Tandem Mass Tag Approach Utilizing Pervanadate BOOST Channels Delivers Deeper Quantitative Characterization of the Tyrosine Phosphoproteome

Abstract

Dynamic tyrosine phosphorylation is fundamental to a myriad of cellular processes. However, the inherently low abundance of tyrosine phosphorylation in the proteome and the inefficient enrichment of phosphotyrosine(pTyr)-containing peptides has led to poor pTyr peptide identification and quantitation, critically hindering researchers' ability to elucidate signaling pathways regulated by tyrosine phosphorylation in systems where cellular material is limited. The most popular approaches to wide-scale characterization of the tyrosine phosphoproteome use pTyr enrichment with pan-specific, anti-pTyr antibodies from a large amount of starting material. Methods that decrease the amount of starting material and increase the characterization depth of the tyrosine phosphoproteome while maintaining quantitative accuracy and precision would enable the discovery of tyrosine phosphorylation networks in rarer cell populations. To achieve these goals, the BOOST (Broad-spectrum Optimization Of Selective Triggering) method leveraging the multiplexing capability of tandem mass tags (TMT) and the use of pervanadate (PV) boost channels (cells treated with the broad-spectrum tyrosine phosphatase inhibitor PV) selectively increased the relative abundance of pTyr-containing peptides. After PV boost channels facilitated selective fragmentation of pTyr-containing peptides, TMT reporter ions delivered accurate quantitation of each peptide for the experimental samples while the quantitation from PV boost channels was ignored. This method yielded up to 6.3-fold boost in pTyr quantification depth of statistically significant data derived from contrived ratios, compared with TMT without PV boost channels or intensity-based label-free (LF) quantitation while maintaining quantitative accuracy and precision, allowing quantitation of over 2300 unique pTyr peptides from only 1 mg of T cell receptor-stimulated Jurkat T cells. The BOOST strategy can potentially be applied in analyses of other post-translational modifications where treatments that broadly elevate the levels of those modifications across the proteome are available. Dynamic tyrosine phosphorylation is fundamental to a myriad of cellular processes. However, the inherently low abundance of tyrosine phosphorylation in the proteome and the inefficient enrichment of phosphotyrosine(pTyr)-containing peptides has led to poor pTyr peptide identification and quantitation, critically hindering researchers' ability to elucidate signaling pathways regulated by tyrosine phosphorylation in systems where cellular material is limited. The most popular approaches to wide-scale characterization of the tyrosine phosphoproteome use pTyr enrichment with pan-specific, anti-pTyr antibodies from a large amount of starting material. Methods that decrease the amount of starting material and increase the characterization depth of the tyrosine phosphoproteome while maintaining quantitative accuracy and precision would enable the discovery of tyrosine phosphorylation networks in rarer cell populations. To achieve these goals, the BOOST (Broad-spectrum Optimization Of Selective Triggering) method leveraging the multiplexing capability of tandem mass tags (TMT) and the use of pervanadate (PV) boost channels (cells treated with the broad-spectrum tyrosine phosphatase inhibitor PV) selectively increased the relative abundance of pTyr-containing peptides. After PV boost channels facilitated selective fragmentation of pTyr-containing peptides, TMT reporter ions delivered accurate quantitation of each peptide for the experimental samples while the quantitation from PV boost channels was ignored. This method yielded up to 6.3-fold boost in pTyr quantification depth of statistically significant data derived from contrived ratios, compared with TMT without PV boost channels or intensity-based label-free (LF) quantitation while maintaining quantitative accuracy and precision, allowing quantitation of over 2300 unique pTyr peptides from only 1 mg of T cell receptor-stimulated Jurkat T cells. The BOOST strategy can potentially be applied in analyses of other post-translational modifications where treatments that broadly elevate the levels of those modifications across the proteome are available. Tyrosine phosphorylation is a major biochemical currency in cellular signaling that regulates vital cellular processes. Dysregulation of these processes underlies many disease states such as immune disorders and oncogenesis. As such, comprehensive mapping of the tyrosine phosphoproteome is essential to understanding the key regulatory mechanisms of these diseases, and for designing improved therapeutic strategies. Mass spectrometry (MS) has become a robust platform for wide-scale quantitative profiling of the phosphorylated tyrosine (pTyr) 1The abbreviations used are:pTyrphosphorylated tyrosineBOOSTBroad-spectrum Optimization Of Selective TriggeringMSmass spectrometryTMTtandem mass tagsPSMpeptide spectrum matchFDRfalse discovery ratePVpervanadatesSH2Src Homology 2 superbinderHPLChigh pressure liquid chromatography. 1The abbreviations used are:pTyrphosphorylated tyrosineBOOSTBroad-spectrum Optimization Of Selective TriggeringMSmass spectrometryTMTtandem mass tagsPSMpeptide spectrum matchFDRfalse discovery ratePVpervanadatesSH2Src Homology 2 superbinderHPLChigh pressure liquid chromatography. proteome, but because tyrosine phosphorylation makes up only 0.05% of the total phosphoproteome in a typical vertebrate cell (1Hunter T. Protein modification: phosphorylation on tyrosine residues.Curr. Opin. Cell Biol. 1989; 1: 1168-1181Crossref PubMed Scopus (85) Google Scholar), MS-based detection of pTyr-containing peptides remains a challenge. pTyr peptide enrichment has improved pTyr proteomics, yet the conventional approach of using pan-specific anti-pTyr antibodies such as P-Tyr-1000 suffers from limitations such as prohibitive cost and low efficiency (2van der Mijn J.C. Labots M. Piersma S.R. Pham T.V. Knol J.C. Broxterman H.J. Verheul H.M. Jimenez C.R. Evaluation of different phospho-tyrosine antibodies for label-free phosphoproteomics.J. Proteomics. 2015; 127: 259-263Crossref PubMed Scopus (31) Google Scholar). Nevertheless, more than 10,000 out of the estimated 300,000 phosphorylatable tyrosine phosphorylation sites (3von Stechow L. Francavilla C. Olsen J.V. Recent findings and technological advances in phosphoproteomics for cells and tissues.Expert Rev. Proteomics. 2015; 12: 469-487Crossref PubMed Scopus (64) Google Scholar) have been identified using the MS-based approach (4Bian Y. Li L. Dong M. Liu X. Kaneko T. Cheng K. Liu H. Voss C. Cao X. Wang Y. Litchfield D. Ye M. Li S.S. Zou H. Ultra-deep tyrosine phosphoproteomics enabled by a phosphotyrosine superbinder.Nat. Chem. Biol. 2016; 12: 959-966Crossref PubMed Scopus (87) Google Scholar). However, these results were achieved using large amounts of starting material (∼5 mg) in combination with the broad-spectrum tyrosine phosphatase inhibitor PV to inflate the total pTyr peptide abundance to non-physiological levels (3von Stechow L. Francavilla C. Olsen J.V. Recent findings and technological advances in phosphoproteomics for cells and tissues.Expert Rev. Proteomics. 2015; 12: 469-487Crossref PubMed Scopus (64) Google Scholar, 5Kettenbach A.N. Gerber S.A. Rapid and reproducible single-stage phosphopeptide enrichment of complex peptide to and phosphoproteomics Chem. PubMed Scopus Google Scholar). more samples without broad-spectrum tyrosine phosphatase using up to mg of to a of the pTyr sites identified by PV (4Bian Y. Li L. Dong M. Liu X. Kaneko T. Cheng K. Liu H. Voss C. Cao X. Wang Y. Litchfield D. Ye M. Li S.S. Zou H. Ultra-deep tyrosine phosphoproteomics enabled by a phosphotyrosine superbinder.Nat. Chem. Biol. 2016; 12: 959-966Crossref PubMed Scopus (87) Google Scholar, Y. M. T. T. and phosphotyrosine identified and phosphorylation networks in cell to PubMed Scopus Google Scholar). a method that starting material while a characterization of the tyrosine phosphoproteome with quantitative accuracy and precision is for the discovery of tyrosine phosphorylation networks in cell where material is limited. phosphorylated tyrosine Optimization Of Selective mass spectrometry tandem mass tags peptide spectrum discovery pervanadate Homology 2 pressure liquid chromatography. phosphorylated tyrosine Optimization Of Selective mass spectrometry tandem mass tags peptide spectrum discovery pervanadate Homology 2 pressure liquid chromatography. The multiplexing of TMT in with selective of pTyr peptides facilitated by PV can be to TMT and quantitation of samples K. T. C. mass a quantification strategy for of complex by Chem. PubMed Scopus Google Scholar, L. J.C. K. the multiplexing of using reporter with Chem. 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The PV and to the quantitation depth of the tyrosine the multiplexing capability of TMT and PV boost as a of the TMT channels to increase the of pTyr peptide for fragmentation PV boost channels are only used for the selective fragmentation of pTyr ions quantitative of the experimental channels can be from reporter to the and as to approach to for pTyr peptides, BOOST a quantitative characterization of the tyrosine phosphoproteome where cellular material is limited. to pTyr proteome quantitation depth have been on the enrichment of pTyr peptides (4Bian Y. Li L. Dong M. Liu X. Kaneko T. Cheng K. Liu H. Voss C. Cao X. Wang Y. Litchfield D. Ye M. Li S.S. Zou H. Ultra-deep tyrosine phosphoproteomics enabled by a phosphotyrosine superbinder.Nat. Chem. Biol. 2016; 12: 959-966Crossref PubMed Scopus (87) Google Scholar, M. Y. Wang Y. Dong Y. H. Zou H. Ye M. robust and approach for tyrosine phosphoproteome Chem. PubMed Scopus Google Scholar, Cao S.S. Li S.S. X. proteome profiling by mass Scopus Google Scholar, Y. Y. Dong M. Wang Y. L. Wang H. Dong Ye M. for the of tyrosine PubMed Scopus Google Scholar), with of total can be using method for the detection of and peptides, with than pTyr sites from mg of pTyr of total D. S.A. profiling using and phosphorylation PubMed Scopus Google Scholar). wide-scale pTyr on the of pan-specific anti-pTyr such as and for pTyr peptide is more than the other enrichment was to have compared with for wide-scale pTyr (4Bian Y. Li L. Dong M. Liu X. Kaneko T. Cheng K. Liu H. Voss C. Cao X. Wang Y. Litchfield D. Ye M. Li S.S. Zou H. Ultra-deep tyrosine phosphoproteomics enabled by a phosphotyrosine superbinder.Nat. Chem. Biol. 2016; 12: 959-966Crossref PubMed Scopus (87) Google Scholar). The most is as the for wide-scale pTyr because was to other enrichment (2van der Mijn J.C. Labots M. Piersma S.R. Pham T.V. Knol J.C. Broxterman H.J. Verheul H.M. Jimenez C.R. Evaluation of different phospho-tyrosine antibodies for label-free phosphoproteomics.J. Proteomics. 2015; 127: 259-263Crossref PubMed Scopus (31) Google Scholar). 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M. K. K. Olsen J.V. of cells in PubMed Scopus Google Scholar, Y. 2 of tyrosine a signaling of Proteomics. 2015; PubMed Scopus Google Scholar, Y. M. T. phosphotyrosine by of phosphotyrosine enrichment and PubMed Scopus Google Scholar, M. profiling of Proteomics. PubMed Scopus Google Scholar). cellular material is the amount for pTyr enrichment has to be to pTyr quantitation than pTyr peptides from mg of Jurkat using a approach Y. Wang Y. Wang Liu X. Liu Li Y. Y. Ye M. approach for of tyrosine PubMed Scopus Google Scholar, M. Y. Wang Y. Dong Y. H. Zou H. Ye M. robust and approach for tyrosine phosphoproteome Chem. PubMed Scopus Google Scholar), identified only pTyr sites from mg of Jurkat (4Bian Y. Li L. Dong M. Liu X. Kaneko T. Cheng K. Liu H. Voss C. Cao X. Wang Y. Litchfield D. Ye M. Li S.S. Zou H. Ultra-deep tyrosine phosphoproteomics enabled by a phosphotyrosine superbinder.Nat. Chem. Biol. 2016; 12: 959-966Crossref PubMed Scopus (87) Google Scholar). the BOOST approach in more than 2300 pTyr peptides from only 1 mg of Jurkat of the of boost channels a with quantitative characterization of boost to the of delivered by PV boost channels as a of reporter the of TMT quantitation, boost by the total reporter in PV boost channels in to that of the experimental channels is that peptides were with boost and were derived from experimental reporter of the reporter from the experimental channels that the of PV boost channels facilitated the selective fragmentation of pTyr ions that would be the detection of quantitation the of PV boost most of the peptides were or of of boost on the for and with the BOOST approach is the that pTyr peptides from PV boost channels to inflate because of in the TMT The reporter for each TMT by the were used as in to for in these the of reporter was in a TMT The of of large data to of Proteomics. 12: PubMed Scopus Google Scholar), of channels PV the channels were a of peptide in those channels would be However, peptide across and the those the boost channels Nevertheless, to in the TMT channels as the boost channels to the of the was to have a more than the The of of large data to of Proteomics. 12: PubMed Scopus Google Scholar). that boost channels were in of the of TMT experimental channels the to a boost be by of the amount of material in the boost as as accuracy is The boost and the of in pTyr quantitation depth across different applied to samples or cell the use of boost channels from a different because of the of samples or to samples with PV where is to cellular material for PV cell of the and cell potentially be used as the boost pTyr the use of cells for PV as the boost such experimental to be with to the of the amount of cellular material in the experimental and boost channels and the of samples in TMT as pTyr enrichment and the of channels to over the experimental would on the experimental and of cellular material for the boost a for such experimental using the Jurkat and a boost to in experimental The use of PV boost channels in a is to the in a where cells facilitated the detection of channels mass spectrometry of cells proteome cell Biol. 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