Chemoselective synthesis and analysis of naturally occurring phosphorylated cysteine peptides.

In contrast to protein O-phosphorylation, studying the function of the less frequent N- and S-phosphorylation events have lagged behind because they have chemical features that prevent their manipulation through standard synthetic and analytical methods. Here we report on the development of a chemoselective synthetic method to phosphorylate Cys side-chains in unprotected peptides. This approach makes use of a reaction between nucleophilic phosphites and electrophilic disulfides accessible by standard methods.

Gas-Phase Rearrangement in Lysine Phosphorylated Peptides During Electron-Transfer Dissociation Tandem Mass Spectrometry.

Tandem mass spectrometry (MS/MS) strategies coupled with collision-induced dissociation (CID) or radical-driven fragmentation techniques such as electron-capture dissociation (ECD) or electron-transfer dissociation (ETD) have been successfully used for comprehensive phosphoproteome analysis. However, the unambiguous characterization of the phosphorylation site remains a significant challenge due to phosphate-related neutral losses and gas-phase rearrangements, which have been observed during CID. In particular, for the analysis of labile N-phosphorylated peptides, ECD and ETD are emerging as a complementary method.

Direct access to site-specifically phosphorylated-lysine peptides from a solid-support.

Phosphorylation is a key process for changing the activity and function of proteins. The impact of phospho-serine (pSer), -threonine (pThr) and -tyrosine (pTyr) is certainly understood for some proteins. Recently, peptides and proteins containing N-phosphorylated amino acids such as phosphoarginine (pArg), phosphohistidine (pHis) and phospholysine (pLys) have gained interest because of their different chemical properties and stability profiles.

Site-specifically phosphorylated lysine peptides.

Protein phosphorylation controls major processes in cells. Although phosphorylation of serine, threonine, and tyrosine and also recently histidine and arginine are well-established, the extent and biological significance of lysine phosphorylation has remained elusive. Research in this area has been particularly limited by the inaccessibility of peptides and proteins that are phosphorylated at specific lysine residues, which are incompatible with solid-phase peptide synthesis (SPPS) due to the intrinsic acid lability of the P(═O)-N phosphoramidate bond.