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 15N and 13C labeling to create three different versions of the same peptide, corresponding to the unlabeled, 15N and 13C 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.
Snijders
Ambrosius P L
AP
Biological and Environmental Systems Group, Department of Chemical and Process Engineering, University of Sheffield, Mappin Street, Sheffield S1 3JD, United Kingdom.
de Vos
Marjon G J
MG
Wright
Phillip C
PC
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry; D002247:Carbon Isotopes; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D010455:Peptides / Q000032:analysis* / Q000737:chemistry; D048229:Sulfolobus solfataricus / Q000378:metabolism
D019843:Archaeal Proteins; D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D010455:Peptides