A picomole-scale method for rapid peptide sequencing through convenient and efficient N-terminal phosphorylation and electrospray ionization mass spectrometry.
Free or resin-bound peptides were phosphorylated at their N-termini by reacting with dimethyl phosphite in the presence of tetrachloromethane and triethylamine, and some of them were labeled using partial deuterium-labeled dimethyl phosphites (molar ratio of m + 6 and m = 1:1 or m + 6, m + 3 and m = 1:2:1) as the phosphorylating agents. The monophosphorylation of the lysine-containing peptides selectively occurred on the amino group of the N-terminus, not the side-chain of lysine residue. The resin-bound phosphoramidate peptides were cleaved by TFA before ESI-MS. The modified peptides were determined by electrospray ionization mass spectrometry, the protonated molecules of the unlabeled phosphoramidate peptides showed the singlet peaks, and those of the labeled phosphoramidate peptides displayed the doublet and/or triplet peaks. Tandem mass spectra (MS/MS) of the chosen protonated molecules gave sequential loss of the amino acid residues from the C-termini of the peptides, providing a convenient and rapid method for peptide sequencing.
Wang
Feng
F
Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing, People's Republic of China.
Fu
Hua
H
Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing, People's Republic of China. fuhua@mail.tsinghua.edu.cn.
Jiang
Yuyang
Y
Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing, People's Republic of China.
Zhao
Yufen
Y
Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing, People's Republic of China.
D000595:Amino Acid Sequence; D008832:Microchemistry / Q000379:methods*; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods*; D010766:Phosphorylation; D020539:Sequence Analysis, Protein / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*