Characterization of Phosphorylated Proteins Using Mass Spectrometry

Author(s): Li-Rong Yu*, Timothy D. Veenstra

Journal Name: Current Protein & Peptide Science

Volume 22 , Issue 2 , 2021

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Graphical Abstract:


Phosphorylation is arguably the most important post-translational modification that occurs within proteins. Phosphorylation is used as a signal to control numerous physiological activities ranging from gene expression to metabolism. Identifying phosphorylation sites within proteins was historically a challenge as it required either radioisotope labeling or the use of phospho-specific antibodies. The advent of mass spectrometry (MS) has had a major impact on the ability to qualitatively and quantitatively characterize phosphorylated proteins. In this article, we describe MS methods for characterizing phosphorylation sites within individual proteins as well as entire proteome samples. The utility of these methods is illustrated in examples that show the information that can be gained using these MS techniques.

Keywords: Phosphorylation, mass spectrometry, peptide mapping, tandem mass spectrometry (MS2), phosphoproteomics, immobilized metal affinity chromatography (IMAC), metal oxide affinity chromatography (MOAC).

Wang, Y.; Wang, L.; Gong, Z. Regulation of acetylation in high mobility group protein B1 cytosol translocation. DNA Cell Biol., 2019, 38(5), 491-499.
[] [PMID: 30874449]
Mestermann, K.; Giavridis, T.; Weber, J.; Rydzek, J.; Frenz, S.; Nerreter, T.; Mades, A.; Sadelain, M.; Einsele, H.; Hudecek, M. The tyrosine kinase inhibitor dasatinib acts as a pharmacologic on/off switch for CAR T cells. Sci. Transl. Med., 2019, 11(499), 1-11.
[] [PMID: 31270272]
Belman, J.P.; Habtemichael, E.N.; Bogan, J.S. A proteolytic pathway that controls glucose uptake in fat and muscle. Rev. Endocr. Metab. Disord., 2014, 15(1), 55-66.
[] [PMID: 24114239]
Salzberg, S.L. Open questions: How many genes do we have? BMC Biol., 2018, 16(1), 94.
[] [PMID: 30124169]
Smith, L.M.; Kelleher, N.L. Consortium for Top Down Proteomics. Proteoform: a single term describing protein complexity. Nat. Methods, 2013, 10(3), 186-187.
[] [PMID: 23443629]
Vanacore, R.; Ham, A.J.; Voehler, M.; Sanders, C.R.; Conrads, T.P.; Veenstra, T.D.; Sharpless, K.B.; Dawson, P.E.; Hudson, B.G. A sulfilimine bond identified in collagen IV. Science, 2009, 325(5945), 1230-1234.
[] [PMID: 19729652]
Garavelli, J.S. The RESID Database of Protein Modifications as a resource and annotation tool. Proteomics, 2004, 4(6), 1527-1533.
[] [PMID: 15174122]
Yakubu, R.R.; Nieves, E.; Weiss, L.M. The methods employed in mass spectrometric analysis of posttranslational modifications (PTMs) and protein-protein interactions (PPIs). Adv. Exp. Med. Biol., 2019, 1140, 169-198.
[] [PMID: 31347048]
Available from:
Manning, D.R.; DiSalvo, J.; Stull, J.T. Protein phosphorylation: quantitative analysis in vivo and in intact cell systems. Mol. Cell. Endocrinol., 1980, 19(1), 1-19.
[] [PMID: 6248400]
Dzandu, J.K.; Johnson, R.M. Membrane protein phosphorylation in intact normal and sickle cell erythrocytes. J. Biol. Chem., 1980, 255(13), 6382-6386.
[PMID: 7391025]
Loyet, K.M.; Stults, J.T.; Arnott, D. Mass spectrometric contributions to the practice of phosphorylation site mapping through 2003: a literature review. Mol. Cell. Proteomics, 2005, 4(3), 235-245.
[] [PMID: 15640519]
Zhou, M.; Meng, Z.; Jobson, A.G.; Pommier, Y.; Veenstra, T.D. Detection of in vitro kinase generated protein phosphorylation sites using gamma[18O4]-ATP and mass spectrometry. Anal. Chem., 2007, 79(20), 7603-7610.
[] [PMID: 17877366]
Goshe, M.B.; Veenstra, T.D.; Panisko, E.A.; Conrads, T.P.; Angell, N.H.; Smith, R.D. Phosphoprotein isotope-coded affinity tags: application to the enrichment and identification of low-abundance phosphoproteins. Anal. Chem., 2002, 74(3), 607-616.
[] [PMID: 11838682]
Molden, R.C.; Goya, J.; Khan, Z.; Garcia, B.A. Stable isotope labeling of phosphoproteins for large-scale phosphorylation rate determination. Mol. Cell. Proteomics, 2014, 13(4), 1106-1118.
[] [PMID: 24532841]
Ducret, A.; Desponts, C.; Desmarais, S.; Gresser, M.J.; Ramachandran, C. A general method for the rapid characterization of tyrosine-phosphorylated proteins by mini two-dimensional gel electrophoresis. Electrophoresis, 2000, 21(11), 2196-2208.
[<2196::AID-ELPS2196>3.0.CO;2-Z] [PMID: 10892730]
Wulfkuhle, J.D.; Aquino, J.A.; Calvert, V.S.; Fishman, D.A.; Coukos, G.; Liotta, L.A.; Petricoin, E.F., III Signal pathway profiling of ovarian cancer from human tissue specimens using reverse-phase protein microarrays. Proteomics, 2003, 3(11), 2085-2090.
[] [PMID: 14595806]
Ponnam, S.; Sevrieva, I.; Sun, Y.B.; Irving, M.; Kampourakis, T. Site-specific phosphorylation of myosin binding protein-C coordinates thin and thick filament activation in cardiac muscle. Proc. Natl. Acad. Sci. USA, 2019, 116(31), 15485-15494.
[] [PMID: 31308242]
Zheng, Y.; Huang, X.; Kelleher, N.L. Epiproteomics: quantitative analysis of histone marks and codes by mass spectrometry. Curr. Opin. Chem. Biol., 2016, 33, 142-150.
[] [PMID: 27371874]
Aslebagh, R.; Wormwood, K.L.; Channaveerappa, D.; Wetie, A.G.N.; Woods, A.G.; Darie, C.C. Identification of posttranslational modifications (PTMs) of proteins by mass spectrometry. Adv. Exp. Med. Biol., 2019, 1140, 199-224.
[] [PMID: 31347049]
Wierenga, S.K.; Zocher, M.J.; Mirus, M.M.; Conrads, T.P.; Goshe, M.B.; Veenstra, T.D. A method to evaluate tryptic digestion efficiency for high-throughput proteome analyses. Rapid Commun. Mass Spectrom., 2002, 16(14), 1404-1408.
[] [PMID: 12112621]
Tabb, D.L.; Huang, Y.; Wysocki, V.H.; Yates, J.R., III Influence of basic residue content on fragment ion peak intensities in low-energy collision-induced dissociation spectra of peptides. Anal. Chem., 2004, 76(5), 1243-1248.
[] [PMID: 14987077]
Areces, L.B.; Matafora, V.; Bachi, A. Analysis of protein phosphorylation by mass spectrometry. Eur. J. Mass. Spectrom. (Chichester), 2004, 10(3), 383-392.
[] [PMID: 15187297]
Dephoure, N.; Gould, K.L.; Gygi, S.P.; Kellogg, D.R. Mapping and analysis of phosphorylation sites: a quick guide for cell biologists. Mol. Biol. Cell, 2013, 24(5), 535-542.
[] [PMID: 23447708]
Palumbo, A.M.; Smith, S.A.; Kalcic, C.L.; Dantus, M.; Stemmer, P.M.; Reid, G.E. Tandem mass spectrometry strategies for phosphoproteome analysis. Mass Spectrom. Rev., 2011, 30(4), 600-625.
[] [PMID: 21294150]
Nolting, D.; Malek, R.; Makarov, A. Ion traps in modern mass spectrometry. Mass Spectrom. Rev., 2019, 38(2), 150-168.
[] [PMID: 29084367]
Berberich, M.J.; Paulo, J.A.; Everley, R.A. MS3-IDQ: Utilizing MS3 spectra beyond quantification yields increased coverage of the phosphoproteome in isobaric tag experiments. J. Proteome Res., 2018, 17(4), 1741-1747.
[] [PMID: 29461835]
Yu, L.R.; Zhu, Z.; Chan, K.C.; Issaq, H.J.; Dimitrov, D.S.; Veenstra, T.D. Improved titanium dioxide enrichment of phosphopeptides from HeLa cells and high confident phosphopeptide identification by cross-validation of MS/MS and MS/MS/MS spectra. J. Proteome Res., 2007, 6(11), 4150-4162.
[] [PMID: 17924679]
Sarbu, M.; Ghiulai, R.M.; Zamfir, A.D. Recent developments and applications of electron transfer dissociation mass spectrometry in proteomics. Amino Acids, 2014, 46(7), 1625-1634.
[] [PMID: 24687149]
Syka, J.E.; Coon, J.J.; Schroeder, M.J.; Shabanowitz, J.; Hunt, D.F. Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry. Proc. Natl. Acad. Sci. USA, 2004, 101(26), 9528-9533.
[] [PMID: 15210983]
Zubarev, R.A. Electron-capture dissociation tandem mass spectrometry. Curr. Opin. Biotechnol., 2004, 15(1), 12-16.
[] [PMID: 15102460]
Mikesh, L.M.; Ueberheide, B.; Chi, A.; Coon, J.J.; Syka, J.E.; Shabanowitz, J.; Hunt, D.F. The utility of ETD mass spectrometry in proteomic analysis. Biochim. Biophys. Acta, 2006, 1764(12), 1811-1822.
[] [PMID: 17118725]
Hevér, H.; Darula, Z.; Medzihradszky, K.F. Characterization of site-specific N-glycosylation. Methods Mol. Biol., 2019, 1934, 93-125.
[] [PMID: 31256376]
Drexler, H.C.A.; Vockel, M.; Polaschegg, C.; Frye, M.; Peters, K.; Vestweber, D. Vascular endothelial receptor tyrosine phosphatase: identification of novel substrates related to junctions and a ternary complex with EPHB4 and TIE2. Mol. Cell. Proteomics, 2019, 18(10), 2058-2077.
[] [PMID: 31427368]
Pinto, S.M.; Nirujogi, R.S.; Rojas, P.L.; Patil, A.H.; Manda, S.S.; Subbannayya, Y.; Roa, J.C.; Chatterjee, A.; Prasad, T.S.; Pandey, A. Quantitative phosphoproteomic analysis of IL-33-mediated signaling. Proteomics, 2015, 15(2-3), 532-544.
[] [PMID: 25367039]
Beausoleil, S.A.; Jedrychowski, M.; Schwartz, D.; Elias, J.E.; Villén, J.; Li, J.; Cohn, M.A.; Cantley, L.C.; Gygi, S.P. Large-scale characterization of HeLa cell nuclear phosphoproteins. Proc. Natl. Acad. Sci. USA, 2004, 101(33), 12130-12135.
[] [PMID: 15302935]
Thingholm, T.E.; Jensen, O.N. Enrichment and characterization of phosphopeptides by immobilized metal affinity chromatography (IMAC) and mass spectrometry. Methods Mol. Biol., 2009, 527, 47-56, xi.
[] [PMID: 19241004]
Yu, L.R.; Veenstra, T. Phosphopeptide enrichment using offline titanium dioxide columns for phosphoproteomics. Methods Mol. Biol., 2013, 1002, 93-103.
[] [PMID: 23625397]
Ficarro, S.B.; McCleland, M.L.; Stukenberg, P.T.; Burke, D.J.; Ross, M.M.; Shabanowitz, J.; Hunt, D.F.; White, F.M. Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nat. Biotechnol., 2002, 20(3), 301-305.
[] [PMID: 11875433]
Liu, H.; Yang, T.; Dai, J.; Zhu, J.; Li, X.; Wen, R.; Yang, X. Hydrophilic modification of titania nanomaterials as a biofunctional adsorbent for selective enrichment of phosphopeptides. Analyst (Lond.), 2015, 140(19), 6652-6659.
[] [PMID: 26299437]
Wang, S.T.; Wang, M.Y.; Su, X.; Yuan, B.F.; Feng, Y.Q. Facile preparation of SiO2/TiO2 composite monolithic capillary column and its application in enrichment of phosphopeptides. Anal. Chem., 2012, 84(18), 7763-7770.
[] [PMID: 22900475]
Beltran, L.; Cutillas, P.R. Advances in phosphopeptide enrichment techniques for phosphoproteomics. Amino Acids, 2012, 43(3), 1009-1024.
[] [PMID: 22821267]
Yang, D.S.; Ding, X.Y.; Min, H.P.; Li, B.; Su, M.X.; Niu, M.M.; Di, B.; Yan, F. Design and synthesis of an immobilized metal affinity chromatography and metal oxide affinity chromatography hybrid material for improved phosphopeptide enrichment. J. Chromatogr. A, 2017, 1505, 56-62.
[] [PMID: 28533032]

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Article Details

Year: 2021
Published on: 23 November, 2020
Page: [148 - 157]
Pages: 10
DOI: 10.2174/1389203721999201123200439
Price: $65

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