Perturbation and interpretation of nitrogen isotope distribution patterns in proteomics.
This study provides a discussion on the applications and limitations of (15)NH(4)(+) metabolic labeling in proteomic studies. The hyperthemophilic crenarchaeon Sulfolobus solfataricus was used as a model organism throughout this study. The distribution of nitrogen was studied in four different experiments in which this distribution was manipulated in a unique way. The experiments included full adaptation to media with relative isotope abundances (RIA) of 0.36%, 50%, and >98% (15)NH(4)(+). The incorporation efficiency was calculated on the basis of a comparison between theoretical and experimental spectra. In the case of full adaptation, incorporation efficiencies reflected the RIA (0.36%, 47.5% and 99% respectively). Labeling efficiencies were calculated on the basis of peak areas in TOF-MS spectra. It is shown that in the case of full adaptation, labeling efficiencies are 100%. In addition, we demonstrate that (15)NH(4)(+) labeling can be used in protein turnover studies, even when labeling is incomplete. In this case, incorporation efficiencies of 88-93% (lower than the RIA) were measured, providing evidence for amino acid recycling. Labeling efficiencies were always between 63% and 94% providing evidence for protein degradation. Finally, it was shown that isotope distributions can be useful in peptide identification.
Snijders
Ambrosius P L
AP
Biological and Environmental Systems Group, Department of Chemical and Process Engineering, University of Sheffield, UK.
de Koning
Bart
B
Wright
Phillip C
PC
D000641:Ammonia / Q000378:metabolism; D030562:Databases, Protein; D004591:Electrophoresis, Polyacrylamide Gel; D009584:Nitrogen / Q000737:chemistry*; D009587:Nitrogen Isotopes / Q000737:chemistry*; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D016944:Sulfolobus / Q000378:metabolism*; D014357:Trypsin / Q000494:pharmacology
D009587:Nitrogen Isotopes; D010455:Peptides; D011506:Proteins; D020543:Proteome; D000641:Ammonia; D014357:Trypsin; D009584:Nitrogen