Journal
ANALYST
Volume 131, Issue 12, Pages 1335-1341Publisher
ROYAL SOC CHEMISTRY
DOI: 10.1039/b610957h
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Funding
- NIDCR NIH HHS [R01 DE014372-03, R01 DE014372, R56 DE014372, DE014372] Funding Source: Medline
- NIGMS NIH HHS [GM60403, R01 GM060403] Funding Source: Medline
- NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH [R56DE014372, R01DE014372] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [R01GM060403] Funding Source: NIH RePORTER
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Spectral counting, a promising method for quantifying relative changes in protein abundance in mass spectrometry-based proteomic analysis, was compared to metabolic stable isotope labeling using N-15/N-14 heavy/light'' peptide pairs. The data were drawn primarily from a Methanococcus maripaludis experiment comparing a wild-type strain with a mutant deficient in a key enzyme relevant to energy metabolism. The dataset contained both proteome and transcriptome measurements. The normalization technique used previously for the isotopic measurements was inappropriate for spectral counting, but a simple adjustment for sampling frequency was sufficient for normalization. This adjustment was satisfactory both for M. maripaludis, an organism that showed relatively little expression change between the wild-type and mutant strains, and Porphyromonas gingivalis, an intracellular pathogen that has demonstrated widespread changes between intracellular and extracellular conditions. Spectral counting showed lower overall sensitivity defined in terms of detecting a two-fold change in protein expression, and in order to achieve the same level of quantitative proteome coverage as the stable isotope method, it would have required approximately doubling the number of mass spectra collected.
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