Quantitative Proteomics of the Archaeon Methanococcus maripaludis Validated by Microarray Analysis and Real Time PCR

For the archaeon Methanococcus maripaludis, a fully sequenced and annotated model species of hydrogenotrophic methanogen, we report validation of quantitative protein level expression ratios on a proteome-wide basis. Using an approach based on quantitative multidimensional capillary HPLC and quadrupole ion trap mass spectrometry, coverage of gene expression approached that currently achievable with transcription microarrays. Comprehensive mass spectrometry-based proteomics and spotted cDNA arrays were used to compare global protein and mRNA levels in a wild-type (S2) and mutant strain (S40) of M. maripaludis. Using linear regression with 652 expression ratios generated by both the proteomic and microarray methods, a product moment correlation coefficient of 0.24 was observed. The correlation improved to 0.61 if only genes showing significant expression changes were included. A novel two-stage method of outlier detection was used for the protein measurements when Dixon’s Q-test by itself failed to give satisfactory results. The log2 transformations of the number of peptides or isotopic peptide pairs associated with each ORF, divided by the predicted molecular weight, were found to have moderately positive correlations with two bioinformatic predictors of gene expression based on codon bias. We detected peptides derived from 939 proteins or 55% of the genome coding capacity. Of these, 60 were overexpressed, and 34 were underexpressed in the mutant. Of the 1722 ORFs encoded in the genome, 1597 or 93% were probed by cDNA arrays. Of these, 50 were more highly expressed, and 45 showed lower expression levels in the mutant relative to the wild type. 15 ORFs were shown to be overexpressed by both methods, and two ORFs were shown to be overexpressed by proteomics and underexpressed by microarray. Molecular & Cellular Proteomics 5:, 868–881. The hydrogenotrophic methanogens are a major group of Archaea that conserve energy from the use of molecular hydrogen or formate to reduce carbon dioxide to methane. Methanococcus maripaludis is a model species of hydrogenotrophic methanogen, having favorable laboratory growth behavior, excellent genetic tools (1), and a fully sequenced genome (2). In another publication (3) we describe the molecular biology of a mutant of M. maripaludis (S40) in which the operon encoding the Ehb hydrogenase was disrupted, and functional changes were observed that led to a model for the role of Ehb in energy metabolism. Here we present validation of the multidimensional capillary HPLC/tandem mass spectrometry approach (also known as MudPIT 1 (4, 5)) to quantitative and qualitative proteomics for M. maripaludis by comparison with mRNA expression. Transcriptome analysis was performed in which the ehb mutant and wild-type strains were compared globally for all known genes under the same culture conditions as for the proteomics. For a subset of genes the proteomic results were further validated by real time quantitative RT-PCR. The relative absence of evidence for posttranscriptional gene regulation and the favorable protein extraction properties of M. maripaludis were suggestive of the hypothesis that, under the experimental conditions reported here, the transcriptome and proteome should both be reasonably complete and show parallel trends for most genes. We also consider codon bias relationships as they relate to the proteomic results, a two-stage algorithm for outlier detection in proteomics, and some of the unique strengths and weaknesses of comprehensive expression ratio calculations based on direct measurement of protein using MudPIT. Our purpose in undertaking these studies was primarily to better understand where the MudPIT approach to global protein measurements fits in to the broader scheme of our armamentarium for