Therefore, syk−/− DT40 B-cell mutants were reconstituted with a OneStrep-tagged version of human Syk and left untreated or stimulated through their BCR for six different time points. Cellular lysates were incubated with a streptactin affinity column and obtained proteins were size-separated by 1-D PAGE. Following in-gel digestion of Syk with endoproteinase trypsin, resulting Alpelisib phosphopeptide products were enriched by TiO2 microcolumns and identified by liquid chromatography (LC)-coupled tandem mass spectrometry (MS/MS) on an orbitrap mass

spectrometer. As shown in Fig. 1, we detected a total of 32 phosphoacceptor sites, 15 of which were on tyrosine, 11 on serine and six on threonine (see Supporting Information data 1 for annotated MS/MS spectra). Our analysis confirmed

all previously published phosphorylation selleck inhibitor events and revealed 19 novel acceptor sites. Notably, almost half of the Syk phosphosites mapped to interdomain B (see Fig. 1) previously implicated in the control of Syk functions 2, 3. Our data show that Syk is extensively modified by phosphorylation on a large number of acceptor sites, which might act individually or in concert to regulate Syk function. To monitor the phosphorylation kinetics of individual acceptor sites we used a quantitative SILAC-based mass-spectrometric approach 29–31. DT40 cells expressing OneStrep-tagged Syk were metabolically labeled in SILAC medium containing arginine and lysine residues with incorporated light or heavy isotopes of carbon and nitrogen.

Different combinations yielded three types of SILAC media. Using 12C6,14N2-Lys and 12C6,14N4-Arg resulted in “light medium” while the combination of 13C6,15N2-Lys and 13C6,15N4-Arg dipyridamole yielded “heavy medium”. “Intermediate medium” was obtained by using 2D4,12C6,14N2-Lys and 13C6,14N4-Arg. Cells cultured in “light medium” were left untreated and those cultured in “intermediate” or “heavy medium” were BCR-stimulated for different time points. This setup had important consequences. Proteins or peptides derived from the differentially labeled cells can be distinguished in the mass spectrometer by virtue of their distinct absolute molecular masses and hence can unambiguously be assigned to one of the three stimulation conditions. Proteins were purified from the three cell cultures via streptactin affinity chromatography, pooled at a 1:1:1 ratio and separated by 1-D PAGE. The gel slice containing the three pools of Syk was excised and subjected to trypsin digestion. TiO2-enriched phosphopeptides were analyzed by LC-MS/MS and individually quantified using MaxQuant software 32. This strategy allowed an unbiased relative quantification of Syk phosphorylation in resting and stimulated cells. Altogether, we monitored the phosphorylation kinetics of 16 phosphosites, which we grouped into three categories (Fig. 2).