Large-scale characterization of HeLa cell nuclear phosphoproteins

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.phosphorylation ͉ mass spectrometry ͉ strong cation exchange chromatography M uch 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 Ͼ500 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 (MS͞MS), 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 Ϸ200 (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 MS͞MS commonly results in the production of a single dominant peak corresponding to a neutral loss of phosphoric acid (H 3 PO 4 , 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 ...

Titanium dioxide as a chemo-affinity solid phase in offline phosphopeptide chromatography prior to HPLC-MS/MS analysis

“…Some phosphopeptide-selective techniques have been developed, such as immobilized metal affinity chromatography (IMAC) [4][5][6] or two-dimensional HPLC systems using strong cation-exchange chromatography (SCX) and reversed-phase (RP) chromatography [7][8][9][10] .…”

“…Edman degradation is a robust and well-established method, but it shows some limitations when analyzing complex samples and cannot fulfill the requirements for sensitive detection of low concentrations of phosphopeptides [1][2][3] . Besides the additional needed labeling of the phosphate residues, it would take more than a hundred fold in time to chemically treat and analyze a complex sample related to other techniques for sequencing and phosphorylation site mapping.Identifying phosphorylation sites in different proteins through mass spectrometry (MS) requires a proper pre-separation and enrichment of the phosphopeptides performed online by the HPLC, because of the often highly complex sample composition.Some phosphopeptide-selective techniques have been developed, such as immobilized metal affinity chromatography (IMAC) [4][5][6] or two-dimensional HPLC systems using strong cation-exchange chromatography (SCX) and reversed-phase (RP) chromatography [7][8][9][10] .IMAC separation uses the affinity of the negatively charged phosphate group in phosphopeptides for positively charged Fe 3+ or Ga 3+ ions, which are bound on the stationary phase of the separation column. However, not only phosphopeptides but also acidic groups in peptides will form complexes.…”

Circles within circles: crosstalk between protein Ser/Thr/Tyr-phosphorylation and Met oxidation

“…Many phosphorylation motifs - kinase recognition signatures - contain hydrophobic Met residue [57,62]. Oxidation of Met to MetSO causes a shift from hydrophobic to hydrophilic in nature, and would thus likely disrupt kinase recognition [50].…”

Proteomic analysis of post translational modifications in cyanobacteria

“…Systematic investigation of PTMs will be greatly helpful to obtain a comprehensive understanding of the signal transduction network and biological processes in cyanobacteria. Proteomic investigations of PTMs have started only since the beginning of the last decade [16][17][18]. Now the study of PTMs via MS-based proteomic strategies constitutes a vibrant and large field of research.…”