DOCSLIB.ORG

Strategies in Relative and Absolute Quantitative Mass Spectrometry Based Proteomics

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Strategies in Relative and Absolute Quantitative Mass Spectrometry Based Proteomics Biol. Chem. 2017; 398(5-6): 687–699 Review Open Access Claudia Lindemanna, Nikolas Thomaneka, Franziska Hundt, Thilo Lerari, Helmut E. Meyer, Dirk Wolters and Katrin Marcus* Strategies in relative and absolute quantitative mass spectrometry based proteomics DOI 10.1515/hsz-2017-0104 Received January 18, 2017; accepted February 28, 2017; previously Introduction published online March 6, 2017 Liquid chromatography-mass spectrometry (LC-MS)- Abstract: Quantitative mass spectrometry approaches are based proteome quantification is an approach often used used for absolute and relative quantification in global pro- to study biological processes. Continuously, different teome studies. To date, relative and absolute quantifica- quantitative techniques coupled to LC-MS are developed tion techniques are available that differ in quantification that can address questions including protein-protein accuracy, proteome coverage, complexity and robustness. interactions (PPI) studies, protein abundances or post- This review focuses on most common relative or absolute translational modifications between two or more physi- quantification strategies exemplified by three experimen- ological states (e.g. treated/non-treated, healthy/disease). tal studies. A label-free relative quantification approach Common techniques can be generally summarized in was performed for the investigation of the membrane pro- relative quantification for comparing whole proteomes teome of sensory cilia to the depth of olfactory receptors or the relative amount of a high number of proteins or in in Mus musculus. A SILAC-based relative quantification absolute quantification to determine the absolute concen- approach was successfully applied for the identification tration of distinct proteins within a sample (see Figure 1) of core components and transient interactors of the per- (Bantscheff et al., 2012; Marcus, 2012). Both protein quan- oxisomal importomer in Saccharomyces cerevisiae. Fur- tification approaches uses either chemical or metabolic thermore, AQUA using stable isotopes was exemplified stable isotope labeling for protein quantification (label- to unraveling the prenylome influenced by novel pre- based) or quantification is performed without the intro- nyltransferase inhibitors. Characteristic enrichment and duction of stable isotopes (label-free). fragmentation strategies for a robust quantification of the Label-free quantification is a cost-efficient and easy prenylome are also summarized. handling relative quantification technique, where the number of measured spectra is determined (spectral count- Keywords: AQUA; label-free; LC-MS/MS; prenylation; pro- ing) or the average MS/MS intensities are analyzed (Liu tein-protein interactions; SILAC. et al., 2004; Asara et al., 2008). The biggest advantage of label-free quantification in comparison to other relative quantification approaches is the possibility to measure and compare an unlimited number of samples and the analysis of untreated proteins or peptides. However, in contrast to other technical labeling techniques, variations are higher, because samples are individually prepared and compari- aClaudia Lindemann and Nikolas Thomanek: These authors son occurs only during data analysis (Bantscheff et al., contributed equally to this article. *Corresponding author: Katrin Marcus, Ruhr-University Bochum, 2012). Label-based approaches granted more accurate Medizinisches Proteom-Center, Universitätsstraße 150, D-44801 relative protein quantification, including metabolic, chemi- Bochum, Germany, e-mail: [email protected] cal and enzymatic labeling. Stable isotope labeling with Claudia Lindemann, Nikolas Thomanek, Thilo Lerari and Helmut E. amino acids in cell culture (SILAC) and heavy 15N-labeling Meyer: Ruhr-University Bochum, Medizinisches Proteom-Center, are two of the best studied metabolic stable isotope-based Universitätsstraße 150, D-44801 Bochum, Germany Franziska Hundt and Dirk Wolters: Ruhr-University Bochum, relative quantification methods so far. Labeling of single Biomolecular Mass Spectrometry, Universitätsstraße 150, D-44801 amino acids like arginine and lysine or the replacement of Bochum, Germany all nitrogens for their heavy 15N variant during cell growth ©2017, Claudia Lindemann et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License. 688 C. Lindemann et al.: Strategies in quantitative proteomics concentration. Using this costly technique, the concentra- tion of only a few peptides of interest can be determined and it is unsuited for large interactome or global proteome studies (Bantscheff et al., 2012). The review focus on most common relative or absolute quantification strategies exem- plified by three different experimental examples: label-free relative quantification, investigation of the membrane pro- teome of sensory cilia to the depth of olfactory receptors in Mus musculus; SILAC-based relative quantification, iden- tification of core components and transient interactors of the peroxisomal importomer in Saccharomyces cerevisiae (S. cerevisiae); and AQUA-based absolute quantification, analysis to unravel the prenylome influenced by e.g. novel prenyltransferase inhibitors. The subsequent parts will give background informa- tion about relative and absolute quantification techniques focused on label-free, SILAC-based and AQUA-based Figure 1: Common relative and absolute protein quantification quantification strategies. workflows. The earlier sample labeling and mixing is performed, the smaller is the technical variance. Time points of introducing the isotopic- labeled standard and/or mixing the sample is illustrated with a Label-free quantification gray circle. Common relative quantification labeling techniques are metabolic, chemical or label-free. When using metabolic labeling, Label-free relative quantification presents a high-through- cell cultures (white and gray) are differently isotopic-labeled during put method in the field of quantitative proteomics. Sample and mixed after cell cultivation. Chemical labeling and mixing of the preparation is managed without the application of labeling samples is performed on protein or peptide level after proteolytic reagents, which makes such approaches simple and cost effi- digestion. Samples are prepared and measured separately in a label-free approach and quantification occurs during data analysis. cient. Relative label-free methods can roughly be subdivided In an absolute label-based quantification approach, isotopic- into counting the number of peptide-to-spectrum matches labeled standard proteins or peptides with a known concentration (PSMs; spectral counting) obtained for each protein, as are added to the sample before LC-MS/MS and data analysis. No more abundant proteins are more likely to be observed in sample mixing takes place. LC-MS/MS, liquid chromatography peptide spectra (Washburn et al., 2001; Maerkens et al., tandem mass spectrometry. Revised from Bantscheff et al. (2012). 2013) or measurement of peptide signal intensities by using the extracted ion chromatogram (XIC) (Wiener et al., 2004; reduce variances and ensure a precise quantification accu- Megger et al., 2013a,b). Spectral counting is based on deter- racy as cells are labeled in a very early step in the work- mination and comparison of the number of tandem (MS/MS) flow. However, a limited number of different samples can fragment-ion spectra of peptides between different samples be compared and metabolic labeling can cause a growth without the consideration of physicochemical peptide prop- defect in some organisms (Crotty et al., 2011; Filiou et al., erties. This approach can easily be used for comparison of 2012). An alternative to this is chemical-based relative quan- a large number of datasets and therefore has gained popu- tification such as the isobaric tags for relative and absolute larity over the last years. Proteomic tools such as absolute quantitation (iTRAQ) or the tandem mass tags (TMT) that protein expression (APEX) and normalized spectral abun- are fused to proteins or peptides after proteolytic digestion. dance factor (NSAF) also incorporate the length of the corre- In comparison to metabolic labeling, chemical labeling is sponding protein for absolute quantification (Megger et al., more cost-efficient and suitable for all sorts of proteins or 2013a,b). The second label-free technique is based on the peptides, however, the variances in the samples are higher, measurement of MS-signal intensity of the area under the as sample mixing is performed at a later step in the sample chromatographic peak of the peptide precursor ion. In this preparation workflow (Thompson et al., 2003; Wiese et al., approach peptide quantification is typically accomplished 2007; Bantscheff et al., 2012). A powerful method to deter- by integration of ion intensities of any given peptide over mine the exact protein or peptide concentration is the its chromatographic elution profile. In differential studies usage of isotopic-labeled absolute quantification (AQUA) the integrated signal response of individual peptides is peptides, which are spiked into the sample in a known compared between LC-MS(/MS) runs of different samples. C. Lindemann et al.: Strategies in quantitative proteomics 689 Subsequently, estimation of differential protein abundance in vitro between the reagent and the peptides of interest is performed by an aggregation of differences measured for leads to a heavy-labeled peptide mixture. After LC-MS/
Load more

Recommended publications
  • Standard Flow Multiplexed Proteomics (Sflompro) – an Accessible and Cost-Effective Alternative to Nanolc Workflows
    bioRxiv preprint doi: https://doi.org/10.1101/2020.02.25.964379; this version posted February 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Standard Flow Multiplexed Proteomics (SFloMPro) – An Accessible and Cost-Effective Alternative to NanoLC Workflows Conor Jenkins1 and Ben Orsburn2* 1Hood College Department of Biology, Frederick, MD 2University of Virginia Medical School, Charlottesville, VA *To whom correspondence should be addressed; [email protected] Abstract Multiplexed proteomics using isobaric tagging allows for simultaneously comparing the proteomes of multiple samples. In this technique, digested peptides from each sample are labeled with a chemical tag prior to pooling sample for LC-MS/MS with nanoflow chromatography (NanoLC). The isobaric nature of the tag prevents deconvolution of samples until fragmentation liberates the isotopically labeled reporter ions. To ensure efficient peptide labeling, large concentrations of labeling reagents are included in the reagent kits to allow scientists to use high ratios of chemical label per peptide. The increasing speed and sensitivity of mass spectrometers has reduced the peptide concentration required for analysis, leading to most of the label or labeled sample to be discarded. In conjunction, improvements in the speed of sample loading, reliable pump pressure, and stable gradient construction of analytical flow HPLCs has continued to improve the sample delivery process to the mass spectrometer. In this study we describe a method for performing multiplexed proteomics without the use of NanoLC by using offline fractionation of labeled peptides followed by rapid “standard flow” HPLC gradient LC-MS/MS.
    [Show full text]
  • Phosphoproteomics: Recent Advances in Analytical Techniques
    International Journal of Pharmaceuticals Analysis, ISSN: 0975-3079, Volume 1, Issue 2, 2009, pp-31-36 Phosphoproteomics: Recent advances in analytical techniques Gomase V.S. and Ramu Akella Department of Biotechnology, Padmashree Dr. D.Y. Patil University, Navi Mumbai, 400614, India Abstract - Post-genomic biology seeks identification and quantification of multiple proteins from complex mixtures and the research is still on. Despite recent progress in high-throughput proteomics, proteomic analysis of post-translationally modified [PTM] proteins remains particularly challenging. Several strategies for isolating phosphoproteins are explored herein. Quantification of phosphoproteins seems to be a novel solution to identify the underlying disease mechanisms, mostly cancer. Keywords - phosphorylation, enrichment, chemical modification, SILAC, mass spectrometry, HILIC Introduction Techniques Phosphoproteomics is a branch of proteomics Of late, there is an increasing interest in that focuses on deriving a comprehensive view of phosphoproteomics as reflected by a the extent & dynamics of protein phosphorylation considerable number of works describing various by way of identifying & characterizing proteins strategies, including the use of different isotopic that contain a phosphate group as a post- technologies [e.g., isotope-coded affinity tag, translational modification. The addition of stable isotope labeling by amino acids in cell phosphate is a key reversible modification culture], protein separation techniques [e.g., gel- catalyzed by protein kinases, and is known as based versus liquid chromatography-based ‘phosphorylation’. Protein kinases account for methods], and chemical modifications [e.g., β- 1.7% of the human genome and 40% of all the elimination and esterification] to identify proteins may be assumed to be phosphorylated phosphoproteins and/or phosphorylation sites in at any given time.
    [Show full text]
  • Relative Quantitation of Protein Digests Using Tandem Mass Tags and Pulsed-Q Dissociation (PQD)
    Application Note: 452 Relative Quantitation of Protein Digests Using Tandem Mass Tags and Pulsed-Q Dissociation (PQD) Jae Schwartz, Terry Zhang, Rosa Viner, Vlad Zabrouskov, Thermo Fisher Scientific, San Jose, CA, USA Introduction LC Separation and MS Analysis Key Words Quantitation of differentially expressed proteins is one of LC Separation • LTQ XL™ the most challenging areas in proteomics. A variety of HPLC: Thermo Scientific Surveyor equipped with quantitation methods have been developed, including Micro AS autosampler • PQD isotope labeling approaches like ICAT®1, SILAC2, iTRAQ™3, Columns: PicoFrit™ column (10 cm x 75 µm i.d.), (New Objective, Inc., Cambridge, MA) • Proteomics AQUA4, and Tandem Mass Tag® (TMT®)5. In contrast to MS-based quantitation methods, iTRAQ- and TMT-labeled Sample: Inject 2 µL TMT-labeled digest mixture • Quantification peptides are identified and quantitated by MS/MS. Pulsed-Q Mobile Phases: A: 0.1% Formic acid in water B: 0.1% Formic acid in acetonitrile • TMT Dissociation (PQD)6,7 has been developed to facilitate quantitating of the low-mass reporter ions in MS/MS spectra Gradient: 10% B 10 minutes, 10% – 30% in 120 minutes of iTRAQ- or TMT-labeled peptides. The PQD technique Flow: 300 nL/min on column enables the detection of low-mass fragments in MS/MS mode MS Analysis including y1- and b1-type fragment ions, and also allows Mass Spectrometer: Thermo Scientific LTQ XL equipped with a the quantitation of peptides using the TMT reporter ions nanospray ion source 8 which appear in the 100 m/z range. Spray Voltage: 2.0 kV Capillary Temperature: 160 °C Goal Full MS: 300-1600 m/z To demonstrate the benefits of the PQD-based quantitation Isolation: 3 Da of isobarically labeled peptides in protein digests.
    [Show full text]
  • Enrichment Strategies for Phosphoproteomics: State-Of-The-Art
    Rev Anal Chem 31 (2012): 29–41 © 2012 by Walter de Gruyter • Berlin • Boston. DOI 10.1515/revac-2011-0025 Enrichment strategies for phosphoproteomics: state-of-the-art Barbora Salovska 1,2, *, Ales Tichy 1,2 , Martina MIP molecularly imprinted polymer Rezacova 1 , Jirina Vavrova 2 and Eva Novotna 2 MOAC metal oxide affi nity chromatography MS mass spectrometry 1 Department of Medical Biochemistry , Faculty of NTA nitriloacetic acid Medicine in Hradec Kralove, Charles University in PTMs post-translational modifi cations Prague, Hradec Kr á lov é , Czech Republic , pS phosphoserine e-mail: [email protected] pT phosphothreonine 2 Department of Radiobiology , Faculty of Military Health pY phosphotyrosine Sciences, University of Defence, Hradec Kralove , RPLC reversed phase liquid chromatography Czech Republic SAX strong anion-exchange chromatography SCX strong cation-exchange chromatography * Corresponding author Introduction Abstract Protein phosphorylation is a key regulator in many biological The human genome involves approximately 30,000 protein- processes, such as homeostasis, cellular signaling and com- coding genes; the human proteome contains several million munication, transcriptional and translational regulation, and different protein effectors. This is due to alternative splic- apoptosis. The defects in this tightly controlled reversible post- ing of genes and post-translational modifi cations (PTMs). translational modifi cation have been described to contribute Several hundred PTMs are currently known, among them to genesis and
    [Show full text]
  • ISOTEC® Stable Isotopes Biomolecular NMR Isotope Labeling Methods for Protein Dynamics Studies
    ISOTEC® Stable Isotopes Biomolecular NMR Isotope Labeling Methods for Protein Dynamics Studies Eric D. Watt and J. Patrick Loria, Ph.D. Department of Chemistry, Yale University, Kline Chemistry Laboratory, 225 Prospect Street, New Haven, CT 06520 Protein structure determination by solution NMR spectroscopy use of 15N-enriched minimal or nutrient-rich growth media that are has long relied on the uniform stable isotopic enrichment with readily available, allowing for easy sample preparation. Uniform 15N 13C and 15N to alleviate resonance overlap and to allow multiple labeling results in an isolated spin system (1H-15N) that lends itself well distance and angular restraints, at as many atomic sites as possible, to relaxation experiments. Every 15N position whether in the protein to facilitate computing the optimal three-dimensional structural backbone or sidechain is separated from another 15N atom by at least (1) 1 model. Recently, the optimization of these labeling techniques has two bonds. Therefore there are no JNN couplings that could lead to increased the range of protein sizes amendable to study, enhanced complicated multiexponential relaxation behavior, which would be the quality of three-dimensional structures, and simplified the analysis difficult to accurately measure and would cloud the interpretation of of experimental data.(2) Similarly, the field of protein dynamics has the associated motions. benefited from advances in isotopic labeling techniques that have However, 15N enrichment by itself does not provide a complete picture allowed researchers to study the motional properties of ever larger of the motions a protein undergoes. Nitrogen makes up only 1/3 of proteins over a broad range of timescales while more accurately the protein backbone and is only sparsely populated in the sidechains describing the protein motions.
    [Show full text]
  • Targeted Quantitative Proteomics Using Selected Reaction Monitoring
    Targeted Quantitative Proteomics Using Selected Reaction Monitoring (SRM)- Mass Spectrometry Coupled with an 18O-labeled Reference as Internal Standards Jong-Seo Kim, Errol Robinson, Boyd L. Champion, Brianne O. Petritis, Thomas L. Fillmore, Ronald J. Moore, Liu Tao, David G. Camp II, Richard D. Smith, and Wei-Jun Qian Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA Methods Results Effect of Q1 resolution Reproducibility 18O labeling efficiency Overview Patient Samples Conclusions Group A: • To address the need of stable isotope Group B: • The utility of 18O-labeled “universal” O area ratio O area Trypsin 18 Red error bars: label-free peak area ratio labeled internal standards for accurate (= each 16O area / average 18O area) reference as internal standards for targeted O/ digestion 16 18 quantification, we introduce the use of an 16 Green error bars: each pair area ratio of O/ O quantitative proteomics has been Patient peptide 18 Pooled reference ratio O area O-labeled reference as comprehensive samples sample 18 successfully demonstrated 16 18 LC-SRM-MS O/ Concentration ratio ( O/ O) internal standards for accurate SRM-MS- 18O labeling 16 – A linear dynamic range of quantification Concen. based quantification instead of using labeled 18 0.01 0.02 0.04 0.1 0.2 1 5 10 25 50 100 4 O-labeled “universal” Ratio ~ 10 in relative concentration synthetic peptides. reference 16O/18O 6.1 12.5 13.7 13.8 5.5 7.7 16.8 2.8 4.6 4.2 1.6 pairs – Better reproducibility than label-free 18 Label • O-labeling efficiency for most peptides is 39.2 23.4 47.5 26.5 19.3 10.6 31.2 3.2 10.7 4.8 3.5 Fig.
    [Show full text]
  • Computational Biology, Protein Chemistry and Mass Spectrometry
    CORE Metadata, citation and similar papers at core.ac.uk Provided by Elsevier - Publisher Connector FEBS Letters 580 (2006) 4764–4770 Minireview Phosphoproteomics toolbox: Computational biology, protein chemistry and mass spectrometry Majbrit Hjerrild*, Steen Gammeltoft Department of Clinical Biochemistry, Glostrup Hospital, Nordre Ringvej, DK-2600 Glostrup, Denmark Received 1 June 2006; revised 21 July 2006; accepted 25 July 2006 Available online 4 August 2006 Edited by Francesc Posas tion, or interaction with other proteins. Regulation of the Abstract Protein phosphorylation is important for regulation of most biological functions and up to 50% of all proteins are cell cycle, membrane transport and permeability, cell adhesion, thought to be modified by protein kinases. Increased knowledge neurotransmission, and metabolism are examples of biological about potential phosphorylation of a protein may increase our functions that are modulated through protein phosphorylation understanding of the molecular processes in which it takes part. [1]. The human genome contains 518 different protein kinases Despite the importance of protein phosphorylation, identification and identification of their biological targets is an active re- of phosphoproteins and localization of phosphorylation sites is search area [2]. Even though the number of identified phospho- still a major challenge in proteomics. However, high-throughput proteins is rapidly increasing especially due to development of methods for identification of phosphoproteins are being devel- high-throughput methods for the identification of phospho- oped, in particular within the fields of bioinformatics and mass proteins, in particular within the fields of bioinformatics and spectrometry. In this review, we present a toolbox of current mass spectrometry, it is believed that only a small fraction of technology applied in phosphoproteomics including compu- tational prediction, chemical approaches and mass spectro- physiological phosphorylation sites has been assigned.
    [Show full text]
  • Quantitative Proteomics Reveals the Selectivity of Ubiquitin-Binding Autophagy Receptors in the Turnover of Damaged Lysosomes by Lysophagy
    bioRxiv preprint doi: https://doi.org/10.1101/2021.07.19.452535; this version posted July 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Quantitative proteomics reveals the selectivity of ubiquitin-binding autophagy receptors in the turnover of damaged lysosomes by lysophagy Vinay Eapen1,*, Sharan Swarup1,*, Melissa Hoyer1,*, Joao Paolo1 and J. Wade Harper 1 1Department of Cell Biology, Harvard Medical School, Boston MA 02115 *, Equal Contribution Send correspondence to: [email protected] ABSTRACT Removal of damaged organelles via the process of selective autophagy constitutes a major form of cellular quality control. Damaged organelles are recognized by a dedicated surveillance machinery, leading to the assembly of an autophagosome around the damaged organelle, prior to fusion with the degradative lysosomal compartment. Lysosomes themselves are also prone to damage and are degraded through the process of lysophagy. While early steps involve recognition of ruptured lysosomal membranes by glycan-binding Galectins and ubiquitylation of transmembrane lysosomal proteins, many steps in the process, and their inter-relationships, remain poorly understood, including the role and identity of cargo receptors required for completion of lysophagy. Here, we employ quantitative organelle capture and proximity biotinylation proteomics of autophagy adaptors, cargo receptors, and Galectins in response to acute lysosomal damage, thereby revealing the landscape of lysosomal proteome remodeling during lysophagy. Among proteins dynamically recruited to damaged lysosomes were ubiquitin-binding autophagic cargo receptors.
    [Show full text]
  • A Roadmap for Interpreting 13C Metabolite Labeling Patterns from Cells
    Available online at www.sciencedirect.com ScienceDirect 13 A roadmap for interpreting C metabolite labeling patterns from cells 1,2 3,* 4,* Joerg M Buescher , Maciek R Antoniewicz , Laszlo G Boros , 5,* 6,* 7,* Shawn C Burgess , Henri Brunengraber , Clary B Clish , 8,* 9,* 10,* Ralph J DeBerardinis , Olivier Feron , Christian Frezza , 1,2,* 11,* 12,* Bart Ghesquiere , Eyal Gottlieb , Karsten Hiller , 13,* 14,* 15,* Russell G Jones , Jurre J Kamphorst , Richard G Kibbey , 16,* 17,* 18,* Alec C Kimmelman , Jason W Locasale , Sophia Y Lunt , 11,* 19,* 20,* Oliver DK Maddocks , Craig Malloy , Christian M Metallo , 21,22,* 23,24,* Emmanuelle J Meuillet , Joshua Munger , 25,* 26,* 27,28,* Katharina No¨ h , Joshua D Rabinowitz , Markus Ralser , 29,* 30,* 31,* Uwe Sauer , Gregory Stephanopoulos , Julie St-Pierre , 32,* 33,* Daniel A Tennant , Christoph Wittmann , 34,35,* 11,* Matthew G Vander Heiden , Alexei Vazquez , 11,* 36,37,* 29,* Karen Vousden , Jamey D Young , Nicola Zamboni and 1,2 Sarah-Maria Fendt 13 Measuring intracellular metabolism has increasingly led to Goodman Cancer Research Centre, Department of Physiology, McGill 13 University, Montreal, QC, Canada important insights in biomedical research. C tracer analysis, 14 13 Institute of Cancer Sciences, University of Glasgow, Glasgow, UK although less information-rich than quantitative C flux 15 Internal Medicine, Cellular and Molecular Physiology, Yale University analysis that requires computational data integration, has been School of Medicine, New Haven, CT, USA 16 established as a time-efficient method to unravel relative Division of Genomic Stability and DNA Repair, Department of Radiation Oncology, Dana-Farber Cancer Institute, Boston, MA, USA pathway activities, qualitative changes in pathway 17 Division of Nutritional Sciences, Cornell University, Ithaca, NY, USA contributions, and nutrient contributions.
    [Show full text]
  • Mass Spectrometry and Proteomics Using R/Bioconductor
    Mass spectrometry and proteomics Using R/Bioconductor Laurent Gatto CSAMA - Bressanone - 25 July 2019 1 / 40 Slides available at: http://bit.ly/20190725csama These slides are available under a creative common CC-BY license. You are free to share (copy and redistribute the material in any medium or format) and adapt (remix, transform, and build upon the material) for any purpose, even commercially . 2 / 40 On the menu Morning lecture: 1. Proteomics in R/Bioconductor 2. How does mass spectrometry-based proteomics work? 3. Quantitative proteomics 4. Quantitative proteomics data processing and analysis Afternoon lab: Manipulating MS data (raw and identification data) Manipulating quantitative proteomics data Data processing and DE 3 / 40 4 / 40 1. Proteomics and mass spectrometry packages, questions and workow in Bioconductor. 5 / 40 2. How does mass spectrometry work? (applies to proteomics and metabolomics) 6 / 40 Overview 7 / 40 How does MS work? 1. Digestion of proteins into peptides - as will become clear later, the features we measure in shotgun (or bottom-up) proteomics are peptides, not proteins. 2. On-line liquid chromatography (LC-MS) 3. Mass spectrometry (MS) is a technology that separates charged molecules (ions, peptides) based on their mass to charge ratio (M/Z). 8 / 40 Chromatography MS is generally coupled to chromatography (liquid LC, but can also be gas- based GC). The time an analytes takes to elute from the chromatography column is the retention time. 9 / 40 An mass spectrometer is composed of three components: 1. The source, that ionises the molecules: examples are Matrix-assisted laser desorption/ionisation (MALDI) or electrospray ionisation (ESI).
    [Show full text]
  • Iodoacetyl Tandem Mass Tags for Cysteine Peptide Modification, Enrichment and Quantitation Ryan D
    Iodoacetyl Tandem Mass Tags for Cysteine Peptide Modification, Enrichment and Quantitation Ryan D. Bomgarden 1, Rosa I. Viner 2, Karsten Kuhn 3, Ian Pike 3, John C. Rogers 1 1Thermo Fisher Scientific, Rockford, IL, USA; 2Thermo Fisher Scientific, San Jose, CA, USA; 3Proteome Sciences, Frankfurt, Germany Overview FIGURE 2. iodoTMT reagents and labeling reaction. A) Mechanism of FIGURE 6. cysTMT and iodoTMT reagent labeling of S-nitrosylated proteins. iodoTMT reagent reaction with cysteine -containing proteins or peptides. B) A549 cell lysates (A) and BSA (B) were blocked with MMTS (lanes 1 –8, Purpose: To develop an iodoacetyl Tandem Mass Tag (iodoTMT) reagent for Structure of iodoTMTsixplex reagents for cysteine labeling, enrichment, and 10–15) or untreated (lanes 9, 16) before 500 mM nitro-glutathione (NO) irreversible cysteine peptide labeling, enrichment, and multiplexed quantitation. isobaric MS quantitation. treatment and TMT reagent labeling in the presence or absence of ascorbate and copper sulfate (CuSO ). Proteins were separated by SDS -PAGE and Methods: Reduced sulfhydryls of protein cysteines were labeled with 4 analyzed by anti-TMT antibody Western blotting or Coomassie stain. iodoTMTzero and/or iodoTMTsixplex reagents. Labeled peptides were enriched A using an immobilized anti -TMT antibody resin before mass spectrometry (MS) A cysTMT iodoTMT analysis. 1 2 3 4 5 6 7 8 9 10111213141516 Results: We developed an iodoTMT reagent set to perform duplex isotopic or sixplex isobaric mass spectrometry (MS) quantitation of cysteine-containing peptides. IodoTMT reagents showed efficient and specific labeling of peptide Anti-TMT cysteine residues with reactivity similar to iodoacetamide. Using an anti-TMT Western Blot Cell Lyate antibody, we characterized the immunoaffinity enrichment of peptides labeled with iodoTMT reagents from complex protein cell lysates and for detection of S-nitrosylated cysteines.
    [Show full text]
  • Quantitative Assay of Targeted Proteome in Tomato Trichome
    Takemori et al. Plant Methods (2019) 15:40 https://doi.org/10.1186/s13007-019-0427-7 Plant Methods METHODOLOGY Open Access Quantitative assay of targeted proteome in tomato trichome glandular cells using a large-scale selected reaction monitoring strategy Ayako Takemori1, Taiken Nakashima2, Hisashi Ômura3, Yuki Tanaka4, Keisuke Nakata1, Hiroshi Nonami1,5,6 and Nobuaki Takemori4,6* Abstract Background: Glandular trichomes found in vascular plants are called natural cell factories because they synthesize and store secondary metabolites in glandular cells. To systematically understand the metabolic processes in glandular cells, it is indispensable to analyze cellular proteome dynamics. The conventional proteomics methods based on mass spectrometry have enabled large-scale protein analysis, but require a large number of trichome samples for in-depth analysis and are not suitable for rapid and sensitive quantifcation of targeted proteins. Results: Here, we present a high-throughput strategy for quantifying targeted proteins in specifc trichome glandular cells, using selected reaction monitoring (SRM) assays. The SRM assay platform, targeting proteins in type VI trichome gland cells of tomato as a model system, demonstrated its efectiveness in quantifying multiple proteins from a lim- ited amount of sample. The large-scale SRM assay uses a triple quadrupole mass spectrometer connected online to a nanofow liquid chromatograph, which accurately measured the expression levels of 221 targeted proteins con- tained in the glandular cell sample recovered from 100 glandular trichomes within 120 min. Comparative quantitative proteomics using SRM assays of type VI trichome gland cells between diferent organs (leaves, green fruits, and calyx) revealed specifc organ-enriched proteins. Conclusions: We present a targeted proteomics approach using the established SRM assays which enables quantif- cation of proteins of interest with minimum sampling efort.
    [Show full text]