Differentially isotope-coded N-terminal protein sulphonation: combining protein identification and quantification.
Most proteomic labelling technologies intend to improve protein quantification and/or facilitate (de novo) peptide sequencing. We present here a novel stable-isotope labelling method to simultaneously identify and quantify protein components in complex mixtures by specifically derivatizing the N-terminus of proteins with 4-sulphophenyl isothiocyanate (SPITC). Our approach combines protein identification with quantification through differential isotope-coded labelling at the protein N-terminus prior to digestion. The isotope spacing of 6 Da (unlabelled vs. six-fold 13C-labelled tag) between derivatized peptide pairs enables the detection on different MS platforms (MALDI and ESI). Optimisation of the reaction conditions using SPITC was performed on three model proteins. Improved detection of the N-terminally derivatized peptide compared to the native analogue was observed in negative-ion MALDI-MS. Simpler fragmentation patterns compared to native peptides facilitated protein identification. The 13C-labelled SPITC resulted in convenient peptide pair spacing without isotopic overlap and hence facilitated relative quantification by MALDI-TOF/TOF and LC-ESI-MS/MS. The combination of facilitated identification and quantification achieved by differentially isotope-coded N-terminal protein tagging with light/heavy SPITC represents, to our knowledge, a new approach to quantitative proteomics.
Guillaume
Elisabeth
E
Department of Bioanalytical Science, Functional Genomics Group, Nestlé Research Centre, Nestec, Lausanne, Switzerland.
Panchaud
Alexandre
A
Affolter
Michael
M
Desvergnes
Valérie
V
Kussmann
Martin
M
D000818:Animals; D001557:Benzenesulfonates / Q000737:chemistry; D002417:Cattle; D002853:Chromatography, Liquid; D006736:Horses; D017879:Isothiocyanates / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D011817:Rabbits; D015203:Reproducibility of Results; D020539:Sequence Analysis, Protein / Q000379:methods; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013451:Sulfonic Acids / Q000737:chemistry; D014357:Trypsin / Q000494:pharmacology
D001557:Benzenesulfonates; D017879:Isothiocyanates; D013451:Sulfonic Acids; C037152:isothiocyanic acid; C015308:4-isothiocyanatobenzenesulfonate; D014357:Trypsin