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
peer-reviewed · PROTEOMICS · 2004
| Date | 2004-06-01 |
| Type | peer-reviewed |
| Venue | PROTEOMICS |
| Publisher | Wiley |
| Contribution | algorithm |
| DOI | 10.1002/pmic.200300698 |
| Citations (OpenAlex) | 45 |
Abstract
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.
Methods and tools
- N-terminal SPITC sulfonation for LC-MS/MS de novo sequencing: Selective N-terminal sulfonation of tryptic peptides with 4-sulfophenyl isothiocyanate, adapted to online capillary LC-MS/MS so the spectra are dominated by y ions and read de novo.
Cites (2)
Cited by (2)
- Benefits of selective peptide derivatization with sulfonating reagent at acidic pH for facile matrix‐assisted laser desorption/ionization de novo sequencing (2016) crossref
- Isotope‐coded N‐terminal sulfonation of peptides allows quantitative proteomic analysis with increased de novo peptide sequencing capability (2004) crossref