synthase I) (tRNA-uridine 0,6 isomerase I) Q96GM5 SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily D member 1 (60 kDa BRG-1/Brm-associated factor subunit A) (BRG1-associated 0,6 factor 60A) (BAF60A) (SWI/SNF complex 60 kDa subunit) O00116 Alkyldihydroxyacetonephosphate synthase, peroxisomal (Alkyl-DHAP synthase) (EC 2.5.1.26) (Aging-associated gene 5 protein) (Alkylglycerone-phosphate synthase) 0,6 P33991 DNA replication licensing factor MCM4 (EC 3.6.4.12) (CDC21 homolog) (P1-CDC21) 0,6 O75367 Core histone macro-H2A.1 (Histone macroH2A1) (mH2A1) (Histone H2A.y) (H2A/y) (Medulloblastoma antigen MU-MB-50.205) 0,6 Q9NX58 Cell growth-regulating nucleolar protein 0,6 O15240 Neurosecretory protein VGF [Cleaved into: Neuroendocrine regulatory peptide-1 (NERP-1); Neuroendocrine regulatory peptide-2 (NERP-2); Antimicrobial peptide 0,6 VGF[554-577]] Q07866 Kinesin light chain 1 (KLC 1) 0,6 P35659 Protein DEK 0,6 P49959 Double-strand break repair protein MRE11A (Meiotic recombination 11 homolog 1) (MRE11 homolog 1) (Meiotic recombination 11 homolog A) (MRE11 homolog A) 0,6 Q6PD62 RNA polymerase-associated protein CTR9 homolog (SH2 domain-binding protein 1) 0,6 P26378 ELAV-like protein 4 (Hu-antigen D) (HuD) (Paraneoplastic encephalomyelitis antigen HuD) 0,6 Q9P2E9 Ribosome-binding protein 1 (180 kDa ribosome receptor homolog) (RRp) (ES/130-related protein) (Ribosome receptor protein) 0,6 Q9BW19 Kinesin-like protein KIFC1 (Kinesin-like protein 2) (Kinesin-related protein HSET) 0,5 P16401 Histone H1.5 (Histone H1a) (Histone H1b) (Histone H1s-3) 0,5 Q15050 Ribosome biogenesis regulatory protein homolog 0,5

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P49750 YLP motif-containing protein 1 (Nuclear protein ZAP3) (ZAP113) 0,5 P18754 Regulator of chromosome condensation (Cell cycle regulatory protein) (Chromosome condensation protein 1) 0,5 Q9BYD3 39S ribosomal protein L4, mitochondrial (L4mt) (MRP-L4) 0,5 Q96G23 Ceramide synthase 2 (CerS2) (LAG1 longevity assurance homolog 2) (SP260) (Tumor metastasis-suppressor gene 1 protein) 0,5 Q99986 Serine/threonine-protein kinase VRK1 (EC 2.7.11.1) (Vaccinia-related kinase 1) 0,5 Q9H7B2 Ribosome production factor 2 homolog (Brix domain-containing protein 1) (Ribosome biogenesis protein RPF2 homolog) 0,5 Q96T88 E3 ubiquitin-protein ligase UHRF1 (EC 6.3.2.-) (Inverted CCAAT box-binding protein of 90 kDa) (Nuclear protein 95) (Nuclear zinc finger protein Np95) (HuNp95) 0,5 (hNp95) (RING finger protein 106) (Transcription factor ICBP90) (Ubiquitin-like PHD and RING finger domain-containing protein 1) (hUHRF1) (Ubiquitin-like-containing PHD and RING finger domains protein 1) Q8IZ81 ELMO domain-containing protein 2 0,5 Q15738 Sterol-4-alpha-carboxylate 3-dehydrogenase, decarboxylating (EC 1.1.1.170) (Protein H105e3) 0,5 Q15181 Inorganic pyrophosphatase (EC 3.6.1.1) (Pyrophosphate phospho-hydrolase) (PPase) 0,5 Q16850 Lanosterol 14-alpha demethylase (LDM) (EC 1.14.13.70) (CYPLI) (Cytochrome P450 51A1) (Cytochrome P450-14DM) (Cytochrome P45014DM) 0,5 (Cytochrome P450LI) (Sterol 14-alpha demethylase) P31350 Ribonucleoside-diphosphate reductase subunit M2 (EC 1.17.4.1) (Ribonucleotide reductase small chain) (Ribonucleotide reductase small subunit) 0,5 Q13492 Phosphatidylinositol-binding clathrin assembly protein (Clathrin assembly lymphoid myeloid leukemia protein) 0,5 Q9BSJ8 Extended synaptotagmin-1 (E-Syt1) (Membrane-bound C2 domain-containing protein) 0,5 Q9Y520 Protein PRRC2C (BAT2 domain-containing protein 1) (HBV X-transactivated gene 2 protein) (HBV XAg-transactivated protein 2) (HLA-B-associated transcript 2-like 2) 0,5 (Proline-rich and coiled-coil-containing protein 2C) Q13823 Nucleolar GTP-binding protein 2 (Autoantigen NGP-1) 0,5 Q9P0M6 Core histone macro-H2A.2 (Histone macroH2A2) (mH2A2) 0,4 Q8NI27 THO complex subunit 2 (Tho2) (hTREX120) 0,4 P42285 Superkiller viralicidic activity 2-like 2 (EC 3.6.4.13) (ATP-dependent RNA helicase SKIV2L2) (TRAMP-like complex helicase) 0,4 O60231 Putative pre-mRNA-splicing factor ATP-dependent RNA helicase DHX16 (EC 3.6.4.13) (ATP-dependent RNA helicase #3) (DEAH-box protein 16) 0,4 P18615 Negative elongation factor E (NELF-E) (RNA-binding protein RD) 0,4 O43847 Nardilysin (EC 3.4.24.61) (N-arginine dibasic convertase) (NRD convertase) (NRD-C) 0,4 Q99543 DnaJ homolog subfamily C member 2 (M-phase phosphoprotein 11) (Zuotin-related factor 1) [Cleaved into: DnaJ homolog subfamily C member 2, N- 0,4 terminally processed] Q9UPQ0 LIM and calponin homology domains-containing protein 1 0,4 P49006 MARCKS-related protein (MARCKS-like protein 1) (Macrophage myristoylated alanine-rich C kinase substrate) (Mac-MARCKS) (MacMARCKS) 0,4 Q15067 Peroxisomal acyl-coenzyme A oxidase 1 (AOX) (EC 1.3.3.6) (Palmitoyl-CoA oxidase) (Straight-chain acyl-CoA oxidase) (SCOX) 0,4 Q7Z2T5 TRMT1-like protein (EC 2.1.1.-) 0,4

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Q9H9J2 39S ribosomal protein L44, mitochondrial (L44mt) (MRP-L44) (EC 3.1.26.-) 0,4 O43852 Calumenin (Crocalbin) (IEF SSP 9302) 0,4 P02794 Ferritin heavy chain (Ferritin H subunit) (EC 1.16.3.1) (Cell proliferation-inducing gene 15 protein) [Cleaved into: Ferritin heavy chain, N-terminally processed] 0,4 P51116 Fragile X mental retardation syndrome-related protein 2 0,4 Q14108 Lysosome membrane protein 2 (85 kDa lysosomal membrane sialoglycoprotein) (LGP85) (CD36 antigen-like 2) (Lysosome membrane protein II) (LIMP II) (Scavenger 0,3 receptor class B member 2) (CD antigen CD36) Q9HDC9 Adipocyte plasma membrane-associated protein (Protein BSCv) 0,3 P08962 CD63 antigen (Granulophysin) (Lysosomal-associated membrane protein 3) (LAMP-3) (Melanoma-associated antigen ME491) (OMA81H) (Ocular melanoma- 0,3 associated antigen) (Tetraspanin-30) (Tspan-30) (CD antigen CD63) O15260 Surfeit locus protein 4 0,3 Q14966 Zinc finger protein 638 (Cutaneous T-cell lymphoma-associated antigen se33-1) (CTCL-associated antigen se33-1) (Nuclear protein 220) (Zinc finger matrin-like 0,3 protein) Q9UIG0 Tyrosine-protein kinase BAZ1B (EC 2.7.10.2) (Bromodomain adjacent to zinc finger domain protein 1B) (Williams syndrome transcription factor) (Williams-Beuren 0,3 syndrome chromosomal region 10 protein) (Williams-Beuren syndrome chromosomal region 9 protein) (hWALp2) O15270 Serine palmitoyltransferase 2 (EC 2.3.1.50) (Long chain base biosynthesis protein 2) (LCB 2) (Long chain base biosynthesis protein 2a) (LCB2a) (Serine-palmitoyl-CoA 0,3 transferase 2) (SPT 2) O75475 PC4 and SFRS1-interacting protein (CLL-associated antigen KW-7) (Dense fine speckles 70 kDa protein) (DFS 70) (Lens epithelium-derived growth factor) 0,2 (Transcriptional coactivator p75/p52) Q9UNN5 FAS-associated factor 1 (hFAF1) (UBX domain-containing protein 12) (UBX domain-containing protein 3A) 2,9 Q92974 Rho guanine nucleotide exchange factor 2 (Guanine nucleotide exchange factor H1) (GEF-H1) (Microtubule-regulated Rho-GEF) (Proliferating cell nucleolar antigen 2,7 p40) P42224 Signal transducer and activator of transcription 1-alpha/beta (Transcription factor ISGF-3 components p91/p84) 2,3 Q01433 AMP deaminase 2 (EC 3.5.4.6) (AMP deaminase isoform L) 2,3 Q92734 Protein TFG (TRK-fused gene protein) 2,2 P49588 Alanine--tRNA ligase, cytoplasmic (EC 6.1.1.7) (Alanyl-tRNA synthetase) (AlaRS) (Renal carcinoma antigen NY-REN-42) 1,9 P09651 Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) (Helix-destabilizing protein) (Single-strand RNA-binding protein) (hnRNP core protein A1) [Cleaved into: 1,8 Heterogeneous nuclear ribonucleoprotein A1, N-terminally processed] O60341 Lysine-specific histone demethylase 1A (EC 1.-.-.-) (BRAF35-HDAC complex protein BHC110) (Flavin-containing amine oxidase domain-containing protein 2) 1,7 P45880 Voltage-dependent anion-selective channel protein 2 (VDAC-2) (hVDAC2) (Outer mitochondrial membrane protein porin 2) 1,7 Q15149 Plectin (PCN) (PLTN) (Hemidesmosomal protein 1) (HD1) (Plectin-1) 1,7 P78527 DNA-dependent protein kinase catalytic subunit (DNA-PK catalytic subunit) (DNA-PKcs) (EC 2.7.11.1) (DNPK1) (p460) 1,6 P08243 Asparagine synthetase [glutamine-hydrolyzing] (EC 6.3.5.4) (Cell cycle control protein TS11) (Glutamine-dependent asparagine synthetase) 1,6 Q9Y2A7 Nck-associated protein 1 (NAP 1) (Membrane-associated protein HEM-2) (p125Nap1) 1,5 Q14204 Cytoplasmic dynein 1 heavy chain 1 (Cytoplasmic dynein heavy chain 1) (Dynein heavy chain, cytosolic) 1,4

156

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P54577 Tyrosine--tRNA ligase, cytoplasmic (EC 6.1.1.1) (Tyrosyl-tRNA synthetase) (TyrRS) [Cleaved into: Tyrosine--tRNA ligase, cytoplasmic, N-terminally processed] 1,4 Q13813 Spectrin alpha chain, non-erythrocytic 1 (Alpha-II spectrin) (Fodrin alpha chain) (Spectrin, non-erythroid alpha subunit) 1,3 P08670 Vimentin 1,3 P21796 Voltage-dependent anion-selective channel protein 1 (VDAC-1) (hVDAC1) (Outer mitochondrial membrane protein porin 1) (Plasmalemmal porin) (Porin 31HL) (Porin 1,2 31HM) P41250 Glycine--tRNA ligase (EC 6.1.1.14) (Diadenosine tetraphosphate synthetase) (AP-4-A synthetase) (Glycyl-tRNA synthetase) (GlyRS) 0,7 P78362 SRSF protein kinase 2 (EC 2.7.11.1) (SFRS protein kinase 2) (Serine/arginine-rich protein-specific kinase 2) (SR-protein-specific kinase 2) [Cleaved 0,7 into: SRSF protein kinase 2 N-terminal; SRSF protein kinase 2 C-terminal] P05556 Integrin beta-1 (Fibronectin receptor subunit beta) (Glycoprotein IIa) (GPIIA) (VLA-4 subunit beta) (CD antigen CD29) 0,6 P98175 RNA-binding protein 10 (G patch domain-containing protein 9) (RNA-binding motif protein 10) (RNA-binding protein S1-1) (S1-1) 0,6 Q99700 Ataxin-2 (Spinocerebellar ataxia type 2 protein) (Trinucleotide repeat-containing gene 13 protein) 0,6 P49756 RNA-binding protein 25 (Arg/Glu/Asp-rich protein of 120 kDa) (RED120) (Protein S164) (RNA-binding motif protein 25) (RNA-binding region-containing protein 7) 0,5 Q05639 Elongation factor 1-alpha 2 (EF-1-alpha-2) (Eukaryotic elongation factor 1 A-2) (eEF1A-2) (Statin-S1) 0,5 Q8IX12 Cell division cycle and apoptosis regulator protein 1 (Cell cycle and apoptosis regulatory protein 1) (CARP-1) (Death inducer with SAP domain) 0,5 P35222 Catenin beta-1 (Beta-catenin) 0,5 P08758 Annexin A5 (Anchorin CII) (Annexin V) (Annexin-5) (Calphobindin I) (CBP-I) (Endonexin II) (Lipocortin V) (Placental anticoagulant protein 4) (PP4) (Placental 0,5 anticoagulant protein I) (PAP-I) (Thromboplastin inhibitor) (Vascular anticoagulant-alpha) (VAC-alpha) P25787 Proteasome subunit alpha type-2 (EC 3.4.25.1) (Macropain subunit C3) (Multicatalytic endopeptidase complex subunit C3) (Proteasome component C3) 0,3 P09936 Ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCH-L1) (EC 3.4.19.12) (EC 6.-.-.-) (Neuron cytoplasmic protein 9.5) (PGP 9.5) (PGP9.5) (Ubiquitin thioesterase L1) 0,3 P35221 Catenin alpha-1 (Alpha E-catenin) (Cadherin-associated protein) (Renal carcinoma antigen NY-REN-13) 0,3 Q06323 Proteasome activator complex subunit 1 (11S regulator complex subunit alpha) (REG-alpha) (Activator of multicatalytic protease subunit 1) (Interferon gamma up- 0,3 regulated I-5111 protein) (IGUP I-5111) (Proteasome activator 28 subunit alpha) (PA28a) (PA28alpha)

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Attachment 7 Cell stress induced 132 protein regulated for WT HtrA2/Omi overexpressing cells. Significant regulation ratio of each protein quantified between two conditions (before and after stress) is listed in the table.

Accession Protein WT 0h/6h

Q8NBL1 Protein O-glucosyltransferase 1 (EC 2.4.1.-) (CAP10-like 46 kDa protein) (hCLP46) (KTEL motif-containing protein 1) (Myelodysplastic syndromes relative protein) (O- ###### glucosyltransferase Rumi homolog) (hRumi) (Protein O-xylosyltransferase) (EC 2.4.2.26) #### Q8IVD9 NudC domain-containing protein 3 5,0 Q13442 28 kDa heat- and acid-stable phosphoprotein (PDGF-associated protein) (PAP) (PDGFA-associated protein 1) (PAP1) 4,4 O96019 Actin-like protein 6A (53 kDa BRG1-associated factor A) (Actin-related protein Baf53a) (ArpNbeta) (BRG1-associated factor 53A) (BAF53A) (INO80 complex subunit K) 4,0 Q12789 General transcription factor 3C polypeptide 1 (TF3C-alpha) (TFIIIC box B-binding subunit) (Transcription factor IIIC 220 kDa subunit) (TFIIIC 220 kDa subunit) (TFIIIC220) 3,9 (Transcription factor IIIC subunit alpha) Q8IUD2 ELKS/Rab6-interacting/CAST family member 1 (ERC-1) (Rab6-interacting protein 2) 3,8 O60282 Kinesin heavy chain isoform 5C (Kinesin heavy chain neuron-specific 2) 3,6 Q92973 Transportin-1 (Importin beta-2) (Karyopherin beta-2) (M9 region interaction protein) (MIP) 3,1 P36915 Guanine nucleotide-binding protein-like 1 (GTP-binding protein HSR1) 3,0 Q9NY33 Dipeptidyl peptidase 3 (EC 3.4.14.4) (Dipeptidyl aminopeptidase III) (Dipeptidyl arylamidase III) (Dipeptidyl peptidase III) (DPP III) (Enkephalinase B) 2,8 P52888 Thimet oligopeptidase (EC 3.4.24.15) (Endopeptidase 24.15) (MP78) 2,7 P36405 ADP-ribosylation factor-like protein 3 2,6 Q5VTR2 E3 ubiquitin-protein ligase BRE1A (BRE1-A) (hBRE1) (EC 6.3.2.-) (RING finger protein 20) 2,5 P49916 DNA ligase 3 (EC 6.5.1.1) (DNA ligase III) (Polydeoxyribonucleotide synthase [ATP] 3) 2,4 Q96HC4 PDZ and LIM domain protein 5 (Enigma homolog) (Enigma-like PDZ and LIM domains protein) 2,3 Q06203 Amidophosphoribosyltransferase (ATase) (EC 2.4.2.14) (Glutamine phosphoribosylpyrophosphate amidotransferase) (GPAT) 2,2 Q14694 Ubiquitin carboxyl-terminal hydrolase 10 (EC 3.4.19.12) (Deubiquitinating enzyme 10) (Ubiquitin thioesterase 10) (Ubiquitin-specific-processing protease 10) 2,2 P20290 Transcription factor BTF3 (Nascent polypeptide-associated complex subunit beta) (NAC-beta) (RNA polymerase B transcription factor 3) 2,2 Q04323 UBX domain-containing protein 1 (SAPK substrate protein 1) (UBA/UBX 33.3 kDa protein) 2,0 O43633 Charged multivesicular body protein 2a (Chromatin-modifying protein 2a) (CHMP2a) (Putative breast adenocarcinoma marker BC-2) (Vacuolar protein sorting-associated 2,0 protein 2-1) (Vps2-1) (hVps2-1) Q3KQU3 MAP7 domain-containing protein 1 (Arginine/proline-rich coiled-coil domain-containing protein 1) (Proline/arginine-rich coiled-coil domain-containing protein 1) 2,0 Q92747 Actin-related protein 2/3 complex subunit 1A (SOP2-like protein) 2,0 Q7L576 Cytoplasmic FMR1-interacting protein 1 (Specifically Rac1-associated protein 1) (Sra-1) (p140sra-1) 2,0

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P08133 Annexin A6 (67 kDa calelectrin) (Annexin VI) (Annexin-6) (Calphobindin-II) (CPB-II) (Chromobindin-20) (Lipocortin VI) (Protein III) (p68) (p70) 2,0 Q6P2E9 Enhancer of mRNA-decapping protein 4 (Autoantigen Ge-1) (Autoantigen RCD-8) (Human enhancer of decapping large subunit) (Hedls) 2,0 Q12792 Twinfilin-1 (Protein A6) (Protein tyrosine kinase 9) 1,9 Q9BYD3 39S ribosomal protein L4, mitochondrial (L4mt) (MRP-L4) 1,8 O60524 Nuclear export mediator factor NEMF (Antigen NY-CO-1) (Serologically defined colon cancer antigen 1) 1,8 Q00341 Vigilin (High density lipoprotein-binding protein) (HDL-binding protein) 1,7 Q9NUL3 Double-stranded RNA-binding protein Staufen homolog 2 1,7 P02794 Ferritin heavy chain (Ferritin H subunit) (EC 1.16.3.1) (Cell proliferation-inducing gene 15 protein) [Cleaved into: Ferritin heavy chain, N-terminally processed] 1,7 O60271 C-Jun-amino-terminal kinase-interacting protein 4 (JIP-4) (JNK-interacting protein 4) (Cancer/testis antigen 89) (CT89) (Human lung cancer oncogene 6 protein) (HLC-6) 1,6 (JNK-associated leucine-zipper protein) (JLP) (Mitogen-activated protein kinase 8-interacting protein 4) (Proliferation-inducing protein 6) (Protein highly expressed in testis) (PHET) (Sperm surface protein) (Sperm-associated antigen 9) (Sperm-specific protein) (Sunday driver 1) Q9NQ29 Putative RNA-binding protein Luc7-like 1 (Putative SR protein LUC7B1) (SR+89) 1,6 Q32MZ4 Leucine-rich repeat flightless-interacting protein 1 (LRR FLII-interacting protein 1) (GC-binding factor 2) (TAR RNA-interacting protein) 1,6 Q96SB3 Neurabin-2 (Neurabin-II) (Protein phosphatase 1 regulatory subunit 9B) (Spinophilin) 1,5 O60763 General vesicular transport factor p115 (Protein USO1 homolog) (Transcytosis-associated protein) (TAP) (Vesicle-docking protein) 1,5 Q9UMS6 Synaptopodin-2 (Genethonin-2) (Myopodin) 1,5 Q92896 Golgi apparatus protein 1 (CFR-1) (Cysteine-rich fibroblast growth factor receptor) (E-selectin ligand 1) (ESL-1) (Golgi sialoglycoprotein MG-160) 1,5 Q96CN7 Isochorismatase domain-containing protein 1 1,4 Q96CX2 BTB/POZ domain-containing protein KCTD12 (Pfetin) (Predominantly fetal expressed T1 domain) 1,4 Q96EP5 DAZ-associated protein 1 (Deleted in azoospermia-associated protein 1) 1,4 O14974 Protein phosphatase 1 regulatory subunit 12A (Myosin phosphatase-targeting subunit 1) (Myosin phosphatase target subunit 1) (Protein phosphatase myosin-binding 1,3 subunit) O14531 Dihydropyrimidinase-related protein 4 (DRP-4) (Collapsin response mediator protein 3) (CRMP-3) (UNC33-like phosphoprotein 4) (ULIP-4) 1,3 P07355 Annexin A2 (Annexin II) (Annexin-2) (Calpactin I heavy chain) (Calpactin-1 heavy chain) (Chromobindin-8) (Lipocortin II) (Placental anticoagulant protein IV) (PAP-IV) 1,3 (Protein I) (p36) Q9Y490 Talin-1 1,3 P18206 Vinculin (Metavinculin) (MV) 1,2 P37108 Signal recognition particle 14 kDa protein (SRP14) (18 kDa Alu RNA-binding protein) 1,2 Q09666 Neuroblast differentiation-associated protein AHNAK (Desmoyokin) 1,2 P24539 ATP synthase F(0) complex subunit B1, mitochondrial (ATP synthase proton-transporting mitochondrial F(0) complex subunit B1) (ATP synthase subunit b) (ATPase subunit 0,8 b)

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Q02978 Mitochondrial 2-oxoglutarate/malate carrier protein (OGCP) (Solute carrier family 25 member 11) 0,8 O60264 SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5 (SWI/SNF-related matrix-associated actin-dependent regulator of 0,8 chromatin A5) (EC 3.6.4.-) (Sucrose nonfermenting protein 2 homolog) (hSNF2H) P67812 Signal peptidase complex catalytic subunit SEC11A (EC 3.4.21.89) (Endopeptidase SP18) (Microsomal signal peptidase 18 kDa subunit) (SPase 18 kDa subunit) (SEC11 0,8 homolog A) (SEC11-like protein 1) (SPC18) O14776 Transcription elongation regulator 1 (TATA box-binding protein-associated factor 2S) (Transcription factor CA150) 0,7 Q02952 A-kinase anchor protein 12 (AKAP-12) (A-kinase anchor protein 250 kDa) (AKAP 250) (Gravin) (Myasthenia gravis autoantigen) 0,7 Q9BTV4 Transmembrane protein 43 (Protein LUMA) 0,7 P05023 Sodium/potassium-transporting ATPase subunit alpha-1 (Na(+)/K(+) ATPase alpha-1 subunit) (EC 3.6.3.9) (Sodium pump subunit alpha-1) 0,7 Q8ND56 Protein LSM14 homolog A (Protein FAM61A) (Protein SCD6 homolog) (Putative alpha-synuclein-binding protein) (AlphaSNBP) (RNA-associated protein 55A) (hRAP55) 0,7 (hRAP55A) Q8TCJ2 Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit STT3B (Oligosaccharyl transferase subunit STT3B) (STT3-B) (EC 2.4.99.18) (Source of 0,7 immunodominant MHC-associated peptides homolog) P33991 DNA replication licensing factor MCM4 (EC 3.6.4.12) (CDC21 homolog) (P1-CDC21) 0,6 Q08170 Serine/arginine-rich splicing factor 4 (Pre-mRNA-splicing factor SRP75) (SRP001LB) (Splicing factor, arginine/serine-rich 4) 0,6 O43847 Nardilysin (EC 3.4.24.61) (N-arginine dibasic convertase) (NRD convertase) (NRD-C) 0,6 P22307 Non-specific lipid-transfer protein (NSL-TP) (EC 2.3.1.176) (Propanoyl-CoA C-acyltransferase) (SCP-chi) (SCPX) (Sterol carrier protein 2) (SCP-2) (Sterol carrier protein X) 0,6 (SCP-X) Q969V3 Nicalin (Nicastrin-like protein) 0,6 O60231 Putative pre-mRNA-splicing factor ATP-dependent RNA helicase DHX16 (EC 3.6.4.13) (ATP-dependent RNA helicase #3) (DEAH-box protein 16) 0,6 P42167 Lamina-associated polypeptide 2, isoforms beta/gamma (Thymopoietin, isoforms beta/gamma) (TP beta/gamma) (Thymopoietin-related peptide isoforms beta/gamma) 0,6 (TPRP isoforms beta/gamma) [Cleaved into: Thymopoietin (TP) (Splenin); Thymopentin (TP5)] Q8IZL8 Proline-, glutamic acid- and leucine-rich protein 1 (Modulator of non-genomic activity of estrogen receptor) (Transcription factor HMX3) 0,6 Q14739 Lamin-B receptor (Integral nuclear envelope inner membrane protein) (LMN2R) 0,6 Q8TAQ2 SWI/SNF complex subunit SMARCC2 (BRG1-associated factor 170) (BAF170) (SWI/SNF complex 170 kDa subunit) (SWI/SNF-related matrix-associated actin-dependent 0,6 regulator of chromatin subfamily C member 2) Q16891 Mitochondrial inner membrane protein (Cell proliferation-inducing gene 4/52 protein) (Mitofilin) (p87/89) 0,6 Q92522 Histone H1x 0,6 O00116 Alkyldihydroxyacetonephosphate synthase, peroxisomal (Alkyl-DHAP synthase) (EC 2.5.1.26) (Aging-associated gene 5 protein) (Alkylglycerone-phosphate synthase) 0,6 Q99848 Probable rRNA-processing protein EBP2 (EBNA1-binding protein 2) (Nucleolar protein p40) 0,6 Q9H307 Pinin (140 kDa nuclear and cell adhesion-related phosphoprotein) (Desmosome-associated protein) (Domain-rich serine protein) (DRS protein) (DRSP) (Melanoma 0,6 metastasis clone A protein) (Nuclear protein SDK3) (SR-like protein) Q8TCS8 Polyribonucleotide nucleotidyltransferase 1, mitochondrial (EC 2.7.7.8) (3'-5' RNA exonuclease OLD35) (PNPase old-35) (Polynucleotide phosphorylase 1) (PNPase 1) 0,6 (Polynucleotide phosphorylase-like protein)

160

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Q8N5K1 CDGSH iron-sulfur domain-containing protein 2 (Endoplasmic reticulum intermembrane small protein) (MitoNEET-related 1 protein) (Miner1) (Nutrient-deprivation 0,6 autophagy factor-1) (NAF-1) Q99426 Tubulin-folding cofactor B (Cytoskeleton-associated protein 1) (Cytoskeleton-associated protein CKAPI) (Tubulin-specific chaperone B) 0,6 P35613 Basigin (5F7) (Collagenase stimulatory factor) (Extracellular matrix metalloproteinase inducer) (EMMPRIN) (Leukocyte activation antigen M6) (OK blood group antigen) 0,6 (Tumor cell-derived collagenase stimulatory factor) (TCSF) (CD antigen CD147) P21912 Succinate dehydrogenase [ubiquinone] iron-sulfur subunit, mitochondrial (EC 1.3.5.1) (Iron-sulfur subunit of complex II) (Ip) 0,6 Q92621 Nuclear pore complex protein Nup205 (205 kDa nucleoporin) (Nucleoporin Nup205) 0,6 Q9HDC9 Adipocyte plasma membrane-associated protein (Protein BSCv) 0,5 Q9BW19 Kinesin-like protein KIFC1 (Kinesin-like protein 2) (Kinesin-related protein HSET) 0,5 P38606 V-type proton ATPase catalytic subunit A (V-ATPase subunit A) (EC 3.6.3.14) (V-ATPase 69 kDa subunit) (Vacuolar ATPase isoform VA68) (Vacuolar proton pump subunit 0,5 alpha) Q96HS1 Serine/threonine-protein phosphatase PGAM5, mitochondrial (EC 3.1.3.16) (Bcl-XL-binding protein v68) (Phosphoglycerate mutase family member 5) 0,5 Q00688 Peptidyl-prolyl cis-trans isomerase FKBP3 (PPIase FKBP3) (EC 5.2.1.8) (25 kDa FK506-binding protein) (25 kDa FKBP) (FKBP-25) (FK506-binding protein 3) (FKBP-3) 0,5 (Immunophilin FKBP25) (Rapamycin-selective 25 kDa immunophilin) (Rotamase) Q9BYD1 39S ribosomal protein L13, mitochondrial (L13mt) (MRP-L13) 0,5 P22695 Cytochrome b-c1 complex subunit 2, mitochondrial (Complex III subunit 2) (Core protein II) (Ubiquinol-cytochrome-c reductase complex core protein 2) 0,5 O43148 mRNA cap guanine-N7 methyltransferase (EC 2.1.1.56) (RG7MT1) (mRNA (guanine-N(7)-)-methyltransferase) (mRNA cap methyltransferase) (hCMT1) (hMet) (hcm1p) 0,5 Q07955 Serine/arginine-rich splicing factor 1 (Alternative-splicing factor 1) (ASF-1) (Splicing factor, arginine/serine-rich 1) (pre-mRNA-splicing factor SF2, P33 subunit) 0,5 Q9Y5S9 RNA-binding protein 8A (Binder of OVCA1-1) (BOV-1) (RNA-binding motif protein 8A) (RNA-binding protein Y14) (Ribonucleoprotein RBM8A) 0,5 P42696 RNA-binding protein 34 (RNA-binding motif protein 34) 0,5 Q96CW1 AP-2 complex subunit mu (AP-2 mu chain) (Adapter-related protein complex 2 subunit mu) (Adaptin-mu2) (Adaptor protein complex AP-2 subunit mu) (Clathrin assembly 0,5 protein complex 2 mu medium chain) (Clathrin coat assembly protein AP50) (Clathrin coat-associated protein AP50) (HA2 50 kDa subunit) (Plasma membrane adaptor AP-2 50 kDa protein) Q07866 Kinesin light chain 1 (KLC 1) 0,5 Q16352 Alpha-internexin (Alpha-Inx) (66 kDa neurofilament protein) (NF-66) (Neurofilament-66) (Neurofilament 5) 0,5 Q05193 Dynamin-1 (EC 3.6.5.5) 0,5 Q13243 Serine/arginine-rich splicing factor 5 (Delayed-early protein HRS) (Pre-mRNA-splicing factor SRP40) (Splicing factor, arginine/serine-rich 5) 0,4 Q13505 Metaxin-1 (Mitochondrial outer membrane import complex protein 1) 0,4 Q9Y3B7 39S ribosomal protein L11, mitochondrial (L11mt) (MRP-L11) 0,4 Q13423 NAD(P) transhydrogenase, mitochondrial (EC 1.6.1.2) (Nicotinamide nucleotide transhydrogenase) (Pyridine nucleotide transhydrogenase) 0,4 P49792 E3 SUMO-protein ligase RanBP2 (EC 6.3.2.-) (358 kDa nucleoporin) (Nuclear pore complex protein Nup358) (Nucleoporin Nup358) (Ran-binding protein 2) (RanBP2) (p270) 0,4 Q9UKA9 Polypyrimidine tract-binding protein 2 (Neural polypyrimidine tract-binding protein) (Neurally-enriched homolog of PTB) (PTB-like protein) 0,4

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P31040 Succinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial (EC 1.3.5.1) (Flavoprotein subunit of complex II) (Fp) 0,3 Q9UFN0 Protein NipSnap homolog 3A (NipSnap3A) (Protein NipSnap homolog 4) (NipSnap4) (Target for Salmonella secreted protein C) (TassC) 0,3 Q9H9Y6 DNA-directed RNA polymerase I subunit RPA2 (RNA polymerase I subunit 2) (EC 2.7.7.6) (DNA-directed RNA polymerase I 135 kDa polypeptide) (RPA135) 0,3 O14497 AT-rich interactive domain-containing protein 1A (ARID domain-containing protein 1A) (B120) (BRG1-associated factor 250) (BAF250) (BRG1-associated factor 250a) 0,3 (BAF250A) (Osa homolog 1) (hOSA1) (SWI-like protein) (SWI/SNF complex protein p270) (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin subfamily F member 1) (hELD) Q7L0Y3 Mitochondrial ribonuclease P protein 1 (Mitochondrial RNase P protein 1) (EC 2.1.1.-) (HBV pre-S2 trans-regulated protein 2) (RNA (guanine-9-)-methyltransferase domain- 0,3 containing protein 1) (Renal carcinoma antigen NY-REN-49) (tRNA methyltransferase 10 homolog C) Q9BV38 WD repeat-containing protein 18 0,3 P30042 ES1 protein homolog, mitochondrial (Protein GT335) (Protein KNP-I) 0,3 P49750 YLP motif-containing protein 1 (Nuclear protein ZAP3) (ZAP113) 0,2 Q9H078 Caseinolytic peptidase B protein homolog (Suppressor of potassium transport defect 3) 0,2 O15240 Neurosecretory protein VGF [Cleaved into: Neuroendocrine regulatory peptide-1 (NERP-1); Neuroendocrine regulatory peptide-2 (NERP-2); Antimicrobial peptide VGF[554- 0,0 577]] Q9UBB4 Ataxin-10 (Brain protein E46 homolog) (Spinocerebellar ataxia type 10 protein) 2,9 Q01433 AMP deaminase 2 (EC 3.5.4.6) (AMP deaminase isoform L) 1,8 P08758 Annexin A5 (Anchorin CII) (Annexin V) (Annexin-5) (Calphobindin I) (CBP-I) (Endonexin II) (Lipocortin V) (Placental anticoagulant protein 4) (PP4) (Placental anticoagulant 1,7 protein I) (PAP-I) (Thromboplastin inhibitor) (Vascular anticoagulant-alpha) (VAC-alpha) P49588 Alanine--tRNA ligase, cytoplasmic (EC 6.1.1.7) (Alanyl-tRNA synthetase) (AlaRS) (Renal carcinoma antigen NY-REN-42) 1,6 P09972 Fructose-bisphosphate aldolase C (EC 4.1.2.13) (Brain-type aldolase) 1,5 P62258 14-3-3 protein epsilon (14-3-3E) 1,5 P04792 Heat shock protein beta-1 (HspB1) (28 kDa heat shock protein) (Estrogen-regulated 24 kDa protein) (Heat shock 27 kDa protein) (HSP 27) (Stress-responsive protein 27) 1,4 (SRP27) P08243 Asparagine synthetase [glutamine-hydrolyzing] (EC 6.3.5.4) (Cell cycle control protein TS11) (Glutamine-dependent asparagine synthetase) 1,3 Q13813 Spectrin alpha chain, non-erythrocytic 1 (Alpha-II spectrin) (Fodrin alpha chain) (Spectrin, non-erythroid alpha subunit) 1,2 P08670 Vimentin 1,2 P45880 Voltage-dependent anion-selective channel protein 2 (VDAC-2) (hVDAC2) (Outer mitochondrial membrane protein porin 2) 0,8 Q15149 Plectin (PCN) (PLTN) (Hemidesmosomal protein 1) (HD1) (Plectin-1) 0,7 O95292 Vesicle-associated membrane protein-associated protein B/C (VAMP-B/VAMP-C) (VAMP-associated protein B/C) (VAP-B/VAP-C) 0,7 Q96ST3 Paired amphipathic helix protein Sin3a (Histone deacetylase complex subunit Sin3a) (Transcriptional corepressor Sin3a) 0,7 O00429 Dynamin-1-like protein (EC 3.6.5.5) (Dnm1p/Vps1p-like protein) (DVLP) (Dynamin family member proline-rich carboxyl-terminal domain less) (Dymple) (Dynamin-like 0,6 protein) (Dynamin-like protein 4) (Dynamin-like protein IV) (HdynIV) (Dynamin-related protein 1) P54577 Tyrosine--tRNA ligase, cytoplasmic (EC 6.1.1.1) (Tyrosyl-tRNA synthetase) (TyrRS) [Cleaved into: Tyrosine--tRNA ligase, cytoplasmic, N-terminally processed] 0,6

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O95831 Apoptosis-inducing factor 1, mitochondrial (EC 1.1.1.-) (Programmed cell death protein 8) 0,5 P78527 DNA-dependent protein kinase catalytic subunit (DNA-PK catalytic subunit) (DNA-PKcs) (EC 2.7.11.1) (DNPK1) (p460) 0,5 Q86YP4 Transcriptional repressor p66-alpha (Hp66alpha) (GATA zinc finger domain-containing protein 2A) 0,5 Q8TB36 Ganglioside-induced differentiation-associated protein 1 (GDAP1) 0,5 P09172 Dopamine beta-hydroxylase (EC 1.14.17.1) (Dopamine beta-monooxygenase) [Cleaved into: Soluble dopamine beta-hydroxylase] 0,4 Q8IX12 Cell division cycle and apoptosis regulator protein 1 (Cell cycle and apoptosis regulatory protein 1) (CARP-1) (Death inducer with SAP domain) 0,3

Attachment 8 Cell stress induced 249 protein regulated for G399S mutant HtrA2/Omi overexpressing cells. Significant regulation ratio of each protein quantified between two conditions (before and after stress) is listed in the table.

Accession Protein G399S 0h/6h

Q9P0M6 Core histone macro-H2A.2 (Histone macroH2A2) (mH2A2) 0,0 Q13505 Metaxin-1 (Mitochondrial outer membrane import complex protein 1) 0,0 Q9BVJ6 U3 small nucleolar RNA-associated protein 14 homolog A (Antigen NY-CO-16) (Serologically defined colon cancer antigen 16) 0,0 Q96HS1 Serine/threonine-protein phosphatase PGAM5, mitochondrial (EC 3.1.3.16) (Bcl-XL-binding protein v68) (Phosphoglycerate mutase family member 5) 0,0 Q01995 Transgelin (22 kDa actin-binding protein) (Protein WS3-10) (Smooth muscle protein 22-alpha) (SM22-alpha) 0,0 Q16891 Mitochondrial inner membrane protein (Cell proliferation-inducing gene 4/52 protein) (Mitofilin) (p87/89) 0,0 Q02978 Mitochondrial 2-oxoglutarate/malate carrier protein (OGCP) (Solute carrier family 25 member 11) 0,0 Q13423 NAD(P) transhydrogenase, mitochondrial (EC 1.6.1.2) (Nicotinamide nucleotide transhydrogenase) (Pyridine nucleotide transhydrogenase) 0,0 Q03252 Lamin-B2 0,0 O96008 Mitochondrial import receptor subunit TOM40 homolog (Protein Haymaker) (Translocase of outer membrane 40 kDa subunit homolog) (p38.5) 0,0 Q9H9B4 Sideroflexin-1 (Tricarboxylate carrier protein) (TCC) 0,1 P53007 Tricarboxylate transport protein, mitochondrial (Citrate transport protein) (CTP) (Solute carrier family 25 member 1) (Tricarboxylate carrier protein) 0,1 O75367 Core histone macro-H2A.1 (Histone macroH2A1) (mH2A1) (Histone H2A.y) (H2A/y) (Medulloblastoma antigen MU-MB-50.205) 0,1 P49006 MARCKS-related protein (MARCKS-like protein 1) (Macrophage myristoylated alanine-rich C kinase substrate) (Mac-MARCKS) (MacMARCKS) 0,1 Q8N766 ER membrane protein complex subunit 1 0,1

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O75955 Flotillin-1 0,1 P22695 Cytochrome b-c1 complex subunit 2, mitochondrial (Complex III subunit 2) (Core protein II) (Ubiquinol-cytochrome-c reductase complex core protein 2) 0,1 P56182 Ribosomal RNA processing protein 1 homolog A (Novel nuclear protein 1) (NNP-1) (Nucleolar protein Nop52) (RRP1-like protein) 0,1 P36542 ATP synthase subunit gamma, mitochondrial (F-ATPase gamma subunit) 0,1 Q9UJZ1 Stomatin-like protein 2, mitochondrial (SLP-2) (EPB72-like protein 2) (Paraprotein target 7) (Paratarg-7) 0,2 P62805 Histone H4 0,2 Q969V3 Nicalin (Nicastrin-like protein) 0,2 P24539 ATP synthase F(0) complex subunit B1, mitochondrial (ATP synthase proton-transporting mitochondrial F(0) complex subunit B1) (ATP synthase subunit b) (ATPase subunit 0,2 b) Q9Y3Y2 Chromatin target of PRMT1 protein (Friend of PRMT1 protein) (Small arginine- and glycine-rich protein) (SRAG) 0,2 Q8N2K0 Monoacylglycerol lipase ABHD12 (EC 3.1.1.23) (2-arachidonoylglycerol hydrolase) (Abhydrolase domain-containing protein 12) 0,2 P08240 Signal recognition particle receptor subunit alpha (SR-alpha) (Docking protein alpha) (DP-alpha) 0,2 Q9UJS0 Calcium-binding mitochondrial carrier protein Aralar2 (Citrin) (Mitochondrial aspartate glutamate carrier 2) (Solute carrier family 25 member 13) 0,2 Q9Y277 Voltage-dependent anion-selective channel protein 3 (VDAC-3) (hVDAC3) (Outer mitochondrial membrane protein porin 3) 0,2 O15173 Membrane-associated progesterone receptor component 2 (Progesterone membrane-binding protein) (Steroid receptor protein DG6) 0,2 P35613 Basigin (5F7) (Collagenase stimulatory factor) (Extracellular matrix metalloproteinase inducer) (EMMPRIN) (Leukocyte activation antigen M6) (OK blood group antigen) 0,2 (Tumor cell-derived collagenase stimulatory factor) (TCSF) (CD antigen CD147) O95864 Fatty acid desaturase 2 (EC 1.14.19.-) (Delta(6) fatty acid desaturase) (D6D) (Delta(6) desaturase) (Delta-6 desaturase) 0,2 O75475 PC4 and SFRS1-interacting protein (CLL-associated antigen KW-7) (Dense fine speckles 70 kDa protein) (DFS 70) (Lens epithelium-derived growth factor) (Transcriptional 0,2 coactivator p75/p52) Q14137 Ribosome biogenesis protein BOP1 (Block of proliferation 1 protein) 0,2 P00403 Cytochrome c oxidase subunit 2 (Cytochrome c oxidase polypeptide II) 0,2 Q9P0I2 ER membrane protein complex subunit 3 (Transmembrane protein 111) 0,3 Q9BYG3 MKI67 FHA domain-interacting nucleolar phosphoprotein (Nucleolar phosphoprotein Nopp34) (Nucleolar protein interacting with the FHA domain of pKI-67) (hNIFK) 0,3 O60264 SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5 (SWI/SNF-related matrix-associated actin-dependent regulator of 0,3 chromatin A5) (EC 3.6.4.-) (Sucrose nonfermenting protein 2 homolog) (hSNF2H) Q8N5K1 CDGSH iron-sulfur domain-containing protein 2 (Endoplasmic reticulum intermembrane small protein) (MitoNEET-related 1 protein) (Miner1) (Nutrient-deprivation 0,3 autophagy factor-1) (NAF-1) P16401 Histone H1.5 (Histone H1a) (Histone H1b) (Histone H1s-3) 0,3 Q9H9J2 39S ribosomal protein L44, mitochondrial (L44mt) (MRP-L44) (EC 3.1.26.-) 0,3 P18754 Regulator of chromosome condensation (Cell cycle regulatory protein) (Chromosome condensation protein 1) 0,3 O95202 LETM1 and EF-hand domain-containing protein 1, mitochondrial (Leucine zipper-EF-hand-containing transmembrane protein 1) 0,3

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P51148 Ras-related protein Rab-5C (L1880) (RAB5L) 0,3 P35659 Protein DEK 0,3 Q9UIG0 Tyrosine-protein kinase BAZ1B (EC 2.7.10.2) (Bromodomain adjacent to zinc finger domain protein 1B) (Williams syndrome transcription factor) (Williams-Beuren 0,3 syndrome chromosomal region 10 protein) (Williams-Beuren syndrome chromosomal region 9 protein) (hWALp2) P06576 ATP synthase subunit beta, mitochondrial (EC 3.6.3.14) 0,3 Q99848 Probable rRNA-processing protein EBP2 (EBNA1-binding protein 2) (Nucleolar protein p40) 0,3 Q9NY93 Probable ATP-dependent RNA helicase DDX56 (EC 3.6.4.13) (ATP-dependent 61 kDa nucleolar RNA helicase) (DEAD box protein 21) (DEAD box protein 56) 0,3 Q00325 Phosphate carrier protein, mitochondrial (Phosphate transport protein) (PTP) (Solute carrier family 25 member 3) 0,3 P24534 Elongation factor 1-beta (EF-1-beta) 0,3 P28288 ATP-binding cassette sub-family D member 3 (70 kDa peroxisomal membrane protein) (PMP70) 0,3 P42696 RNA-binding protein 34 (RNA-binding motif protein 34) 0,3 Q9NTJ5 Phosphatidylinositide phosphatase SAC1 (EC 3.1.3.-) (Suppressor of actin mutations 1-like protein) 0,3 Q9UBU9 Nuclear RNA export factor 1 (Tip-associated protein) (Tip-associating protein) (mRNA export factor TAP) 0,3 Q6UWP7 Lysocardiolipin acyltransferase 1 (EC 2.3.1.-) (EC 2.3.1.51) (1-acylglycerol-3-phosphate O-acyltransferase 8) (1-AGP acyltransferase 8) (1-AGPAT 8) (Acyl-CoA:lysocardiolipin 0,3 acyltransferase 1) Q15050 Ribosome biogenesis regulatory protein homolog 0,3 P42167 Lamina-associated polypeptide 2, isoforms beta/gamma (Thymopoietin, isoforms beta/gamma) (TP beta/gamma) (Thymopoietin-related peptide isoforms beta/gamma) 0,3 (TPRP isoforms beta/gamma) [Cleaved into: Thymopoietin (TP) (Splenin); Thymopentin (TP5)] Q14108 Lysosome membrane protein 2 (85 kDa lysosomal membrane sialoglycoprotein) (LGP85) (CD36 antigen-like 2) (Lysosome membrane protein II) (LIMP II) (Scavenger 0,3 receptor class B member 2) (CD antigen CD36) Q96GQ7 Probable ATP-dependent RNA helicase DDX27 (EC 3.6.4.13) (DEAD box protein 27) 0,3 Q14562 ATP-dependent RNA helicase DHX8 (EC 3.6.4.13) (DEAH box protein 8) (RNA helicase HRH1) 0,3 Q99623 Prohibitin-2 (B-cell receptor-associated protein BAP37) (D-prohibitin) (Repressor of estrogen receptor activity) 0,4 Q9BQ39 ATP-dependent RNA helicase DDX50 (EC 3.6.4.13) (DEAD box protein 50) (Gu-beta) (Nucleolar protein Gu2) 0,4 Q8NBL1 Protein O-glucosyltransferase 1 (EC 2.4.1.-) (CAP10-like 46 kDa protein) (hCLP46) (KTEL motif-containing protein 1) (Myelodysplastic syndromes relative protein) (O- 0,4 glucosyltransferase Rumi homolog) (hRumi) (Protein O-xylosyltransferase) (EC 2.4.2.26) Q14165 Malectin 0,4 P05023 Sodium/potassium-transporting ATPase subunit alpha-1 (Na(+)/K(+) ATPase alpha-1 subunit) (EC 3.6.3.9) (Sodium pump subunit alpha-1) 0,4 O00116 Alkyldihydroxyacetonephosphate synthase, peroxisomal (Alkyl-DHAP synthase) (EC 2.5.1.26) (Aging-associated gene 5 protein) (Alkylglycerone-phosphate synthase) 0,4 O43615 Mitochondrial import inner membrane translocase subunit TIM44 0,4 Q8TCT9 Minor histocompatibility antigen H13 (EC 3.4.23.-) (Intramembrane protease 1) (IMP-1) (IMPAS-1) (hIMP1) (Presenilin-like protein 3) (Signal peptide peptidase) 0,4 Q9Y3B7 39S ribosomal protein L11, mitochondrial (L11mt) (MRP-L11) 0,4

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P82979 SAP domain-containing ribonucleoprotein (Cytokine-induced protein of 29 kDa) (Nuclear protein Hcc-1) (Proliferation-associated cytokine-inducible protein CIP29) 0,4 P50402 Emerin 0,4 Q14692 Ribosome biogenesis protein BMS1 homolog (Ribosome assembly protein BMS1 homolog) 0,4 P35908 Keratin, type II cytoskeletal 2 epidermal (Cytokeratin-2e) (CK-2e) (Epithelial keratin-2e) (Keratin-2 epidermis) (Keratin-2e) (K2e) (Type-II keratin Kb2) 0,4 Q92544 Transmembrane 9 superfamily member 4 0,4 Q07065 Cytoskeleton-associated protein 4 (63-kDa cytoskeleton-linking membrane protein) (Climp-63) (p63) 0,4 P51571 Translocon-associated protein subunit delta (TRAP-delta) (Signal sequence receptor subunit delta) (SSR-delta) 0,4 P30084 Enoyl-CoA hydratase, mitochondrial (EC 4.2.1.17) (Enoyl-CoA hydratase 1) (Short-chain enoyl-CoA hydratase) (SCEH) 0,4 Q96A33 Coiled-coil domain-containing protein 47 0,4 Q9NR45 Sialic acid synthase (N-acetylneuraminate synthase) (EC 2.5.1.56) (N-acetylneuraminate-9-phosphate synthase) (EC 2.5.1.57) (N-acetylneuraminic acid phosphate synthase) 0,4 (N-acetylneuraminic acid synthase) Q9BTV4 Transmembrane protein 43 (Protein LUMA) 0,4 P67812 Signal peptidase complex catalytic subunit SEC11A (EC 3.4.21.89) (Endopeptidase SP18) (Microsomal signal peptidase 18 kDa subunit) (SPase 18 kDa subunit) (SEC11 0,4 homolog A) (SEC11-like protein 1) (SPC18) Q96GM5 SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily D member 1 (60 kDa BRG-1/Brm-associated factor subunit A) (BRG1-associated 0,4 factor 60A) (BAF60A) (SWI/SNF complex 60 kDa subunit) Q10471 Polypeptide N-acetylgalactosaminyltransferase 2 (EC 2.4.1.41) (Polypeptide GalNAc transferase 2) (GalNAc-T2) (pp-GaNTase 2) (Protein-UDP 0,4 acetylgalactosaminyltransferase 2) (UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 2) [Cleaved into: Polypeptide N-acetylgalactosaminyltransferase 2 soluble form] Q9Y5M8 Signal recognition particle receptor subunit beta (SR-beta) (Protein APMCF1) 0,4 P35527 Keratin, type I cytoskeletal 9 (Cytokeratin-9) (CK-9) (Keratin-9) (K9) 0,5 Q9BW19 Kinesin-like protein KIFC1 (Kinesin-like protein 2) (Kinesin-related protein HSET) 0,5 Q9NZ01 Very-long-chain enoyl-CoA reductase (EC 1.3.1.93) (Synaptic glycoprotein SC2) (Trans-2,3-enoyl-CoA reductase) (TER) 0,5 P49257 Protein ERGIC-53 (ER-Golgi intermediate compartment 53 kDa protein) (Gp58) (Intracellular mannose-specific lectin MR60) (Lectin mannose-binding 1) 0,5 Q99986 Serine/threonine-protein kinase VRK1 (EC 2.7.11.1) (Vaccinia-related kinase 1) 0,5 Q9H7B2 Ribosome production factor 2 homolog (Brix domain-containing protein 1) (Ribosome biogenesis protein RPF2 homolog) 0,5 P07910 Heterogeneous nuclear ribonucleoproteins C1/C2 (hnRNP C1/C2) 0,5 O95232 Luc7-like protein 3 (Cisplatin resistance-associated-overexpressed protein) (Luc7A) (Okadaic acid-inducible phosphoprotein OA48-18) (cAMP regulatory element-associated 0,5 protein 1) (CRE-associated protein 1) (CREAP-1) P07355 Annexin A2 (Annexin II) (Annexin-2) (Calpactin I heavy chain) (Calpactin-1 heavy chain) (Chromobindin-8) (Lipocortin II) (Placental anticoagulant protein IV) (PAP-IV) 0,5 (Protein I) (p36) Q13442 28 kDa heat- and acid-stable phosphoprotein (PDGF-associated protein) (PAP) (PDGFA-associated protein 1) (PAP1) 0,5 Q9Y399 28S ribosomal protein S2, mitochondrial (MRP-S2) (S2mt) 0,5

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Q9C0E8 Protein lunapark 0,5 P51116 Fragile X mental retardation syndrome-related protein 2 0,5 Q13823 Nucleolar GTP-binding protein 2 (Autoantigen NGP-1) 0,5 P62851 40S ribosomal protein S25 0,5 Q9BRX8 Redox-regulatory protein FAM213A (Peroxiredoxin-like 2 activated in M-CSF stimulated monocytes) (Protein PAMM) 0,5 Q14739 Lamin-B receptor (Integral nuclear envelope inner membrane protein) (LMN2R) 0,5 P37108 Signal recognition particle 14 kDa protein (SRP14) (18 kDa Alu RNA-binding protein) 0,5 O15511 Actin-related protein 2/3 complex subunit 5 (Arp2/3 complex 16 kDa subunit) (p16-ARC) 0,5 Q9NX58 Cell growth-regulating nucleolar protein 0,5 Q53GQ0 Estradiol 17-beta-dehydrogenase 12 (EC 1.1.1.62) (17-beta-hydroxysteroid dehydrogenase 12) (17-beta-HSD 12) (3-ketoacyl-CoA reductase) (KAR) (EC 1.3.1.-) 0,5 Q9BX40 Protein LSM14 homolog B (Protein FAM61B) (RNA-associated protein 55B) (hRAP55B) 0,5 Q9P2N5 RNA-binding protein 27 (RNA-binding motif protein 27) 0,5 P25705 ATP synthase subunit alpha, mitochondrial 0,5 Q07020 60S ribosomal protein L18 0,5 O75223 Gamma-glutamylcyclotransferase (EC 2.3.2.4) (Cytochrome c-releasing factor 21) 0,5 Q15738 Sterol-4-alpha-carboxylate 3-dehydrogenase, decarboxylating (EC 1.1.1.170) (Protein H105e3) 0,5 O43602 Neuronal migration protein doublecortin (Doublin) (Lissencephalin-X) (Lis-X) 0,5 O14828 Secretory carrier-associated membrane protein 3 (Secretory carrier membrane protein 3) 0,6 P09012 U1 small nuclear ribonucleoprotein A (U1 snRNP A) (U1-A) (U1A) 0,6 Q13523 Serine/threonine-protein kinase PRP4 homolog (EC 2.7.11.1) (PRP4 kinase) (PRP4 pre-mRNA-processing factor 4 homolog) 0,6 Q7L1Q6 Basic leucine zipper and W2 domain-containing protein 1 (Protein Orf) 0,6 P12004 Proliferating cell nuclear antigen (PCNA) (Cyclin) 0,6 Q96CX2 BTB/POZ domain-containing protein KCTD12 (Pfetin) (Predominantly fetal expressed T1 domain) 0,6 Q92522 Histone H1x 0,6 Q9BYD2 39S ribosomal protein L9, mitochondrial (L9mt) (MRP-L9) 0,6 Q05682 Caldesmon (CDM) 0,6 Q14232 Translation initiation factor eIF-2B subunit alpha (eIF-2B GDP-GTP exchange factor subunit alpha) 0,6 O15347 High mobility group protein B3 (High mobility group protein 2a) (HMG-2a) (High mobility group protein 4) (HMG-4) 0,7 Q06787 Fragile X mental retardation protein 1 (FMRP) (Protein FMR-1) 0,7

167

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O43395 U4/U6 small nuclear ribonucleoprotein Prp3 (Pre-mRNA-splicing factor 3) (hPrp3) (U4/U6 snRNP 90 kDa protein) 0,7 Q29RF7 Sister chromatid cohesion protein PDS5 homolog A (Cell proliferation-inducing gene 54 protein) (Sister chromatid cohesion protein 112) (SCC-112) 0,7 P13984 General transcription factor IIF subunit 2 (EC 3.6.4.12) (ATP-dependent helicase GTF2F2) (General transcription factor IIF 30 kDa subunit) (Transcription initiation factor IIF 1,3 subunit beta) (TFIIF-beta) (Transcription initiation factor RAP30) A6NKG5 Retrotransposon-like protein 1 (Mammalian retrotransposon derived protein 1) (Paternally expressed gene 11 protein) (Retrotransposon-derived protein PEG11) 1,3 P38606 V-type proton ATPase catalytic subunit A (V-ATPase subunit A) (EC 3.6.3.14) (V-ATPase 69 kDa subunit) (Vacuolar ATPase isoform VA68) (Vacuolar proton pump subunit 1,3 alpha) O14776 Transcription elongation regulator 1 (TATA box-binding protein-associated factor 2S) (Transcription factor CA150) 1,4 P61201 COP9 signalosome complex subunit 2 (SGN2) (Signalosome subunit 2) (Alien homolog) (JAB1-containing signalosome subunit 2) (Thyroid receptor-interacting protein 15) 1,4 (TR-interacting protein 15) (TRIP-15) Q7Z2T5 TRMT1-like protein (EC 2.1.1.-) 1,5 O14744 Protein arginine N-methyltransferase 5 (EC 2.1.1.-) (72 kDa ICln-binding protein) (Histone-arginine N-methyltransferase PRMT5) (EC 2.1.1.125) (Jak-binding protein 1) (Shk1 1,5 kinase-binding protein 1 homolog) (SKB1 homolog) (SKB1Hs) [Cleaved into: Protein arginine N-methyltransferase 5, N-terminally processed] Q15181 Inorganic pyrophosphatase (EC 3.6.1.1) (Pyrophosphate phospho-hydrolase) (PPase) 1,5 P33991 DNA replication licensing factor MCM4 (EC 3.6.4.12) (CDC21 homolog) (P1-CDC21) 1,5 P49915 GMP synthase [glutamine-hydrolyzing] (EC 6.3.5.2) (GMP synthetase) (Glutamine amidotransferase) 1,6 Q02543 60S ribosomal protein L18a 1,7 P42704 Leucine-rich PPR motif-containing protein, mitochondrial (130 kDa leucine-rich protein) (LRP 130) (GP130) 1,7 Q15291 Retinoblastoma-binding protein 5 (RBBP-5) (Retinoblastoma-binding protein RBQ-3) 1,7 Q9Y4L1 Hypoxia up-regulated protein 1 (150 kDa oxygen-regulated protein) (ORP-150) (170 kDa glucose-regulated protein) (GRP-170) 1,8 P17174 Aspartate aminotransferase, cytoplasmic (cAspAT) (EC 2.6.1.1) (EC 2.6.1.3) (Cysteine aminotransferase, cytoplasmic) (Cysteine transaminase, cytoplasmic) (cCAT) 1,8 (Glutamate oxaloacetate transaminase 1) (Transaminase A) Q9BTT0 Acidic leucine-rich nuclear phosphoprotein 32 family member E (LANP-like protein) (LANP-L) 1,9 Q9Y490 Talin-1 1,9 Q9Y5L0 Transportin-3 (Importin-12) (Imp12) (Transportin-SR) (TRN-SR) 1,9 P62899 60S ribosomal protein L31 1,9 Q9Y2Q3 Glutathione S-transferase kappa 1 (EC 2.5.1.18) (GST 13-13) (GST class-kappa) (GSTK1-1) (hGSTK1) (Glutathione S-transferase subunit 13) 1,9 P08237 ATP-dependent 6-phosphofructokinase, muscle type (ATP-PFK) (PFK-M) (EC 2.7.1.11) (6-phosphofructokinase type A) (Phosphofructo-1-kinase isozyme A) (PFK-A) 2,0 (Phosphohexokinase) P53992 Protein transport protein Sec24C (SEC24-related protein C) 2,0 Q8WWY3 U4/U6 small nuclear ribonucleoprotein Prp31 (Pre-mRNA-processing factor 31) (Serologically defined breast cancer antigen NY-BR-99) (U4/U6 snRNP 61 kDa protein) 2,0 (Protein 61K) (hPrp31) Q9NSD9 Phenylalanine--tRNA ligase beta subunit (EC 6.1.1.20) (Phenylalanyl-tRNA synthetase beta subunit) (PheRS) 2,1

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P52888 Thimet oligopeptidase (EC 3.4.24.15) (Endopeptidase 24.15) (MP78) 2,2 Q9H4M9 EH domain-containing protein 1 (PAST homolog 1) (hPAST1) (Testilin) 2,2 P46782 40S ribosomal protein S5 [Cleaved into: 40S ribosomal protein S5, N-terminally processed] 2,2 Q8IVD9 NudC domain-containing protein 3 2,2 P78347 General transcription factor II-I (GTFII-I) (TFII-I) (Bruton tyrosine kinase-associated protein 135) (BAP-135) (BTK-associated protein 135) (SRF-Phox1-interacting protein) 2,2 (SPIN) (Williams-Beuren syndrome chromosomal region 6 protein) Q6PI48 Aspartate--tRNA ligase, mitochondrial (EC 6.1.1.12) (Aspartyl-tRNA synthetase) (AspRS) 2,2 Q6YP21 Kynurenine--oxoglutarate transaminase 3 (EC 2.6.1.7) (Cysteine-S-conjugate beta-lyase 2) (EC 4.4.1.13) (Kynurenine aminotransferase III) (KATIII) (Kynurenine--glyoxylate 2,2 transaminase) (EC 2.6.1.63) (Kynurenine--oxoglutarate transaminase III) Q9ULV4 Coronin-1C (Coronin-3) (hCRNN4) 2,3 O43847 Nardilysin (EC 3.4.24.61) (N-arginine dibasic convertase) (NRD convertase) (NRD-C) 2,3 P48735 Isocitrate dehydrogenase [NADP], mitochondrial (IDH) (EC 1.1.1.42) (ICD-M) (IDP) (NADP(+)-specific ICDH) (Oxalosuccinate decarboxylase) 2,4 O00410 Importin-5 (Imp5) (Importin subunit beta-3) (Karyopherin beta-3) (Ran-binding protein 5) (RanBP5) 2,4 Q14677 Clathrin interactor 1 (Clathrin-interacting protein localized in the trans-Golgi region) (Clint) (Enthoprotin) (Epsin-4) (Epsin-related protein) (EpsinR) 2,4 Q92747 Actin-related protein 2/3 complex subunit 1A (SOP2-like protein) 2,4 Q15435 Protein phosphatase 1 regulatory subunit 7 (Protein phosphatase 1 regulatory subunit 22) 2,4 Q00341 Vigilin (High density lipoprotein-binding protein) (HDL-binding protein) 2,4 Q92878 DNA repair protein RAD50 (hRAD50) (EC 3.6.-.-) 2,4 Q9NYU2 UDP-glucose:glycoprotein glucosyltransferase 1 (UGT1) (hUGT1) (EC 2.4.1.-) (UDP--Glc:glycoprotein glucosyltransferase) (UDP-glucose ceramide glucosyltransferase-like 1) 2,5 Q9Y678 Coatomer subunit gamma-1 (Gamma-1-coat protein) (Gamma-1-COP) 2,5 O14974 Protein phosphatase 1 regulatory subunit 12A (Myosin phosphatase-targeting subunit 1) (Myosin phosphatase target subunit 1) (Protein phosphatase myosin-binding 2,6 subunit) O60763 General vesicular transport factor p115 (Protein USO1 homolog) (Transcytosis-associated protein) (TAP) (Vesicle-docking protein) 2,7 Q13492 Phosphatidylinositol-binding clathrin assembly protein (Clathrin assembly lymphoid myeloid leukemia protein) 2,7 P30520 Adenylosuccinate synthetase isozyme 2 (AMPSase 2) (AdSS 2) (EC 6.3.4.4) (Adenylosuccinate synthetase, acidic isozyme) (Adenylosuccinate synthetase, liver isozyme) (L- 2,7 type adenylosuccinate synthetase) (IMP--aspartate ligase 2) Q9UNH7 Sorting nexin-6 (TRAF4-associated factor 2) [Cleaved into: Sorting nexin-6, N-terminally processed] 2,7 Q9Y2X7 ARF GTPase-activating protein GIT1 (ARF GAP GIT1) (Cool-associated and tyrosine-phosphorylated protein 1) (CAT-1) (CAT1) (G protein-coupled receptor kinase-interactor 2,7 1) (GRK-interacting protein 1) O15260 Surfeit locus protein 4 2,8 Q8TCS8 Polyribonucleotide nucleotidyltransferase 1, mitochondrial (EC 2.7.7.8) (3'-5' RNA exonuclease OLD35) (PNPase old-35) (Polynucleotide phosphorylase 1) (PNPase 1) 2,8 (Polynucleotide phosphorylase-like protein)

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O15270 Serine palmitoyltransferase 2 (EC 2.3.1.50) (Long chain base biosynthesis protein 2) (LCB 2) (Long chain base biosynthesis protein 2a) (LCB2a) (Serine-palmitoyl-CoA 2,8 transferase 2) (SPT 2) P18206 Vinculin (Metavinculin) (MV) 2,8 P29144 Tripeptidyl-peptidase 2 (TPP-2) (EC 3.4.14.10) (Tripeptidyl aminopeptidase) (Tripeptidyl-peptidase II) (TPP-II) 2,8 Q8NI36 WD repeat-containing protein 36 (T-cell activation WD repeat-containing protein) (TA-WDRP) 2,9 Q15437 Protein transport protein Sec23B (SEC23-related protein B) 2,9 P27708 CAD protein [Includes: Glutamine-dependent carbamoyl-phosphate synthase (EC 6.3.5.5); Aspartate carbamoyltransferase (EC 2.1.3.2); Dihydroorotase (EC 3.5.2.3)] 2,9 O00743 Serine/threonine-protein phosphatase 6 catalytic subunit (PP6C) (EC 3.1.3.16) [Cleaved into: Serine/threonine-protein phosphatase 6 catalytic subunit, N-terminally 3,1 processed] Q01813 ATP-dependent 6-phosphofructokinase, platelet type (ATP-PFK) (PFK-P) (EC 2.7.1.11) (6-phosphofructokinase type C) (Phosphofructo-1-kinase isozyme C) (PFK-C) 3,3 (Phosphohexokinase) Q08J23 tRNA (cytosine(34)-C(5))-methyltransferase (EC 2.1.1.203) (Myc-induced SUN domain-containing protein) (Misu) (NOL1/NOP2/Sun domain family member 2) (Substrate of 3,4 AIM1/Aurora kinase B) (tRNA (cytosine-5-)-methyltransferase) (tRNA methyltransferase 4 homolog) (hTrm4) O75369 Filamin-B (FLN-B) (ABP-278) (ABP-280 homolog) (Actin-binding-like protein) (Beta-filamin) (Filamin homolog 1) (Fh1) (Filamin-3) (Thyroid autoantigen) (Truncated actin- 3,4 binding protein) (Truncated ABP) Q9HCC0 Methylcrotonoyl-CoA carboxylase beta chain, mitochondrial (MCCase subunit beta) (EC 6.4.1.4) (3-methylcrotonyl-CoA carboxylase 2) (3-methylcrotonyl-CoA carboxylase 3,8 non-biotin-containing subunit) (3-methylcrotonyl-CoA:carbon dioxide ligase subunit beta) P25325 3-mercaptopyruvate sulfurtransferase (MST) (EC 2.8.1.2) 3,9 P53618 Coatomer subunit beta (Beta-coat protein) (Beta-COP) 3,9 Q9UPN3 Microtubule-actin cross-linking factor 1, isoforms 1/2/3/5 (620 kDa actin-binding protein) (ABP620) (Actin cross-linking family protein 7) (Macrophin-1) (Trabeculin-alpha) 3,9 Q6P2E9 Enhancer of mRNA-decapping protein 4 (Autoantigen Ge-1) (Autoantigen RCD-8) (Human enhancer of decapping large subunit) (Hedls) 4,7 P30566 Adenylosuccinate lyase (ASL) (EC 4.3.2.2) (Adenylosuccinase) (ASase) 4,9 P48637 Glutathione synthetase (GSH synthetase) (GSH-S) (EC 6.3.2.3) (Glutathione synthase) 5,1 Q92888 Rho guanine nucleotide exchange factor 1 (115 kDa guanine nucleotide exchange factor) (p115-RhoGEF) (p115RhoGEF) (Sub1.5) 5,1 A5YKK6 CCR4-NOT transcription complex subunit 1 (CCR4-associated factor 1) (Negative regulator of transcription subunit 1 homolog) (NOT1H) (hNOT1) 5,2 Q15785 Mitochondrial import receptor subunit TOM34 (hTom34) (Translocase of outer membrane 34 kDa subunit) 5,6 Q7L576 Cytoplasmic FMR1-interacting protein 1 (Specifically Rac1-associated protein 1) (Sra-1) (p140sra-1) 7,2 P24928 DNA-directed RNA polymerase II subunit RPB1 (RNA polymerase II subunit B1) (EC 2.7.7.6) (DNA-directed RNA polymerase II subunit A) (DNA-directed RNA polymerase III 7,5 largest subunit) (RNA-directed RNA polymerase II subunit RPB1) (EC 2.7.7.48) Q7Z6Z7 E3 ubiquitin-protein ligase HUWE1 (EC 6.3.2.-) (ARF-binding protein 1) (ARF-BP1) (HECT, UBA and WWE domain-containing protein 1) (Homologous to E6AP carboxyl 8,8 terminus homologous protein 9) (HectH9) (Large structure of UREB1) (LASU1) (Mcl-1 ubiquitin ligase E3) (Mule) (Upstream regulatory element-binding protein 1) (URE-B1) (URE-binding protein 1) O60271 C-Jun-amino-terminal kinase-interacting protein 4 (JIP-4) (JNK-interacting protein 4) (Cancer/testis antigen 89) (CT89) (Human lung cancer oncogene 6 protein) (HLC-6) 13,5 (JNK-associated leucine-zipper protein) (JLP) (Mitogen-activated protein kinase 8-interacting protein 4) (Proliferation-inducing protein 6) (Protein highly expressed in testis) (PHET) (Sperm surface protein) (Sperm-associated antigen 9) (Sperm-specific protein) (Sunday driver 1)

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Q02952 A-kinase anchor protein 12 (AKAP-12) (A-kinase anchor protein 250 kDa) (AKAP 250) (Gravin) (Myasthenia gravis autoantigen) ###### #### Q05193 Dynamin-1 (EC 3.6.5.5) ###### #### P05556 Integrin beta-1 (Fibronectin receptor subunit beta) (Glycoprotein IIa) (GPIIA) (VLA-4 subunit beta) (CD antigen CD29) 0,0 Q9Y6C9 Mitochondrial carrier homolog 2 (Met-induced mitochondrial protein) 0,1 P20700 Lamin-B1 0,1 P05141 ADP/ATP translocase 2 (ADP,ATP carrier protein 2) (ADP,ATP carrier protein, fibroblast isoform) (Adenine nucleotide translocator 2) (ANT 2) (Solute carrier family 25 0,1 member 5) [Cleaved into: ADP/ATP translocase 2, N-terminally processed] Q9UKV3 Apoptotic chromatin condensation inducer in the nucleus (Acinus) 0,1 P18583 Protein SON (Bax antagonist selected in saccharomyces 1) (BASS1) (Negative regulatory element-binding protein) (NRE-binding protein) (Protein DBP-5) (SON3) 0,2 Q8TB36 Ganglioside-induced differentiation-associated protein 1 (GDAP1) 0,2 O00567 Nucleolar protein 56 (Nucleolar protein 5A) 0,2 P35232 Prohibitin 0,2 Q9Y3E5 Peptidyl-tRNA hydrolase 2, mitochondrial (PTH 2) (EC 3.1.1.29) (Bcl-2 inhibitor of transcription 1) 0,2 O95292 Vesicle-associated membrane protein-associated protein B/C (VAMP-B/VAMP-C) (VAMP-associated protein B/C) (VAP-B/VAP-C) 0,3 P02545 Prelamin-A/C [Cleaved into: Lamin-A/C (70 kDa lamin) (Renal carcinoma antigen NY-REN-32)] 0,3 P21796 Voltage-dependent anion-selective channel protein 1 (VDAC-1) (hVDAC1) (Outer mitochondrial membrane protein porin 1) (Plasmalemmal porin) (Porin 31HL) (Porin 0,3 31HM) Q86YP4 Transcriptional repressor p66-alpha (Hp66alpha) (GATA zinc finger domain-containing protein 2A) 0,4 P45880 Voltage-dependent anion-selective channel protein 2 (VDAC-2) (hVDAC2) (Outer mitochondrial membrane protein porin 2) 0,4 P12236 ADP/ATP translocase 3 (ADP,ATP carrier protein 3) (ADP,ATP carrier protein, isoform T2) (ANT 2) (Adenine nucleotide translocator 3) (ANT 3) (Solute carrier family 25 0,4 member 6) [Cleaved into: ADP/ATP translocase 3, N-terminally processed] P35222 Catenin beta-1 (Beta-catenin) 0,4 P09651 Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) (Helix-destabilizing protein) (Single-strand RNA-binding protein) (hnRNP core protein A1) [Cleaved into: 0,4 Heterogeneous nuclear ribonucleoprotein A1, N-terminally processed] Q9H2P0 Activity-dependent neuroprotector homeobox protein (Activity-dependent neuroprotective protein) 0,5 P49720 Proteasome subunit beta type-3 (EC 3.4.25.1) (Proteasome chain 13) (Proteasome component C10-II) (Proteasome theta chain) 0,5 O75340 Programmed cell death protein 6 (Apoptosis-linked gene 2 protein) (Probable calcium-binding protein ALG-2) 0,5 Q86UE4 Protein LYRIC (3D3/LYRIC) (Astrocyte elevated gene-1 protein) (AEG-1) (Lysine-rich CEACAM1 co-isolated protein) (Metadherin) (Metastasis adhesion protein) 0,5 P04792 Heat shock protein beta-1 (HspB1) (28 kDa heat shock protein) (Estrogen-regulated 24 kDa protein) (Heat shock 27 kDa protein) (HSP 27) (Stress-responsive protein 27) 0,6 (SRP27) P62258 14-3-3 protein epsilon (14-3-3E) 0,7

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P08758 Annexin A5 (Anchorin CII) (Annexin V) (Annexin-5) (Calphobindin I) (CBP-I) (Endonexin II) (Lipocortin V) (Placental anticoagulant protein 4) (PP4) (Placental anticoagulant 0,7 protein I) (PAP-I) (Thromboplastin inhibitor) (Vascular anticoagulant-alpha) (VAC-alpha) P08243 Asparagine synthetase [glutamine-hydrolyzing] (EC 6.3.5.4) (Cell cycle control protein TS11) (Glutamine-dependent asparagine synthetase) 1,7 O43707 Alpha-actinin-4 (F-actin cross-linking protein) (Non-muscle alpha-actinin 4) 1,7 P12814 Alpha-actinin-1 (Alpha-actinin cytoskeletal isoform) (F-actin cross-linking protein) (Non-muscle alpha-actinin-1) 1,8 Q92974 Rho guanine nucleotide exchange factor 2 (Guanine nucleotide exchange factor H1) (GEF-H1) (Microtubule-regulated Rho-GEF) (Proliferating cell nucleolar antigen p40) 1,9 Q13616 Cullin-1 (CUL-1) 2,0 Q14204 Cytoplasmic dynein 1 heavy chain 1 (Cytoplasmic dynein heavy chain 1) (Dynein heavy chain, cytosolic) 2,1 Q9Y2A7 Nck-associated protein 1 (NAP 1) (Membrane-associated protein HEM-2) (p125Nap1) 2,1 P42224 Signal transducer and activator of transcription 1-alpha/beta (Transcription factor ISGF-3 components p91/p84) 2,2 O00232 26S proteasome non-ATPase regulatory subunit 12 (26S proteasome regulatory subunit RPN5) (26S proteasome regulatory subunit p55) 2,2 P43246 DNA mismatch repair protein Msh2 (hMSH2) (MutS protein homolog 2) 2,3 Q6Y7W6 PERQ amino acid-rich with GYF domain-containing protein 2 (GRB10-interacting GYF protein 2) (Trinucleotide repeat-containing gene 15 protein) 2,5 O60341 Lysine-specific histone demethylase 1A (EC 1.-.-.-) (BRAF35-HDAC complex protein BHC110) (Flavin-containing amine oxidase domain-containing protein 2) 2,8 Q14203 Dynactin subunit 1 (150 kDa dynein-associated polypeptide) (DAP-150) (DP-150) (p135) (p150-glued) 2,8 O00429 Dynamin-1-like protein (EC 3.6.5.5) (Dnm1p/Vps1p-like protein) (DVLP) (Dynamin family member proline-rich carboxyl-terminal domain less) (Dymple) (Dynamin-like 2,9 protein) (Dynamin-like protein 4) (Dynamin-like protein IV) (HdynIV) (Dynamin-related protein 1) P49588 Alanine--tRNA ligase, cytoplasmic (EC 6.1.1.7) (Alanyl-tRNA synthetase) (AlaRS) (Renal carcinoma antigen NY-REN-42) 3,0 Q8IX12 Cell division cycle and apoptosis regulator protein 1 (Cell cycle and apoptosis regulatory protein 1) (CARP-1) (Death inducer with SAP domain) 3,2 Q01433 AMP deaminase 2 (EC 3.5.4.6) (AMP deaminase isoform L) 3,4 Q13813 Spectrin alpha chain, non-erythrocytic 1 (Alpha-II spectrin) (Fodrin alpha chain) (Spectrin, non-erythroid alpha subunit) 3,4 Q8IV08 Phospholipase D3 (PLD 3) (EC 3.1.4.4) (Choline phosphatase 3) (HindIII K4L homolog) (Hu-K4) (Phosphatidylcholine-hydrolyzing phospholipase D3) 3,5 P06400 Retinoblastoma-associated protein (p105-Rb) (pRb) (Rb) (pp110) 6,2 Q15149 Plectin (PCN) (PLTN) (Hemidesmosomal protein 1) (HD1) (Plectin-1) 10,6 Q96TA1 Niban-like protein 1 (Meg-3) (Melanoma invasion by ERK) (MINERVA) (Protein FAM129B) ###### ####

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Curriculum Vitea

Education

08/2011 – 03/2015 Biochemistry Ph.D at Medical Proteom-Center, Ruhr-University, Bochum

10/2008 – 06/2011 Chemistry master, TU-Dortmund

09/2006 – 06/2008 Chemistry bachelor degree at Hebei University accomplished

09/2005 – 06/2006 Intensive German language course at Goethe Institute, Beijing

09/2003 – 07/2005 Chemistry bachelor (4 years regular) Hebei University, Baoding

09/2000 – 06/2003 No.1 high school, Tangshan

09/1997 – 06/2000 No.35 middle school, Tangshan

09/1991 – 06/1997 Elementary school, Tangshan

Language

Chinese (Mandarin) Native speaker

German Advanced, fluent in spoken and written

English Advanced, fluent in spoken and written

Publications

1). Low-bias phosphopeptide enrichment from scarce samples using plastic antibodies. Chen et al., Sci. Rep. 5:11438 (2015).

2). Comparison of pY-MIP, TiO2 and Immunoprecipitation for site and side chain selective Phosphopeptide enrichment using Imprinted Polymer Receptors. (Chen et al., manuscript submitted to J.Proteome Res.)

3). Jiafei Zhang, Jing Chen, Robert Misch, David W. Agar

Carbon dioxide absorption in biphasic amine solvents with enhanced low temperature solvent regeneration. Chemical Engineering Transactions, 2010, Vol. 21, 169-147

Leisure time activities

Tae bo, jogging, piano playing, language learning, movies

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