In depth qualitative and quantitative profiling of tyrosine phosphorylation using a combination of phosphopeptide immuno-affinity purification and stable isotope dimethyl labeling

P.J. Boersema; L.Y. Foong; V.M. Ding; S. Lemeer; B. van Breukelen; R. Philp; J. Boekhorst; B. Snel; J. den Hertog; A.B. Choo; A.J.R. Heck

doi:10.1074/mcp.M900291-MCP200

In depth qualitative and quantitative profiling of tyrosine phosphorylation using a combination of phosphopeptide immuno-affinity purification and stable isotope dimethyl labeling

P.J. Boersema, L.Y. Foong, V.M. Ding, S. Lemeer, B. van Breukelen, R. Philp, J. Boekhorst, B. Snel, J. den Hertog, A.B. Choo, A.J.R. Heck

Hubrecht Institute

Research output: Contribution to journal/periodical › Article › Scientific › peer-review

151 Citations (Scopus)

Abstract

Several mass spectrometry based assays have emerged for the quantitative profiling of cellular tyrosine phosphorylation. Ideally, these methods should reveal the exact sites of tyrosine phosphorylation, be quantitative and not cost-prohibitive. The latter is often an issue as typically several milligrams of (stable isotope labeled) starting protein material is required to enable the detection of low abundant phosphotyrosine peptides. Here, we adopted and refined a peptide centric immuno-affinity purification approach for the quantitative analysis of tyrosine phosphorylation by combining it with a cost-effective stable isotope dimethyl labeling method. We were able to identify by mass spectrometry, using just two LC-MS/MS runs, more than 1100 unique non-redundant phosphopeptides in HeLa cells from about 4 mg of starting material without requiring any further affinity enrichment as close to 80% of the identified peptides were tyrosine phosphorylated peptides. Stable isotope dimethyl labeling could be incorporated prior to the immuno-affinity purification, even for the used large quantities (mg) of peptide material, enabling the quantification of differences in tyrosine phosphorylation upon pervanadate treatment or EGF stimulation. Analysis of the EGF-stimulated HeLa cells, a frequently used model system for tyrosine phosphorylation, resulted in the quantification of 73 regulated unique phosphotyrosine peptides. The quantitative data was found to be exceptionally consistent with the literature, evidencing that such a targeted quantitative phosphoproteomics approach can provide reproducible results. In general, the combination of immuno-affinity purification of tyrosine phosphorylated peptides with large scale stable isotope dimethyl labeling provides a cost-effective approach that can alleviate variation in sample preparation and analysis as samples can be combined early on. Using this approach a rather complete qualitative and quantitative picture of tyrosine phosphorylation signaling events can be generated.

Original language	English
Journal	Molecular and Cellular Proteomics
DOIs	https://doi.org/10.1074/mcp.M900291-MCP200
Publication status	Published - 2009

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10.1074/mcp.M900291-MCP200

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Boersema, P. J., Foong, L. Y., Ding, V. M., Lemeer, S., van Breukelen, B., Philp, R., Boekhorst, J., Snel, B., den Hertog, J., Choo, A. B., & Heck, A. J. R. (2009). In depth qualitative and quantitative profiling of tyrosine phosphorylation using a combination of phosphopeptide immuno-affinity purification and stable isotope dimethyl labeling. Molecular and Cellular Proteomics. https://doi.org/10.1074/mcp.M900291-MCP200

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title = "In depth qualitative and quantitative profiling of tyrosine phosphorylation using a combination of phosphopeptide immuno-affinity purification and stable isotope dimethyl labeling",

abstract = "Several mass spectrometry based assays have emerged for the quantitative profiling of cellular tyrosine phosphorylation. Ideally, these methods should reveal the exact sites of tyrosine phosphorylation, be quantitative and not cost-prohibitive. The latter is often an issue as typically several milligrams of (stable isotope labeled) starting protein material is required to enable the detection of low abundant phosphotyrosine peptides. Here, we adopted and refined a peptide centric immuno-affinity purification approach for the quantitative analysis of tyrosine phosphorylation by combining it with a cost-effective stable isotope dimethyl labeling method. We were able to identify by mass spectrometry, using just two LC-MS/MS runs, more than 1100 unique non-redundant phosphopeptides in HeLa cells from about 4 mg of starting material without requiring any further affinity enrichment as close to 80% of the identified peptides were tyrosine phosphorylated peptides. Stable isotope dimethyl labeling could be incorporated prior to the immuno-affinity purification, even for the used large quantities (mg) of peptide material, enabling the quantification of differences in tyrosine phosphorylation upon pervanadate treatment or EGF stimulation. Analysis of the EGF-stimulated HeLa cells, a frequently used model system for tyrosine phosphorylation, resulted in the quantification of 73 regulated unique phosphotyrosine peptides. The quantitative data was found to be exceptionally consistent with the literature, evidencing that such a targeted quantitative phosphoproteomics approach can provide reproducible results. In general, the combination of immuno-affinity purification of tyrosine phosphorylated peptides with large scale stable isotope dimethyl labeling provides a cost-effective approach that can alleviate variation in sample preparation and analysis as samples can be combined early on. Using this approach a rather complete qualitative and quantitative picture of tyrosine phosphorylation signaling events can be generated.",

author = "P.J. Boersema and L.Y. Foong and V.M. Ding and S. Lemeer and {van Breukelen}, B. and R. Philp and J. Boekhorst and B. Snel and {den Hertog}, J. and A.B. Choo and A.J.R. Heck",

note = "Reporting year: 2009 Metis note: M900291-mcp200",

year = "2009",

doi = "10.1074/mcp.M900291-MCP200",

language = "English",

journal = "Molecular and Cellular Proteomics",

issn = "1535-9476",

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Boersema, PJ, Foong, LY, Ding, VM, Lemeer, S, van Breukelen, B, Philp, R, Boekhorst, J, Snel, B, den Hertog, J, Choo, AB & Heck, AJR 2009, 'In depth qualitative and quantitative profiling of tyrosine phosphorylation using a combination of phosphopeptide immuno-affinity purification and stable isotope dimethyl labeling', Molecular and Cellular Proteomics. https://doi.org/10.1074/mcp.M900291-MCP200

TY - JOUR

T1 - In depth qualitative and quantitative profiling of tyrosine phosphorylation using a combination of phosphopeptide immuno-affinity purification and stable isotope dimethyl labeling

AU - Boersema, P.J.

AU - Foong, L.Y.

AU - Ding, V.M.

AU - Lemeer, S.

AU - van Breukelen, B.

AU - Philp, R.

AU - Boekhorst, J.

AU - Snel, B.

AU - den Hertog, J.

AU - Choo, A.B.

AU - Heck, A.J.R.

N1 - Reporting year: 2009 Metis note: M900291-mcp200

PY - 2009

Y1 - 2009

N2 - Several mass spectrometry based assays have emerged for the quantitative profiling of cellular tyrosine phosphorylation. Ideally, these methods should reveal the exact sites of tyrosine phosphorylation, be quantitative and not cost-prohibitive. The latter is often an issue as typically several milligrams of (stable isotope labeled) starting protein material is required to enable the detection of low abundant phosphotyrosine peptides. Here, we adopted and refined a peptide centric immuno-affinity purification approach for the quantitative analysis of tyrosine phosphorylation by combining it with a cost-effective stable isotope dimethyl labeling method. We were able to identify by mass spectrometry, using just two LC-MS/MS runs, more than 1100 unique non-redundant phosphopeptides in HeLa cells from about 4 mg of starting material without requiring any further affinity enrichment as close to 80% of the identified peptides were tyrosine phosphorylated peptides. Stable isotope dimethyl labeling could be incorporated prior to the immuno-affinity purification, even for the used large quantities (mg) of peptide material, enabling the quantification of differences in tyrosine phosphorylation upon pervanadate treatment or EGF stimulation. Analysis of the EGF-stimulated HeLa cells, a frequently used model system for tyrosine phosphorylation, resulted in the quantification of 73 regulated unique phosphotyrosine peptides. The quantitative data was found to be exceptionally consistent with the literature, evidencing that such a targeted quantitative phosphoproteomics approach can provide reproducible results. In general, the combination of immuno-affinity purification of tyrosine phosphorylated peptides with large scale stable isotope dimethyl labeling provides a cost-effective approach that can alleviate variation in sample preparation and analysis as samples can be combined early on. Using this approach a rather complete qualitative and quantitative picture of tyrosine phosphorylation signaling events can be generated.

AB - Several mass spectrometry based assays have emerged for the quantitative profiling of cellular tyrosine phosphorylation. Ideally, these methods should reveal the exact sites of tyrosine phosphorylation, be quantitative and not cost-prohibitive. The latter is often an issue as typically several milligrams of (stable isotope labeled) starting protein material is required to enable the detection of low abundant phosphotyrosine peptides. Here, we adopted and refined a peptide centric immuno-affinity purification approach for the quantitative analysis of tyrosine phosphorylation by combining it with a cost-effective stable isotope dimethyl labeling method. We were able to identify by mass spectrometry, using just two LC-MS/MS runs, more than 1100 unique non-redundant phosphopeptides in HeLa cells from about 4 mg of starting material without requiring any further affinity enrichment as close to 80% of the identified peptides were tyrosine phosphorylated peptides. Stable isotope dimethyl labeling could be incorporated prior to the immuno-affinity purification, even for the used large quantities (mg) of peptide material, enabling the quantification of differences in tyrosine phosphorylation upon pervanadate treatment or EGF stimulation. Analysis of the EGF-stimulated HeLa cells, a frequently used model system for tyrosine phosphorylation, resulted in the quantification of 73 regulated unique phosphotyrosine peptides. The quantitative data was found to be exceptionally consistent with the literature, evidencing that such a targeted quantitative phosphoproteomics approach can provide reproducible results. In general, the combination of immuno-affinity purification of tyrosine phosphorylated peptides with large scale stable isotope dimethyl labeling provides a cost-effective approach that can alleviate variation in sample preparation and analysis as samples can be combined early on. Using this approach a rather complete qualitative and quantitative picture of tyrosine phosphorylation signaling events can be generated.

U2 - 10.1074/mcp.M900291-MCP200

DO - 10.1074/mcp.M900291-MCP200

M3 - Article

SN - 1535-9476

JO - Molecular and Cellular Proteomics

JF - Molecular and Cellular Proteomics

ER -

In depth qualitative and quantitative profiling of tyrosine phosphorylation using a combination of phosphopeptide immuno-affinity purification and stable isotope dimethyl labeling

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