Large-scale characterization of HeLa cell nuclear phosphoproteins.
Journal: 2004/September - Proceedings of the National Academy of Sciences of the United States of America
ISSN: 0027-8424
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Determining the site of a regulatory phosphorylation event is often essential for elucidating specific kinase-substrate relationships, providing a handle for understanding essential signaling pathways and ultimately allowing insights into numerous disease pathologies. Despite intense research efforts to elucidate mechanisms of protein phosphorylation regulation, efficient, large-scale identification and characterization of phosphorylation sites remains an unsolved problem. In this report we describe an application of existing technology for the isolation and identification of phosphorylation sites. By using a strategy based on strong cation exchange chromatography, phosphopeptides were enriched from the nuclear fraction of HeLa cell lysate. From 967 proteins, 2,002 phosphorylation sites were determined by tandem MS. This unprecedented large collection of sites permitted a detailed accounting of known and unknown kinase motifs and substrates.
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Proc Natl Acad Sci U S A 101(33): 12130-12135

Large-scale characterization of HeLa cell nuclear phosphoproteins

Department of Cell Biology, Taplin Biological Mass Spectrometry Facility, Dana–Farber Cancer Institute, and Beth Israel Deaconess Medical Center and Department of Systems Biology, Harvard Medical School, Boston, MA 02115
To whom correspondence should be addressed at: Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115. E-mail: ude.dravrah.smh@igyg_nevets.
S.A.B. and M.J. contributed equally to this work.
Contributed by Lewis C. Cantley, July 2, 2004
Contributed by Lewis C. Cantley, July 2, 2004

Abstract

Determining the site of a regulatory phosphorylation event is often essential for elucidating specific kinase–substrate relationships, providing a handle for understanding essential signaling pathways and ultimately allowing insights into numerous disease pathologies. Despite intense research efforts to elucidate mechanisms of protein phosphorylation regulation, efficient, large-scale identification and characterization of phosphorylation sites remains an unsolved problem. In this report we describe an application of existing technology for the isolation and identification of phosphorylation sites. By using a strategy based on strong cation exchange chromatography, phosphopeptides were enriched from the nuclear fraction of HeLa cell lysate. From 967 proteins, 2,002 phosphorylation sites were determined by tandem MS. This unprecedented large collection of sites permitted a detailed accounting of known and unknown kinase motifs and substrates.

Keywords: phosphorylation, mass spectrometry, strong cation exchange chromatography
Abstract

Much of eukaryotic protein regulation occurs when protein kinases add a phosphate moiety in an ATP-dependent manner to a Ser, Thr, or Tyr residue of a substrate protein. Not surprisingly, malfunctions in this critical cellular process have been implicated as causal factors in diseases, such as diabetes, cancer, and Alzheimer's. With >500 identified kinases and thousands of potential substrates, these proteins remain attractive drug targets. Large-scale identification of phosphorylated kinase substrates will certainly enhance our understanding of diverse biological phenomena, potentially leading to targeted intervention in any number of disease paradigms.

The identification of phosphorylation sites is most robustly accomplished by MS (1, 2). With tandem MS (MS/MS), phosphopeptides are fragmented to determine their sequence and to pinpoint the specific Ser, Thr, or Tyr modified by a protein kinase. Despite many reports of thousands of identified proteins from a single biological sample, the large-scale determination of phosphorylation sites is just emerging. To date, the three largest reported repositories of identified sites are from yeast and plant studies [383 (3),125 (4) and ≈200 (5)], whereas the most phosphorylation sites identified from a single human sample stands at 64 (6). Clearly, to study the rich biology relying on protein phosphorylation will require more effective methodologies.

Uninformative fragmentation is a fundamental obstacle to phosphorylation site analysis, regardless of the scale of an experiment. Fragmentation of phosphopeptides by collision-induced dissociation by MS/MS commonly results in the production of a single dominant peak corresponding to a neutral loss of phosphoric acid (H3PO4, 98 Da) from the phosphopeptide (for example, see Fig. 2B). The lack of informative fragmentation at the peptide backbone severely reduces the ability of database searching algorithms to unambiguously identify the phosphopeptide. Furthermore, when a phosphopeptide is identified, it is often not possible to assign the site to a particular Ser, Thr, or Tyr residue because of the lack of informative fragmentation (2).

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Analysis of human nuclear phosphorylation sites by multidimensional LC coupled to MS/MS/MS. (A) Eight milligrams of nuclear extract from asynchronous HeLa cells were separated by SDS/PAGE. The entire gel was excised into 10 regions and proteolyzed with trypsin followed by phosphopeptide enrichment by SCX LC. Early eluting fractions were subjected to amino acid sequence analysis by reverse-phase LC-MS/MS with data-dependent MS/MS/MS acquisition. Phosphorylation sites (n = 2,002) were identified by the sequest algorithm, acquisition of MS/MS/MS spectra, and manual validation. Prep, preparation. (B) Example of a MS/MS spectrum of a phosphopeptide showing a typical extensive neutral loss of phosphoric acid. (C)MS/MS/MS spectrum of the neutral loss precursor ion from B. Abundant peptide bond fragmentation permitted the unambiguous identification of this peptide from the protein cell division cycle 2-related protein kinase 7, with a phosphorylated Ser residue marked by an asterisk.

Further hindering phosphorylation studies, the phosphorylated form of a protein frequently has low stoichiometry relative to its unphosphorylated counterpart. Considering the already low expression levels of most proteins regulated by phosphorylation, it is obvious that “shotgun” sequencing strategies can easily miss these rare peptides. It is essential to employ some type of enrichment strategy to overcome the tremendous complexity of a proteolyzed lysate. Efforts to isolate phosphopeptides in the past have used either chemical modification of phosphate groups (79), phosphate-specific MS-based methods (1013), or affinity-based methods (antibody or metal ion chromatography) (3, 6, 1416). Regardless of the enrichment procedure, amino acid sequence analysis and site determination were accomplished by MS/MS. Each technique has been successful for the analysis of a few proteins (<30), but only immobilized metal affinity chromatography has shown the potential for the identification of more than a few sites from complex mixtures (3, 6).

In this report, we show that strong cation exchange (SCX) chromatography provides an additional, robust enrichment tool for phosphopeptides. SCX is often used as a primary separation strategy for complex peptide mixtures before analysis by reversephase liquid chromatography (LC)-MS/MS (17), in which peptides elute according to their solution state charge. The most common implementation utilizes an on-line system in which complex mixtures are adsorbed to a bi-phasic column packed with SCX and reverse-phase resins. Several salt “bumps” are then initiated to release discrete peptide fractions to the reverse-phase column for analysis. Although this strategy has been widely successful for the analysis of complex peptide mixtures, the ability of SCX chromatography to enrich phosphopeptides has not been reported likely because of weak retention of most tryptic phosphopeptides.

Here, we describe a SCX-based strategy to enrich for the phosphopeptide component of a proteome and a method to enhance phosphopeptide identification. We demonstrate the technique by isolating and identifying >2,000 phosphorylation sites from HeLa cell nuclei.

Accession numbers were derived from the following sources: †, GenBank; ‡, Protein Information Resource; §, SwissProt. Sites of phosphorylation in the peptides are indicated by an asterisk. A literature search of phosphorylation sites was done by using the Human Protein Reference Database (30).

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Acknowledgments

We thank I. Jardine, I. Mylchreest, J. Shofstahl, J. Schwartz, and E. Hemenway (Thermo Electron) for integrating the MS/MS/MS algorithm idea and providing early access. We also thank past and present members of the Gygi lab for useful discussions and critical reading, R. Duarte (Harvard Medical School) for producing phosphorylation intensity maps, J. Rush (Cell Signaling Technology, Beverly, MA) for synthetic peptides, and S. Gerber (Harvard Medical School) for HeLa cell lysate. This work was supported in part by National Institutes of Health Grants HG00041 (to S.P.G.), GM67945 (to S.P.G.), and GMS6203 (to L.C.C.).

Acknowledgments

Notes

Abbreviations: SCX, strong cation exchange; MS/MS, tandem MS; MS/MS/MS, third stage of MS; LC, liquid chromatography.

Notes
Abbreviations: SCX, strong cation exchange; MS/MS, tandem MS; MS/MS/MS, third stage of MS; LC, liquid chromatography.

Footnotes

Tomaino, R., Rush, J., Steen, H., Licklider, L., Hemenway, E., Shofstahl, J., Schwartz, J., Mylchreest, I. &amp; Gygi, S. P., 50th American Society for Mass Spectrometry Conference on Mass Spectrometry and Allied Topics, June 2–8, 2002, Orlando, FL, abstr. ThOEpm.

Footnotes

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