Highly Informative Proteome Analysis by Combining Improved N-Terminal Sulfonation for de novo Peptide Sequencing and Online Capillary Reverse-Phase Liquid Chromatography/Tandem Mass Spectrometry

다국어 초록 (Multilingual Abstract)

Recently, various chemical modifications of peptides have been incorporated into mass spectrometric analyses of proteome samples, predominantly in conjunction with MALDI MS, to facilitate de novo sequencing of peptides. In this work, we investigate systematically the utility of N-terminal sulfonation of tryptic peptides by 4-sulfophenyl isothiocyanate(SPITC) for proteome analysis by capillary reverse-phase liquid chromatography/tandem mass spectrometry(cRPLC/MS/MS). The experimental conditions for the sulfonation were carefully adjusted so that the SPITC reacts selectively with the N-terminal amino groups, even in the presence of the ε-amino groups of lysine residues. Mass spectrometric analyses of the modified peptides by cRPLC/MS/MS indicated that SPITC derivatization proceeded toward near completion under the experimental conditions employed here. The SPITC-derivatized peptides underwent facile fragmentation, predominantly resulting in y-series ions in the MS/MS spectra. Combining SPITC derivatization and cRPLC/MS/MS analyses facilitated the acquisition of sequence information for lysine-terminated tryptic peptides as well as arginine-terminated peptides without the need for additional peptide pre-treatment, such as guanidination of lysine amino group. This process alleviated the biased detection of arginine-terminated peptides that is often observed in MALDI MS experiments. We will discuss the utility of the technique as a viable method for proteome analyses and present examples of its application in analyzing samples having different levels of complexity.