Isotope‐coded N‐terminal sulfonation of peptides allows quantitative proteomic analysis with increased de novo peptide sequencing capability

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Abstract
Recently various methods for the N‐terminal sulfonation of peptides have been developed for the mass spectrometric analyses of proteomic samples to facilitate de novo sequencing of the peptides produced. This paper describes the isotope‐coded N‐terminal sulfonation (ICenS) of peptides; this procedure allows both de novo peptide sequencing and quantitative proteomics to be studied simultaneously. As N‐terminal sulfonation reagents, 13C‐labeled 4‐sulfophenyl[13C6]isothiocyanate (13C‐SPITC) and unlabeled 4‐sulfophenyl isothiocyanate (12C‐SPITC) were synthesized. The experimental and reference peptide mixtures were derivatized independently using 13C‐SPITC and 12C‐SPITC and then combined to generate an isotopically labeled peptide mixture in which each isotopic pair differs in mass by 6 Da. Capillary reverse‐phase liquid chromatography/tandem mass spectrometry experiments on the resulting peptide mixtures revealed several immediate advantages of ICenS in addition to the de novo sequencing capability of N‐terminal sulfonation, namely, differentiation between N‐terminal sulfonated peptides and unmodified peptides in mass spectra, differentiation between N‐ and C‐terminal fragments in tandem mass spectra of multiply protonated peptides by comparing fragmentations of the isotopic pairs, and relative peptide quantification between proteome samples. We demonstrate that the combination of N‐terminal sulfonation and isotope coding in the mass spectrometric analysis of proteomic samples is a viable method that overcomes many problems associated with current N‐terminal sulfonation methods. Copyright © 2004 John Wiley & Sons, Ltd.