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A Standardized and Reproducible Proteomics Protocol for Bottom-Up Quantitative Analysis of Protein Samples Using SP3 and Mass Spectrometry

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Proteomics for Biomarker Discovery

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1959))

Abstract

The broad utility of mass spectrometry (MS) for investigating the proteomes of a diverse array of sample types has significantly expanded the use of this technology in biological studies. This widespread use has resulted in a substantial collection of protocols and acquisition approaches designed to obtain the highest-quality data for each experiment. As a result, distilling this information to develop a standard operating protocol for essential workflows, such as bottom-up quantitative shotgun whole proteome analysis, can be complex for users new to MS technology. Further complicating this matter, in-depth description of the methodological choices is seldom given in the literature. In this work, we describe a workflow for quantitative whole proteome analysis that is suitable for biomarker discovery, giving detailed consideration to important stages, including (1) cell lysis and protein cleanup using SP3 paramagnetic beads, (2) quantitative labeling, (3) offline peptide fractionation, (4) MS analysis, and (5) data analysis and interpretation. Special attention is paid to providing comprehensive details for all stages of this proteomics workflow to enhance transferability to external labs. The standardized protocol described here will provide a simplified resource to the proteomics community toward efficient adaptation of MS technology in proteomics studies.

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References

  1. Larance M, Lamond AI (2015) Multidimensional proteomics for cell biology. Nat Rev Mol Cell Biol 16(5):269–280. https://doi.org/10.1038/nrm3970

    Google Scholar 

  2. Gillet LC, Leitner A, Aebersold R (2016) Mass spectrometry applied to bottom-up proteomics: entering the high-throughput era for hypothesis testing. Annu Rev Anal Chem (Palo Alto, Calif) 9(1):449–472. https://doi.org/10.1146/annurev-anchem-071015-041535

    Google Scholar 

  3. Weston LA, Bauer KM, Hummon AB (2013) Comparison of bottom-up proteomic approaches for LC-MS analysis of complex proteomes. Anal Methods 5(18):4615–4621. https://doi.org/10.1039/C3AY40853A

    Google Scholar 

  4. Zhang Y, Fonslow BR, Shan B et al (2013) Protein analysis by shotgun/bottom-up proteomics. Chem Rev 113(4):2343–2394. https://doi.org/10.1021/cr3003533

    Google Scholar 

  5. Hughes CS, Foehr S, Garfield DA et al (2014) Ultrasensitive proteome analysis using paramagnetic bead technology. Mol Syst Biol 10:757. https://doi.org/10.15252/msb.20145625

    Google Scholar 

  6. Hughes CS, McConechy MK, Cochrane DR et al (2016) Quantitative profiling of single formalin fixed tumour sections: proteomics for translational research. Sci Rep 6:34949. https://doi.org/10.1038/srep34949

    Google Scholar 

  7. Moggridge S, Sorensen PH, Morin GB et al (2018) Extending the compatibility of the SP3 paramagnetic bead processing approach for proteomics. J Proteome Res 17(4):1730–1740. https://doi.org/10.1021/acs.jproteome.7b00913

    Google Scholar 

  8. Bakalarski CE, Kirkpatrick DS (2016) A biologist’s field guide to multiplexed quantitative proteomics. Mol Cell Proteomics 15(5):1489–1497. https://doi.org/10.1074/mcp.O115.056986

    Google Scholar 

  9. Domon B, Aebersold R (2010) Options and considerations when selecting a quantitative proteomics strategy. Nat Biotechnol 28(7):710–721. https://doi.org/10.1038/nbt.1661

    Google Scholar 

  10. Ong SE, Mann M (2005) Mass spectrometry-based proteomics turns quantitative. Nat Chem Biol 1(5):252–262. https://doi.org/10.1038/nchembio736

    Google Scholar 

  11. Paulo JA, O’Connell JD, Gaun A et al (2015) Proteome-wide quantitative multiplexed profiling of protein expression: carbon-source dependency in Saccharomyces cerevisiae. Mol Biol Cell 26(22):4063–4074. https://doi.org/10.1091/mbc.E15-07-0499

    Google Scholar 

  12. Mertins P, Mani DR, Ruggles KV et al (2016) Proteogenomics connects somatic mutations to signalling in breast cancer. Nature 534(7605):55–62. https://doi.org/10.1038/nature18003

    Google Scholar 

  13. Zecha J, Meng C, Zolg DP et al (2018) Peptide level turnover measurements enable the study of proteoform dynamics. Mol Cell Proteomics 17(5):974–992. https://doi.org/10.1074/mcp.RA118.000583

    Google Scholar 

  14. Kuljanin M, Dieters-Castator DZ, Hess DA et al (2017) Comparison of sample preparation techniques for large-scale proteomics. Proteomics 17(1–2). https://doi.org/10.1002/pmic.201600337

  15. Yang F, Shen Y, Camp DG et al (2012) High-pH reversed-phase chromatography with fraction concatenation for 2D proteomic analysis. Expert Rev Proteomics 9(2):129–134. https://doi.org/10.1586/epr.12.15

    Google Scholar 

  16. Spicer V, Ezzati P, Neustaeter H et al (2016) 3D HPLC-MS with reversed-phase separation functionality in all three dimensions for large-scale bottom-up proteomics and peptide retention data collection. Anal Chem 88(5):2847–2855. https://doi.org/10.1021/acs.analchem.5b04567

    Google Scholar 

  17. Eliuk S, Makarov A (2015) Evolution of Orbitrap mass spectrometry instrumentation. Annu Rev Anal Chem (Palo Alto, Calif) 8:61–80. https://doi.org/10.1146/annurev-anchem-071114-040325

    Google Scholar 

  18. Beck S, Michalski A, Raether O et al (2015) The impact II, a very high-resolution quadrupole time-of-flight instrument (QTOF) for deep shotgun proteomics. Mol Cell Proteomics 14(7):2014–2029. https://doi.org/10.1074/mcp.M114.047407

    Google Scholar 

  19. Andrews GL, Simons BL, Young JB et al (2011) Performance characteristics of a new hybrid quadrupole time-of-flight tandem mass spectrometer (TripleTOF 5600). Anal Chem 83(13):5442–5446. https://doi.org/10.1021/ac200812d

    Google Scholar 

  20. Schilling B, Gibson BW, Hunter CL (2017) Generation of high-quality SWATH® acquisition data for label-free quantitative proteomics studies using TripleTOF® mass spectrometers. In: Comai L, Katz J, Mallick P (eds) Proteomics. Methods in molecular biology, vol 1550. Humana Press, New York, NY, pp 223–233. https://doi.org/10.1007/978-1-4939-6747-6_16

    Google Scholar 

  21. Senko MW, Remes PM, Canterbury JD et al (2013) Novel parallelized quadrupole/linear ion trap/Orbitrap tribrid mass spectrometer improving proteome coverage and peptide identification rates. Anal Chem 85(24):11710–11714. https://doi.org/10.1021/ac403115c

    Google Scholar 

  22. Nesvizhskii AI, Vitek O, Aebersold R (2007) Analysis and validation of proteomic data generated by tandem mass spectrometry. Nat Methods 4(10):787–797. https://doi.org/10.1038/nmeth1088

    Google Scholar 

  23. Schmidt A, Forne I, Imhof A (2014) Bioinformatic analysis of proteomics data. BMC Syst Biol 8(Suppl 2):S3. https://doi.org/10.1186/1752-0509-8-S2-S3

    Google Scholar 

  24. Vaudel M, Barsnes H, Berven FS et al (2011) SearchGUI: an open-source graphical user interface for simultaneous OMSSA and X!Tandem searches. Proteomics 11(5):996–999. https://doi.org/10.1002/pmic.201000595

    Google Scholar 

  25. Vaudel M, Burkhart JM, Zahedi RP et al (2015) PeptideShaker enables reanalysis of MS-derived proteomics data sets. Nat Biotechnol 33(1):22–24. https://doi.org/10.1038/nbt.3109

    Google Scholar 

  26. Kovalchik KA, Moggridge S, Chen DDY et al (2018) Parsing and quantification of raw Orbitrap mass spectrometer data using RawTools. J Proteome Res 17(6):2237–2247. https://doi.org/10.1021/acs.jproteome.8b00072. PMID: 30462513

  27. Hughes CS, Spicer V, Krokhin OV et al (2017) Investigating acquisition performance on the Orbitrap fusion when using tandem MS/MS/MS scanning with isobaric tags. J Proteome Res 16(5):1839–1846. https://doi.org/10.1021/acs.jproteome.7b00091

    Google Scholar 

  28. Hughes CS, Zhu C, Spicer V et al (2017) Evaluating the characteristics of reporter ion signal acquired in the Orbitrap analyzer for isobaric mass tag proteome quantification experiments. J Proteome Res 16(5):1831–1838. https://doi.org/10.1021/acs.jproteome.7b00092

    Google Scholar 

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Acknowledgments

C.S.H. would like to acknowledge valuable discussions with Lida Radan.

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Authors and Affiliations

  1. Canada’s Michael Smith Genome Sciences Centre, BC Cancer, Vancouver, BC, Canada

    Christopher S. Hughes

  2. Department of Molecular Oncology, British Columbia Cancer Research Centre, Vancouver, BC, Canada

    Poul H. Sorensen

  3. Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada

    Gregg B. Morin

Authors
  1. Christopher S. Hughes
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  2. Poul H. Sorensen
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  3. Gregg B. Morin
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Corresponding author

Correspondence to Gregg B. Morin .

Editor information

Editors and Affiliations

  1. Université Grenoble Alpes, CEA, Inserm, BGE U1038, Grenoble, France

    Virginie Brun

  2. Université Grenoble Alpes, CEA, Inserm, BGE U1038, Grenoble, France

    Yohann Couté

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Hughes, C.S., Sorensen, P.H., Morin, G.B. (2019). A Standardized and Reproducible Proteomics Protocol for Bottom-Up Quantitative Analysis of Protein Samples Using SP3 and Mass Spectrometry. In: Brun, V., Couté, Y. (eds) Proteomics for Biomarker Discovery. Methods in Molecular Biology, vol 1959. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9164-8_5

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  • DOI: https://doi.org/10.1007/978-1-4939-9164-8_5

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9163-1

  • Online ISBN: 978-1-4939-9164-8

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