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Proteomics in Systems Biology pp 1–13Cite as

Multiplexed Quantitative Proteomics for High-Throughput Comprehensive Proteome Comparisons of Human Cell Lines

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1394))

Abstract

The proteome is the functional entity of the cell, and perturbations of a cellular system almost always cause changes in the proteome. These changes are a molecular fingerprint, allowing characterization and a greater understanding of the effect of the perturbation on the cell as a whole. Monitoring these changes has therefore given great insight into cellular responses to stress and disease states, and analytical platforms to comprehensively analyze the proteome are thus extremely important tools in biological research. Mass spectrometry has evolved as the most relevant technology to characterize proteomes in a comprehensive way. However, due to a lack of throughput capacity of mass spectrometry-based proteomics, researchers frequently use measurement of mRNA levels to approximate proteome changes. Growing evidence of substantial differences between mRNA and protein levels as well as recent improvements in mass spectrometry-based proteomics are heralding an increased use of mass spectrometry for comprehensive proteome mapping. Here we describe the use of multiplexed quantitative proteomics using isobaric labeling with tandem mass tags (TMT) for the simultaneous quantitative analysis of five cancer cell proteomes in biological duplicates in one mass spectrometry experiment.

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References

  1. Kim M, Pinto S, Getnet D et al (2014) A draft map of the human proteome. Nature 509:575–581

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Wilhelm M, Schlegl J, Hahne H et al (2014) Mass-spectrometry-based draft of the human proteome. Nature 509:582–587

    Article  CAS  PubMed  Google Scholar 

  3. Wang Z, Gerstein M, Snyder M (2009) RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 10:57–63

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Li G, Burkhardt D, Gross C et al (2014) Quantifying absolute protein synthesis rates reveals principles underlying allocation of cellular resources. Cell 157:624–635

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Torres E, Dephoure N, Panneerselvam A et al (2010) Identification of aneuploidy-tolerating mutations. Cell 143:71–83

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Stingele S, Stoehr G, Peplowska K et al (2012) Global analysis of genome, transcriptome and proteome reveals the response to aneuploidy in human cells. Mol Syst Biol 8:608

    Article  PubMed  PubMed Central  Google Scholar 

  7. Dephoure N, Hwang S, O’Sullivan C et al (2014) Quantitative proteomic analysis reveals posttranslational responses to aneuploidy in yeast. Elife 3, e03023

    Article  PubMed  PubMed Central  Google Scholar 

  8. Wu Y, Williams E, Dubuis S et al (2014) Multilayered genetic and omics dissection of mitochondrial activity in a mouse reference population. Cell 158:1415–1430

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Zhang B, Wang J, Wang X et al (2014) Proteogenomic characterization of human colon and rectal cancer. Nature 513:382–387

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Gygi S, Rist B, Gerber S et al (1999) Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat Biotechnol 17:994–999

    Article  CAS  PubMed  Google Scholar 

  11. Ong S, Blagoev B, Kratchmarova I et al (2002) Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics 1:376–386

    Article  CAS  PubMed  Google Scholar 

  12. Syka J, Marto J, Bai D et al (2004) Novel linear quadrupole ion trap/FT mass spectrometer: performance characterization and use in the comparative analysis of histone H3 post-translational modification. J Proteome Res 3:621–626

    Article  CAS  PubMed  Google Scholar 

  13. Olsen J, de Godoy L, Li G et al (2005) Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass injection into a C-trap. Mol Cell Proteomics 12:2010–2021

    Article  Google Scholar 

  14. Hsu J, Huang S, Chow N et al (2003) Stable-isotope dimethyl labeling for quantitative proteomics. Anal Chem 75:6843–6852

    Article  CAS  PubMed  Google Scholar 

  15. Wilson-Grady J, Haas W, Gygi S (2013) Quantitative comparison of the fasted and re-fed mouse liver phosphoproteomes using lower pH reductive dimethylation. Methods 61:277–286

    Article  CAS  PubMed  Google Scholar 

  16. Wu Y, Wang F, Liu Z et al (2014) Five-plex isotope dimethyl labeling for quantitative proteomics. Chem Commun (Camb) 50:1708–1710

    Article  CAS  Google Scholar 

  17. Blagoev B, Ong S, Kratchmarova I et al (2004) Temporal analysis of phosphotyrosine-dependent signaling networks by quantitative proteomics. Nat Biotechnol 22:1139–1145

    Article  CAS  PubMed  Google Scholar 

  18. Thompson A, Schäfer J, Kuhn K et al (2003) Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS. Anal Chem 75:1895–1904

    Article  CAS  PubMed  Google Scholar 

  19. Ross P, Huang Y, Marchese J et al (2004) Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteomics 3:1154–1169

    Article  CAS  PubMed  Google Scholar 

  20. Choe L, D’Ascenzo M, Relkin N et al (2007) 8‐Plex quantitation of changes in cerebrospinal fluid protein expression in subjects undergoing intravenous immunoglobulin treatment for Alzheimer’s disease. Proteomics 7:3651–3660

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. McAlister G, Huttlin E, Haas W et al (2012) Increasing the multiplexing capacity of TMTs using reporter ion isotopologues with isobaric masses. Anal Chem 84:7469–7478

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Weekes M, Tomasec P, Huttlin E et al (2014) Quantitative temporal viromics: an approach to investigate host-pathogen interaction. Cell 157:1460–1472

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Wenger C, Lee M, Hebert A et al (2011) Gas-phase purification enables accurate, multiplexed proteome quantification with isobaric tagging. Nat Methods 8:933–935

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Ting L, Rad R, Gygi S et al (2011) MS3 eliminates ratio distortion in isobaric multiplexed quantitative proteomics. Nat Methods 8:937–940

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. McAlister G, Nusinow D, Jedrychowski M et al (2014) MultiNotch MS3 enables accurate, sensitive, and multiplexed detection of differential expression across cancer cell line proteomes. Anal Chem 86:7150–7158

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Eng J, McCormack A, Yates J (1994) An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J Am Soc Mass Spectrom 5:976–989

    Article  CAS  PubMed  Google Scholar 

  27. Peng J, Elias J, Thoreen C et al (2003) Evaluation of multidimensional chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for large-scale protein analysis: the yeast proteome. J Proteome Res 1:43–50

    Article  Google Scholar 

  28. Elias J, Gygi S (2007) Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nat Methods 4:207–214

    Article  CAS  PubMed  Google Scholar 

  29. Elias J, Gygi S (2009) Target-decoy search strategy for mass spectrometry-based proteomics. Methods Mol Biol 604:55–71

    Article  Google Scholar 

  30. Huttlin E, Jedrychowski M, Elias J et al (2010) A tissue-specific atlas of mouse protein phosphorylation and expression. Cell 143:1174–1189

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Wühr M, Haas W, McAlister G et al (2012) Accurate multiplexed proteomics at the MS2 level using the complement reporter ion cluster. Anal Chem 84:9214–9221

    Article  PubMed  PubMed Central  Google Scholar 

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Author information

Authors and Affiliations

  1. Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA, USA

    Amanda Edwards & Wilhelm Haas

  2. Department of Medicine, Harvard Medical School, Boston, MA, USA

    Amanda Edwards & Wilhelm Haas

Authors
  1. Amanda Edwards
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  2. Wilhelm Haas
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Corresponding author

Correspondence to Wilhelm Haas .

Editor information

Editors and Affiliations

  1. University of Regensburg Institute of Functional Genomics, Regensburg, Germany

    Jörg Reinders

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Edwards, A., Haas, W. (2016). Multiplexed Quantitative Proteomics for High-Throughput Comprehensive Proteome Comparisons of Human Cell Lines. In: Reinders, J. (eds) Proteomics in Systems Biology. Methods in Molecular Biology, vol 1394. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3341-9_1

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  • DOI: https://doi.org/10.1007/978-1-4939-3341-9_1

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3339-6

  • Online ISBN: 978-1-4939-3341-9

  • eBook Packages: Springer Protocols

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