Phosphopeptide derivatization signatures to identify serine and threonine phosphorylated peptides by mass spectrometry

The development of rapid, global methods for monitoring states of protein p hosphorylation would provide greater insight for understanding many fundame ntal biological processes. Current best practices use mass spectrometry (MS ) to profile digests of purified proteins for evidence of phosphorylation. However, this approach is beset by inherent difficulties in both identifyin g phosphopeptides from within a complex mixture containing many other unmod ified peptides and ionizing phosphopeptides in positive-ion MS. We have mod ified an approach that uses barium hydroxide to rapidly eliminate the phosp horyl group of serine and threonine modified amino acids, creating dehydroa mino acids that are susceptible to nucleophilic derivatization. By derivati zing a protein digest with a mixture of two different alkanethiols, phospho peptide-specific derivatives were readily distinguished by MS due to their characteristic ion-pair signature. The resulting tagged ion pairs accommoda te simple and rapid screening for phosphopeptides in a protein digest, obvi ating the use of isotopically labeled samples for qualitative phosphopeptid e detection. MALDI-MS is used in a first pass manner to detect derivatized phosphopeptides, while the remaining sample is available for tandem MS to r eveal the site of derivatization and, thus, phosphorylation. We demonstrate d the technique by identifying phosphopeptides from fl-casein and ovalbumin . The approach was further used to examine in vitro phosphorylation of reco mbinant human HSP22 by protein kinase C, revealing phosphorylation of Thr-6 3.