Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae

Abstract

Protein kinases are coded by more than 2,000 genes and thus constitute the largest single enzyme family in the human genome. Most cellular processes are in fact regulated by the reversible phosphorylation of proteins on serine, threonine, and tyrosine residues. At least 30% of all proteins are thought to contain covalently bound phosphate. Despite the importance and widespread occurrence of this modification, identification of sites of protein phosphorylation is still a challenge, even when performed on highly purified protein. Reported here is methodology that should make it possible to characterize most, if not all, phosphoproteins from a whole-cell lysate in a single experiment. Proteins are digested with trypsin and the resulting peptides are then converted to methyl esters, enriched for phosphopeptides by immobilized metal-affinity chromatography (IMAC), and analyzed by nanoflow HPLC/electrospray ionization mass spectrometry. More than 1,000 phosphopeptides were detected when the methodology was applied to the analysis of a whole-cell lysate from Saccharomyces cerevisiae. A total of 216 peptide sequences defining 383 sites of phosphorylation were determined. Of these, 60 were singly phosphorylated, 145 doubly phosphorylated, and 11 triply phosphorylated. Comparison with the literature revealed that 18 of these sites were previously identified, including the doubly phosphorylated motif pTXpY derived from the activation loop of two mitogen-activated protein (MAP) kinases. We note that the methodology can easily be extended to display and quantify differential expression of phosphoproteins in two different cell systems, and therefore demonstrates an approach for "phosphoprofiling" as a measure of cellular states.

Automatic Validation of Phosphopeptide Identifications from Tandem Mass Spectra

“…Average intensity of b-ions; (3). Average intensity of y-ions; (4). Average intensity of the 2L unassigned peaks; (5).…”

An isotope labeling strategy for quantifying the degree of phosphorylation at multiple sites in proteins

“…The Fischer esterification was quantitative with no peaks assignable to partial derivatization products as was the case for all ten peptides observed using this method (data not shown). Complete removal of HCl is critical to prevent ester hydrolysis upon addition of aqueous solvent (see modifications to the procedure of Ficarro et al [12] described in the Experimental section). Two of the peptides with ambiguous phosphorylation sites overlap because of a missed cleavage.…”

“…Both aliquots for each protein were lyophilized and then differentially isotopically labeled by bulk Fischer esterification in either natural abundance-or deutero-methanol that is 2 M in anhydrous HCl. The esterification protocol is essentially that described by Ficarro and coworkers [12], where HCl is generated in situ by addition of acetylchloride to the methanol, on ice, with stirring, prior to its addition to the peptide mixture. The reaction was allowed to proceed for 2 h at room temperature at which point the methanol was removed under vacuum (Savant Speed Vac, GMI, Albertville, MN).…”

Fine regulation of Saccharomyces cerevisiae MAPK pathways by post‐translational modifications

“…Quantitative phosphoproteomic approaches based on stable isotope labelling of amino acids in cell culture (SILAC) coupled to phosphopeptide enrichment and high-resolution liquid chromatography mass spectrometry (LC-MS/MS) has allowed the characterization of a massive amount of phosphopeptides on a whole-proteome scale and, more importantly, to assess differential phosphorylation events [22,48]. Since Ficarro et al [16] pioneered this field in yeast, numerous groups have produced a wealth of data that can be consulted in public databases, such as PhosphoPep (www.phosphopep.org; [6]), which has been integrated into SGD (Saccharomyces Genome Database; www.yeastgenome.org). Remarkably, SILAC-based quantitative phosphoproteomics have been applied to the study of differential phosphorylation upon pheromone treatment [24] and osmotic shock [54], aiming to detect changes in the yeast phosphoproteome attributable to the mating pheromone response and HOG MAPK pathways, respectively.…”