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.
Recently, various chemical modifications of peptides have been incorporated into mass spectrometric analyses of proteome samples, predominantly in conjunction with matrix-assisted laser desorption/ionization mass spectrometry (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 SPITC reacts selectively with the N-terminal amino groups, even in the presence of the epsilon-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 pretreatment, 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.
Lee
Yong Ho
YH
Department of Chemistry and Center for Electro- and Photo-Responsive Molecules, Korea University, Seoul, South Korea.
Kim
Min-Sik
MS
Choie
Woo-Suk
WS
Min
Hye-Ki
HK
Lee
Sang-Won
SW
D000595:Amino Acid Sequence; D001557:Benzenesulfonates / Q000494:pharmacology; D020374:Chromatography, Micellar Electrokinetic Capillary; D017879:Isothiocyanates / Q000494:pharmacology; D013058:Mass Spectrometry; D010455:Peptides / Q000032:analysis* / Q000737:chemistry; D020543:Proteome / Q000032:analysis*; D020539:Sequence Analysis, Protein
D001557:Benzenesulfonates; D017879:Isothiocyanates; D010455:Peptides; D020543:Proteome; C015308:4-isothiocyanatobenzenesulfonate