Isotope-coded N-terminal sulfonation of peptides allows quantitative proteomic analysis with increased de novo peptide sequencing capability.
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.
Lee
Yong Ho
YH
Department of Chemistry and Center for Electro- and Photo-Responsive Molecules, Korea University, 1, 5-ka, Anam-dong, Seongbuk-ku, Seoul 136-701, South Korea.
Han
Hoon
H
Chang
Seok-Bok
SB
Lee
Sang-Won
SW
D000595:Amino Acid Sequence; D000818:Animals; D002247:Carbon Isotopes / Q000737:chemistry; D002417:Cattle; D002645:Chickens; D006736:Horses; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis* / Q000737:chemistry*; D020543:Proteome / Q000032:analysis* / Q000737:chemistry*; D011817:Rabbits; D020539:Sequence Analysis, Protein / Q000379:methods*; D013450:Sulfones / Q000737:chemistry*
D002247:Carbon Isotopes; D010455:Peptides; D020543:Proteome; D013450:Sulfones