A new approach for detecting C-terminal amidation of proteins and peptides by mass spectrometry in conjunction with chemical derivatization.
We describe a mass spectrometric method for distinguishing between free and modified forms of the C-terminal carboxyl group of peptides and proteins, in combination with chemical approaches for the isolation of C-terminal peptides and site-specific derivatization of the C-terminal carboxyl group. This method could most advantageously be exploited to discriminate between peptides having C-terminal carboxyl groups in the free (COOH) and amide (CONH(2)) forms by increasing their mass difference from 1 to 14 Da by selectively converting the free carboxyl group into methylamide (CONHCH(3)). This method has been proven to be applicable to peptides containing aspartic and glutamic acids, because all the carboxyl groups except the C-terminal one are inert to derivatization, according to oxazolone chemistry. The efficiency of the method is illustrated by a comparison of the peaks of processed peptides obtained from a mixture of adrenomedullin, calcitonin, and BSA. Among these components of the mixture, only the C-terminal peptide of BSA exhibited the mass shift of 13 Da upon treatment, eventually unambiguously validating the C-terminal amide structures of adrenomedullin and calcitonin. The possibility of extending this method for the analysis of C-terminal PTMs is also discussed.
Kuyama
Hiroki
H
Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan. kuyama@protein.osaka-u.ac.jp
Nakajima
Chihiro
C
Nakazawa
Takashi
T
Nishimura
Osamu
O
Tsunasawa
Susumu
S
D000577:Amides / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D010455:Peptides / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000577:Amides; D010455:Peptides; D011506:Proteins