Analysis of phosphorylation sites on proteins from Saccharomyces cerevisiae by electron transfer dissociation (ETD) mass spectrometry

Abstract

We present a strategy for the analysis of the yeast phosphoproteome that uses endo-Lys C as the proteolytic enzyme, immobilized metal affinity chromatography for phosphopeptide enrichment, a 90-min nanoflow-HPLC/electrospray-ionization MS/MS experiment for phosphopeptide fractionation and detection, gas phase ion/ion chemistry, electron transfer dissociation for peptide fragmentation, and the Open Mass Spectrometry Search Algorithm for phosphoprotein identification and assignment of phosphorylation sites. From a 30-g (Ϸ600 pmol) sample of total yeast protein, we identify 1,252 phosphorylation sites on 629 proteins. Identified phosphoproteins have expression levels that range from <50 to 1,200,000 copies per cell and are encoded by genes involved in a wide variety of cellular processes. We identify a consensus site that likely represents a motif for one or more uncharacterized kinases and show that yeast kinases, themselves, contain a disproportionately large number of phosphorylation sites. Detection of a pHis containing peptide from the yeast protein, Cdc10, suggests an unexpected role for histidine phosphorylation in septin biology. From diverse functional genomics data, we show that phosphoproteins have a higher number of interactions than an average protein and interact with each other more than with a random protein. They are also likely to be conserved across large evolutionary distances.yeast phosphoproteome ͉ network analysis I n an earlier study of the yeast phosphoproteome (1), we digested proteins from a whole cell lysate with trypsin, used immobilized metal-affinity chromatography (IMAC) to enrich the sample for phosphopeptides, and analyzed the resulting mixture by nano-flow HPLC interfaced to electrospray ionization tandem mass spectrometry (MS/MS). Low-energy collision-activated dissociation (CAD) was used to fragment the peptide backbone and to produce ions of types b and y (Fig. 1) required for successful sequence analysis and identification of phosphorylation sites. We detected Ͼ1,000 phosphopeptides but defined only 383 sites of phosphorylation, largely because the CAD process often promoted elimination of phosphoric acid from Ser and Thr residues without breaking the amide bonds along the peptide backbone. The resulting MS/MS spectra were essentially devoid of sequence information.To circumvent this problem, we have modified the LTQ mass spectrometer for ion/ion chemistry and now fragment both peptides (2) and intact proteins (3) by electron transfer dissociation (ETD). In this process, fluoranthene radical-anions are generated in a chemical ionization source and used as reagents to transfer an electron to a multiply charged peptide generated by electrospray ionization. This reaction is highly exothermic, reduces the peptide charge by one, and triggers fragmentation of the peptide backbone to produce a homologous series of complementary fragment ions of type c and z⅐ (Fig. 1). Subtraction of m/z values for fragments within a given ion series that differ by a single amino acid affords the mass...

Posttranslational regulation impacts the fate of duplicated genes

“…There have been several large-scale in vivo studies (15)(16)(17)(18)(19)(20) of the phosphoproteome of S. cerevisiae, using highly reproducible techniques of mass spectrometric analysis. Because the identified phosphopeptides could match one or more open reading frames (ORFs), we generated two phosphorylation datasets.…”

Analytical strategies for phosphoproteomics

“…The implementation of ETD in lowercost instruments makes it more readily used, however, the drawback of using instruments such as the quadrupole ITs as opposed to the FT-ICR, is reduced resolution and accuracy [119]. Chi and co-workers combined IMAC for phosphopeptide enrichment with ETD in a study of the yeast phosphoproteome, identifying 1252 phosphorylation sites [120], Figure 5. MS 2 , MS 3 , and MSA data obtained from a phosphopeptide with the amino acid sequence, pSRpTPLLPR.…”

SLoMo: Automated Site Localization of Modifications from ETD/ECD Mass Spectra

“…[1][2][3][4][5] Until recently these analyses relied heavily on manual validation to confirm correct site localization. 6 This approach is both time-consuming and labor intensive, making it impractical for large data sets.…”