Abstract
Isobaric tagging reagents have become an invaluable tool for multiplexed quantitative proteomic analysis. These reagents can label multiple, distinct peptide samples from virtually any source material (e.g., tissue, cell line, purified proteins), allowing users the opportunity to assess changes in peptide abundances across many different time points or experimental conditions. Here, we describe the application of isobaric peptide labeling, specifically 8plex isobaric tags for relative and absolute quantitation (8plex iTRAQ), for quantitative phosphoproteomic analysis of cultured cells or tissue suspensions. For this particular protocol, labeled samples are pooled, fractionated by strong cation exchange chromatography, enriched for phosphopeptides, and analyzed by tandem mass spectrometry (LC-MS/MS) for both peptide identification and quantitation.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Nuwaysir LM, Stults JT (1993) Electrospray ionization mass spectrometry of phosphopeptides isolated by on-line immobilized metal-ion affinity chromatography. J Am Soc Mass Spectrom 4(8):662–669
Kange R, Selditz U, Granberg M, Lindberg U, Ekstrand G, Ek B, Gustafsson M (2005) Comparison of different IMAC techniques used for enrichment of phosphorylated peptides. J Biomol Tech 16(2):91–103
Thingholm TE, Jensen ON (2009) Enrichment and characterization of phosphopeptides by immobilized metal affinity chromatography (IMAC) and mass spectrometry. Methods Mol Biol 527:47–56, Xi
Larsen MR, Thingholm TE, Jensen ON, Roepstorff P, Jorgensen TJ (2005) Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol Cell Proteomics 4(7):873–886
Klemm C, Otto S, Wolf C, Haseloff RF, Beyermann M, Krause E (2006) Evaluation of the titanium dioxide approach for MS analysis of phosphopeptides. J Mass Spectrom 41(12):1623–1632
Schlosser A, Pipkorn R, Bossemeyer D, Lehmann WD (2001) Analysis of protein phosphorylation by a combination of elastase digestion and neutral loss tandem mass spectrometry. Anal Chem 73(2):170–176
Bateman RH, Carruthers R, Hoyes JB, Jones C, Langridge JI, Millar A, Vissers JP (2002) A novel precursor ion discovery method on a hybrid quadrupole orthogonal acceleration time-of-flight (Q-TOF) mass spectrometer for studying protein phosphorylation. J Am Soc Mass Spectrom 13(7):792–803
Schroeder MJ, Shabanowitz J, Schwartz JC, Hunt DF, Coon JJ (2004) A neutral loss activation method for improved phosphopeptide sequence analysis by quadrupole ion trap mass spectrometry. Anal Chem 76(13):3590–3598
Stensballe A, Jensen ON, Olsen JV, Haselmann KF, Zubarev RA (2000) Electron capture dissociation of singly and multiply phosphorylated peptides. Rapid Commun Mass Spectrom 14(19):1793–1800
Molina H, Horn DM, Tang N, Mathivanan S, Pandey A (2007) Global proteomic profiling of phosphopeptides using electron transfer dissociation tandem mass spectrometry. Proc Natl Acad Sci U S A 104(7):2199–2204
Jedrychowski MP, Huttlin EL, Haas W, Sowa ME, Rad R, Gygi SP (2011) Evaluation of HCD- and CID-type fragmentation within their respective detection platforms for murine phosphoproteomics. Mol Cell Proteomics 10(12):M111
Altelaar AF, Frese CK, Preisinger C, Hennrich ML, Schram AW, Timmers HT, Heck AJ, Mohammed S (2013) Benchmarking stable isotope labeling based quantitative proteomics. J Proteomics 88:14–26
Dephoure N, Gygi SP (2012) Hyperplexing: a method for higher-order multiplexed quantitative proteomics provides a map of the dynamic response to rapamycin in yeast. Sci Signal 5(217):rs2
Everley RA, Kunz RC, McAllister FE, Gygi SP (2013) Increasing throughput in targeted proteomics assays: 54-plex quantitation in a single mass spectrometry run. Anal Chem 85(11):5340–5346
Thompson A, Schafer J, Kuhn K, Kienle S, Schwarz J, Schmidt G, Neumann T, Johnstone R, Mohammed AK, Hamon C (2003) Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS. Anal Chem 75(8):1895–1904
Ross PL, Huang YN, Marchese JN, Williamson B, Parker K, Hattan S, Khainovski N, Pillai S, Dey S, Daniels S, Purkayastha S, Juhasz P, Martin S, Bartlet-Jones M, He F, Jacobson A, Pappin DJ (2004) Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteomics 3(12):1154–1169
Pichler P, Kocher T, Holzmann J, Mazanek M, Taus T, Ammerer G, Mechtler K (2010) Peptide labeling with isobaric tags yields higher identification rates using iTRAQ 4-plex compared to TMT 6-plex and iTRAQ 8-plex on LTQ Orbitrap. Anal Chem 82(15):6549–6558
Pottiez G, Wiederin J, Fox HS, Ciborowski P (2012) Comparison of 4-plex to 8-plex iTRAQ quantitative measurements of proteins in human plasma samples. J Proteome Res 11(7):3774–3781
Wenger CD, Lee MV, Hebert AS, McAlister GC, Phanstiel DH, Westphall MS, Coon JJ (2011) Gas-phase purification enables accurate, multiplexed proteome quantification with isobaric tagging. Nat Methods 8(11):933–935
Ting L, Rad R, Gygi SP, Haas W (2011) MS3 eliminates ratio distortion in isobaric multiplexed quantitative proteomics. Nat Methods 3(11):937–940
Viner R, Bomgarden R, Blank M, Rogers J (2013) Increasing the Multiplexing of Protein Quantitation from 6- to 10-Plex with Reporter Ion Isotopologues. Thermo Scientific Poster Note PN ASMS13, W617
Hoffert JD, Pisitkun T, Saeed F, Song JH, Chou CL, Knepper MA (2012) Dynamics of the G protein-coupled vasopressin V2 receptor signaling network revealed by quantitative phosphoproteomics. Mol Cell Proteomics 11(2):M111
Diedrich JK, Pinto AF, Yates JR III (2013) Energy dependence of HCD on peptide fragmentation: stepped collisional energy finds the sweet spot. J Am Soc Mass Spectrom 24(11):1690–1699
Saeed F, Pisitkun T, Hoffert JD, Wang G, Gucek M, Knepper MA (2012) An efficient dynamic programming algorithm for phosphorylation site assignment of large-scale mass spectrometry data. Proceedings (IEEE Int Conf Bioinformatics Biomed), pp 618–625
Beausoleil SA, Villen J, Gerber SA, Rush J, Gygi SP (2006) A probability-based approach for high-throughput protein phosphorylation analysis and site localization. Nat Biotechnol 24(10):1285–1292
Slotta DJ, McFarland MA, Markey SP (2010) MassSieve: panning MS/MS peptide data for proteins. Proteomics 10(16):3035–3039
Tchapyjnikov D, Li Y, Pisitkun T, Hoffert JD, Yu MJ, Knepper MA (2010) Proteomic profiling of nuclei from native renal inner medullary collecting duct cells using LC-MS/MS. Physiol Genomics 40(3):167–183
Benjamini Y, Hochberg Y (1995) Controlling the false discovery rate: a practical and powerful approach to multiple testing. J Roy Statist Soc Ser B 57:289–300
Acknowledgments
This work was supported by the Intramural Programs of the National Heart, Lung, and Blood Institute (Project Z01-HL-001285) and by the National Research University Project, Office of Higher Education Commission (WCU-006-HR-57).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Cheng, L., Pisitkun, T., Knepper, M.A., Hoffert, J.D. (2016). Peptide Labeling Using Isobaric Tagging Reagents for Quantitative Phosphoproteomics. In: von Stechow, L. (eds) Phospho-Proteomics. Methods in Molecular Biology, vol 1355. Springer, New York, NY. https://doi.org/10.1007/978-1-4939-3049-4_4
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3049-4_4
Publisher Name: Springer, New York, NY
Print ISBN: 978-1-4939-3048-7
Online ISBN: 978-1-4939-3049-4
eBook Packages: Springer Protocols