Novel approach for peptide quantitation and sequencing based on ¹⁵N and ¹³C metabolic labeling

Here we describe a method for protein identification and quantification using stable isotopes via in vivo metabolic labeling of the hyperthermophilic crenarchaeon Sulfolobus solfataricus. Stable isotope labeling for quantitative proteomics is becoming increasingly popular; however, its usefulness in protein identification has not been fully exploited. We use both N-15 and C-13 labeling to create three different versions of the same peptide, corresponding to the unlabeled, N-15 and C-13 labeled versions. The peptide then appears as three different peaks in a TOF-MS scan and three corresponding sets of MS/MS spectra are obtained. With this information, the elemental carbon and nitrogen compositions for each peptide and each fragment can be calculated. When this is used as a constraint in database searching and/or de novo sequencing, the confidence of a match is increased (for an example intact peptide from 34 choices to 1). This makes the method a useful proteomic tool for both sequenced and unsequenced organisms. Furthermore, it allows for accurate protein quantitation (standard deviations over > 4 peptides per protein were within 10%) of three phenotypes in one MS experiment. Abundances for each peptide are calculated by determining the relative areas of each of the three peaks in the TOF-MS spectrum.