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Applicability of Multidimensional Fractionation to Affinity Purification Mass Spectrometry Samples and Protein Phosphatase 4 Substrate Identification

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Applicability of Multidimensional Fractionation to Affinity Purification Mass Spectrometry Samples and Protein Phosphatase 4 Substrate Identification Applicability of Multidimensional Fractionation to Affinity Purification Mass Spectrometry Samples and Protein Phosphatase 4 Substrate Identification by Wade Hampton Dunham A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Molecular Genetics University of Toronto © Copyright by Wade Dunham 2012 ii Applicability of Multidimensional Fractionation to Affinity Purification Mass Spectrometry Samples and Protein Phosphatase 4 Substrate Identification Wade Dunham Master of Science Department of Molecular Genetics University of Toronto 2012 Abstract Affinity-purification coupled to mass spectrometry (AP-MS) is gaining widespread use for the identification of protein-protein interactions. It is unclear however, whether typical AP sample complexity is limiting for the identification of all protein components using standard one-dimensional LC-MS/MS. Multidimensional sample separation is a useful for reducing sample complexity prior to MS analysis, and increases peptide and protein coverage of complex samples, yet the applicability of this approach to AP-MS samples remains unknown. Here I present work to show that multidimensional separation of AP-MS samples is not a cost-effective method for identifying increased peptide or protein coverage in these sample types. As such this approach was not adapted for the identification of putative Phosphoprotein Phosphatase 4 (PP4c) substrates. Instead, affinity purification coupled to one- dimensional LC-MS/MS was used to identify putative PP4c substrates, and semi- quantitative methods applied to identify possible PP4c targeted phosphosites in PP2A subfamily phosphatase inhibited (okadaic acid treated) cells. iii Acknowledgments I would like to acknowledge and thank everyone in the Gingras lab, in particular my supervisor Anne-Claude, for allowing me to take on complex projects and to be an integral part in the pursuit of high impact publications. I would also like to thank our lab manager Marilyn, for patiently letting me know where I can find my much needed reagents, although initially, I likely asked her the same question at least once a month, and Brett for his initial training and work, which helped me to secure my first publication. I must also thank both of my committee members; Drs. Ben Blencowe and Thomas Kislinger for their helpful insight and guidance over the course of my projects. The past two and a half years have definitely been a great learning experience. Thanks to everybody! iv Table of Contents Chapter 1: Introduction…………………………………………………………..……..1 1.1 General Introduction and thesis overview……..………………………………….1 1.2 Identification of Proteins by Mass Spectrometry………………………………....2 1.3 Affinity-purification Coupled to Mass Spectrometry (AP-MS)………………......6 1.4 Affinity-purification using epitope tags…………………………………………...9 1.5 Background contaminants in AP-MS……………………………………………13 1.5.1 Strategies to remove the contaminants from the sample before mass spectrometry……………………………………………………………...14 1.5.2 Strategies to remove the contaminants during or after MS analysis: Label-free approaches……….…………………………………………..15 1.6 Fractionation of mass spectrometry samples………………………….…………17 1.7 Utilizing MS for identification of protein phosphorylation……………………...19 1.7.1 Phosphopeptide enrichment approaches and identification……..……….20 1.7.2 Label free phosphopeptide quantification and phosphosite localization…………………………………………………………..…...24 1.8 PP2A subfamily phosphatases…………………………………………..……….31 1.8.1 PP4c biology and regulation………...…………….…………………......31 1.8.2 PP4c interactions and substrate identification……..…………………….39 1.8.3 PP4c regulation of mRNA transcription and splicing……..……...……...49 1.9 Thesis objectives…………………………………………………………………52 Chapter 2: A cost-benefit analysis of multidimensional fractionation of affinity purification-mass spectrometry samples……………………………..….54 2.1 Methods………………………………………………………………………….55 2.1.1 Generation and culture of stably transfected Flp-In T-REx 293 cell lines……………………………………………………..55 2.1.2 Affinity purification……………………………………………………...56 2.1.3 One dimensional (1D) LC-MS/MS analysis……………………………..57 2.1.4 Multidimensional LC-MS/MS analysis……………………………….…57 v 2.1.4.1 MudPIT…………………………………………………………..58 2.1.4.2 RP/RP…………………………………………………………….58 2.1.4.3 GeLC……………………………………………………………..59 2.1.5 Data Analysis………………………………………………….……..…..59 2.2 Results……………………………………………………………………………60 2.2.1 Reproducibility of protein identifications made by AP-MS……………..64 2.2.2 Effect of fractionating affinity purified samples on spectral count, unique peptide, and protein identification…..…………………………………...69 2.2.3 Effect of fractionating affinity purified samples on protein complex component identification.………………………………………………..75 2.2.4 Applying Significance Analysis of INTeractome (SAINT) to fractionated affinity purified samples…………………………………………………82 2.3 Discussion………………………………………………………………………..84 2.3.1 Multidimensional fractionation of AP-MS samples appears to allow for a better depth of coverage of low level background proteins and not core protein complex components....……………………………………...…..84 2.3.2 Primary benefits and disadvantages of multidimensional fractionation of AP-MS samples…..……..……………………………………………….85 2.3.3 Applicability of multidimensional fractionation of AP-MS samples to the expansion of the PP4c network and substrate identification………….....86 2.3.4 Conclusions………………………………………………………………87 Chapter 3: PP4c interactor/subunit phosphosite identification…………………......88 3.1 Methods………………………………………………………………………….88 3.1.1 Generation and culture of stably transfected Flp-In T-REx 293 cell lines………………………………………………..........88 3.1.2 Affinity purification……………………………………………………...91 3.1.3 Enrichment of phosphopeptides…………………………………….....…91 3.1.4 LC-MS/MS analysis…………………………………………………..…92 3.1.5 Data Analysis……………………………………………………….....…93 3.2 Results……………………………………………………………………………94 vi 3.2.1 PP4c interactor/subunit phosphosite identification………………...…….94 3.2.2 Phosphosite detection reproducibility……………….……………….....104 3.2.3 Changes in PP4c interactor phosphorylation upon PP2A subfamily phosphatase inhibition…………...………………….……………….…114 3.2.4 Reproducibility of phosphosite quantification across biological replicates…………….…………...………………….……………….…136 3.3 Discussion……………………………………………………………………....141 3.3.1 Discerning whether PP4c interactors are possible substrates for the enzyme……..…………………………………………………………...141 Chapter 4: Thesis Summary and Future Directions…………………………….….144 4.1 Thesis Summary……………………………………………………………...…144 4.2 Future Directions…………………………………………………………….....145 4.2.1 PP4c substrate identification……...……………...…………………......145 4.3 Conclusions………………………………………………………………….….150 References……………………………………………..…………………………….…152 vii List of Tables Table 1-1. Select peptide, protein, and dual affinity tags successfully used for purification of recombinant proteins in AP-MS studies………………………………………………10 Table 1-2. Classification of human protein phosphatases……………………………….32 Table 1-3. Gene and protein identifiers for PP4c, PP4c regulatory subunits, and interacting proteins investigated and discussed in detail in this thesis…………..………38 Table 2-1. Summary of the mass spectrometry data for this project.…....………..……..63 Table 2-2. Spectral counts, unique peptides, and non-redundant protein identification (A) for all proteins identified in COPS5 samples after background contaminant removal, (B) for the COPS5 interactors reported in BioGRID and detected in our samples, or (C) for all proteins prior to background contaminant removal..…………………………………65 Table 2-3. Spectral counts, unique peptides, and non-redundant protein identification for two biological replicate analyses of EIF4A2 and RAF1 (A) for all proteins after background contaminant removal, (B) for the interaction partners reported in BioGRID, or (C) for all protein hits prior to background contaminant removal..………………..…70 Table 2-4. Spectral counts, unique peptides, and non-redundant protein identifications for MEPCE samples (A) for all proteins identified after background contaminant removal, (B) for the interactors reported in BioGRID, and (C) for all protein hits prior to background contaminant removal………………………………………………………..72 Table 2-5. Paired t-test analysis comparing enrichment of spectra, unique peptides and protein identification by RP-RP analysis of FLAG-eIF4A2, RAF1, and MEPCE samples, after background removal (A), or for BioGRID annotated interactors only (B)………...74 Table 2-6. Proteins identified in "Core" interaction network of FLAG-COPS5 purifications, that are not annotated as COPS5 interactors in BioGRID…………...……76 viii Table 2-7. Fold increase in spectral counts (A), or unique peptides (B) for BioGRID- annotated COPS5 interactors..……………………………………………………...……78 Table 2-8. A) Fold increase in spectral counts or unique peptides shown as the ratio of 2D/1D for BioGRID-annotated EIF4A2 interactors for each of the biological replicates analyzed (replicates annotated 1 and 2). B) Fold increase in spectral counts or unique peptides shown as the ratio of 2D/1D for BioGRID-annotated RAF1 interactors for each of the biological replicates analyzed (replicates annotated 1 and 2)...…….…………….80 Table 2-9. Fold increase in spectral counts or unique peptides shown as the ratio of 2D/1D for BioGRID-annotated MEPCE interactors………………………………..…...81 Table 3-1. FLAG tagged constructs and stable cell lines generated for identification of puataive PP4c substrates………………………………………………………………....90 Table 3-2. Summary table listing phosphopeptides
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