One pot synthesis and multiple MS/MS fragmentation study of phospholysine peptides.

Rationale Compared with phosphorylation of arginine and histidine, the study of phosphorylation of lysine lags far behind. The major challenges towards the current study of phosphorylation of lysine include synthesis and unambiguous phosphosite identification. This study provided a simple chemical synthesis method to construct phospholysine peptides (pLys peptides), and investigated their fragmentation under multiple activation types. Methods Herein, we developed a synthetic method for pLys peptides in aqueous solution within one pot. Two peptides were lysine-phosphorylated using this method. The purified pLys peptides were firstly characterized by NMR, then were subjected to nano-liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) analysis under multiple fragmentation method including collision-induced dissociation (CID), higher energy collisional dissociation (HCD), electron transfer dissociation (ETD), electron transfer/higher energy collisional dissociation (EThcD) and ultraviolet photodissociation (UVPD) fragmentation to investigate the relevant diagnostic ions. Results Two pLys peptides were synthesized with moderate yield follow an easy-handled experimental protocol. NMR spectra showed the phosphorylation was on e-NH2 of lysine but not other groups. As for the fragmentation, in general, pLys immonium ions and phosphate-related neutral losses were ubiquitous among spectra derived from these activation types except for ETD. Using these ions as diagnostic ions, the misassigned phosphosites by algorithm could be recorrected. UVPD-generated spectra owned good sequence-coverage and abundant fragment ions, with pLys immonium ions and neutral losses of weak intensity. Conclusions a synthetic method for pLys peptides in aqueous solution within one pot was developed. The characteristic pLys immonium ions and phosphate-related neutral loss could serve as the diagnostic ions for unambiguous phosphosite identification of pLys peptides. In addition, UVPD was promising for the identification of pLys peptides.