Comparison of spectral counting and metabolic stable isotope labeling for use with quantitative microbial proteomics

Abstract

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 ¹⁵N/¹⁴N “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.