Skip to main content
SpringerLink
Log in
Book cover

Shotgun Proteomics pp 247–257Cite as

Mass Spectrometry-Based Proteomics for Analysis of Hydrophilic Phosphopeptides

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2259))

Abstract

Protein phosphorylation is a critical posttranslational modification (PTM), with cell signaling networks being tightly regulated by protein phosphorylation. Despite recent technological advances in reversed-phase liquid chromatography (RPLC)-mass spectrometry (MS)-based proteomics, comprehensive phosphoproteomic coverage in complex biological systems remains challenging, especially for hydrophilic phosphopeptides that often have multiple phosphorylation sites. Herein, we describe an MS-based phosphoproteomics protocol for effective quantitative analysis of hydrophilic phosphopeptides. This protocol was built upon a simple tandem mass tag (TMT)-labeling method for significantly increasing peptide hydrophobicity, thus effectively enhancing RPLC-MS analysis of hydrophilic peptides. Through phosphoproteomic analyses of MCF7 cells, this method was demonstrated to greatly increase the number of identified hydrophilic phosphopeptides and improve MS signal detection. With the TMT labeling method, we were able to identify a previously unreported phosphopeptide from the G protein-coupled receptor (GPCR) CXCR3, QPpSSSR, which is thought to be important in regulating receptor signaling. This protocol is easy to adopt and implement and thus should have broad utility for effective RPLC-MS analysis of the hydrophilic phosphoproteome as well as other highly hydrophilic analytes.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   99.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   129.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Graves JD, Krebs EG (1999) Protein phosphorylation and signal transduction. Pharmacol Ther 82:111–121

    Article  CAS  Google Scholar 

  2. Day EK, Sosale NG, Lazzara MJ (2016) Cell signaling regulation by protein phosphorylation: a multivariate, heterogeneous, and context-dependent process. Curr Opin Biotech 40:185–192

    Article  CAS  Google Scholar 

  3. Dermit M, Dokal A, Cutillas PR (2017) Approaches to identify kinase dependencies in cancer signalling networks. FEBS Lett 591:2577–2592

    Article  CAS  Google Scholar 

  4. Ardito F, Giuliani M, Perrone D, Troiano G, Lo Muzio L (2017) The crucial role of protein phosphorylation in cell signaling and its use as targeted therapy. Int J Mol Med 40:271–280

    Article  CAS  Google Scholar 

  5. Riley NM, Coon JJ (2016) Phosphoproteomics in the age of rapid and deep proteome profiling. Anal Chem 88:74–94

    Article  CAS  Google Scholar 

  6. Tsai CF, Wang YT, Yen HY, Tsou CC, Ku WC, Lin PY, Chen HY, Nesvizhskii AI, Ishihama Y, Chen YJ (2015) Large-scale determination of absolute phosphorylation stoichiometries in human cells by motif-targeting quantitative proteomics. Nat Commun 6:6622

    Article  CAS  Google Scholar 

  7. Humphrey SJ, Azimifar SB, Mann M (2015) High-throughput phosphoproteomics reveals in vivo insulin signaling dynamics. Nat Biotechnol 33:990–995

    Article  CAS  Google Scholar 

  8. Bekker-Jensen DB, Kelstrup CD, Batth TS, Larsen SC, Haldrup C, Bramsen JB, Sorensen KD, Hoyer S, Orntoft TF, Andersen CL, Nielsen ML, Olsen JV (2017) An optimized shotgun strategy for the rapid generation of comprehensive human proteomes. Cell Syst 4:587–599 e584

    Article  CAS  Google Scholar 

  9. Wakabayashi M, Kyono Y, Sugiyama N, Ishihama Y (2015) Extended coverage of singly and multiply phosphorylated peptides from a single titanium dioxide microcolumn. Anal Chem 87:10213–10221

    Article  CAS  Google Scholar 

  10. Mertins P, Tang LC, Krug K, Clark DJ, Gritsenko MA, Chen L, Clauser KR, Clauss TR, Shah P, Gillette MA, Petyuk VA, Thomas SN, Mani DR, Mundt F, Moore RJ, Hu Y, Zhao R, Schnaubelt M, Keshishian H, Monroe ME, Zhang Z, Udeshi ND, Mani D, Davies SR, Townsend RR, Chan DW, Smith RD, Zhang H, Liu T, Carr SA (2018) Reproducible workflow for multiplexed deep-scale proteome and phosphoproteome analysis of tumor tissues by liquid chromatography-mass spectrometry. Nat Protoc 13:1632–1661

    Article  CAS  Google Scholar 

  11. Hogrebe A, von Stechow L, Bekker-Jensen DB, Weinert BT, Kelstrup CD, Olsen JV (2018) Benchmarking common quantification strategies for large-scale phosphoproteomics. Nat Commun 9:1045

    Article  Google Scholar 

  12. Yi L, Tsai CF, Dirice E, Swensen AC, Chen J, Shi T, Gritsenko MA, Chu RK, Piehowski PD, Smith RD, Rodland KD, Atkinson MA, Mathews CE, Kulkarni RN, Liu T, Qian WJ (2019) A boosting to amplify signal with isobaric labeling (BASIL) strategy for comprehensive quantitative phosphoproteomic characterization of small populations of cells. Anal Chem 91:5794–5801

    Article  CAS  Google Scholar 

  13. Giansanti P, Aye TT, van den Toorn H, Peng M, van Breukelen B, Heck AJ (2015) An augmented multiple-protease-based human phosphopeptide atlas. Cell Rep 11:1834–1843

    Article  CAS  Google Scholar 

  14. Olsen JV, Ong SE, Mann M (2004) Trypsin cleaves exclusively C-terminal to arginine and lysine residues. Mol Cell Proteomics 3:608–614

    Article  CAS  Google Scholar 

  15. Tsai CF, Smith JS, Krajewski K, Zhao R, Moghieb AM, Nicora CD, Xiong X, Moore RJ, Liu T, Smith RD, Jacobs JM, Rajagopal S, Shi T (2019) Tandem mass tag labeling facilitates reversed-phase liquid chromatography-mass spectrometry analysis of hydrophilic phosphopeptides. Anal Chem 91:11606–11613

    Article  CAS  Google Scholar 

  16. Cox J, Mann M (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26:1367–1372

    Article  CAS  Google Scholar 

  17. Tyanova S, Temu T, Cox J (2016) The MaxQuant computational platform for mass spectrometry-based shotgun proteomics. Nat Protoc 11:2301–2319

    Article  CAS  Google Scholar 

  18. Zecha J, Satpathy S, Kanashova T, Avanessian SC, Kane MH, Clauser KR, Mertins P, Carr SA, Kuster B (2019) TMT labeling for the masses: a robust and cost-efficient, in-solution labeling approach. Mol Cell Proteomics 18:1468–1478

    Article  CAS  Google Scholar 

Download references

Acknowledgments

Portions of the research were supported by P41GM103493 (R.D.S), R21CA223715 (T.S.), T32GM7171 (J.S.S.), the Duke Medical Scientist Training Program (J.S.S.), R01GM122798 (S.R.), and Burroughs Wellcome Career Award for Medical Scientists (S.R.).

The experimental work described herein was performed in the Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, a national scientific user facility sponsored by the United States of America Department of Energy under Contract DE-AC05-76RL0 1830.

Author information

Authors and Affiliations

  1. Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA

    Chia-Feng Tsai, Kendall Martin, Tao Liu, Richard D. Smith, Tujin Shi & Jon M. Jacobs

  2. Department of Biochemistry, Duke University, Durham, NC, USA

    Jeffrey S. Smith, Dylan S. Eiger & Sudarshan Rajagopal

  3. Department of Medicine, Duke University, Durham, NC, USA

    Jeffrey S. Smith, Dylan S. Eiger & Sudarshan Rajagopal

Authors
  1. Chia-Feng Tsai
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jeffrey S. Smith
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Dylan S. Eiger
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kendall Martin
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Tao Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Richard D. Smith
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Tujin Shi
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Sudarshan Rajagopal
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Jon M. Jacobs
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jon M. Jacobs .

Editor information

Editors and Affiliations

  1. Department of Food Technology, Institute of Marine Research (IIM), Spanish National Research Council (CSIC), Vigo, Spain

    Mónica Carrera

  2. Department of Food Technology, Institute of Marine Research (IIM), Spanish National Research Council (CSIC), Vigo, Spain

    Jesús Mateos

Rights and permissions

Reprints and permissions

Copyright information

© 2021 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Tsai, CF. et al. (2021). Mass Spectrometry-Based Proteomics for Analysis of Hydrophilic Phosphopeptides. In: Carrera, M., Mateos, J. (eds) Shotgun Proteomics. Methods in Molecular Biology, vol 2259. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1178-4_16

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-1178-4_16

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1177-7

  • Online ISBN: 978-1-0716-1178-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation