Perturbation and Interpretation of Nitrogen Isotope Distribution Patterns in Proteomics

This study provides a discussion on the applications and limitations of 15NH4 + 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 experiment...

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Bibliographic Details
Published in:Journal of proteome research 2005-11, Vol.4 (6), p.2185-2191
Main Authors: Snijders, Ambrosius P. L, de Koning, Bart, Wright, Phillip C
Format: Article
Language:English
Subjects:
Ammonia - metabolism
Databases, Protein
Electrophoresis, Polyacrylamide Gel
Nitrogen - chemistry
Nitrogen Isotopes - chemistry
Peptide Mapping
Peptides - chemistry
Proteins - chemistry
Proteome
Proteomics - methods
Sequence Analysis, Protein
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Sulfolobus - metabolism
Trypsin - pharmacology
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Summary:This study provides a discussion on the applications and limitations of 15NH4 + 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% 15NH4 +. 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 15NH4 + 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. Keywords: metabolic labeling • protein turnover • proteomics • archaea • Sulfolobus solfataricus
ISSN:1535-3893
1535-3907
DOI:10.1021/pr050260l