Shotgun Proteomics of <i>Methanococcus jannaschii</i> and Insights into Methanogenesis

<i>Methanococcus jannaschii</i> is an autotrophic hyperthermophilic archaeon isolated from an oceanic hydrothermal vent. Its primary pathway for energy production is methanogenesis from H<sub>2</sub> and CO<sub>2</sub>. High-throughput Multidimensional Protein Identification Technology based on microcapillary LC/LC/MS/MS was used to investigate the proteome of <i>M. jannaschii</i> and the methanogenesis pathway in cells grown in complex medium with high H<sub>2</sub> supply. A total of 963 proteins have been unambiguously identified. The identified proteins represent ∼54% of the whole genome of <i>M. jannaschii</i>. About 44% of the identified proteins are either conserved hypothetical or hypothetical proteins. We identified 83−95% of the proteins predicted to be involved in amino acid biosynthesis, cellular processes, central intermediary metabolism, energy metabolism, protein synthesis, transcription, and purine, pyridine, nucleoside, and nucleotide synthesis. Over 40% of these proteins have better than 50% sequence coverage. Approximately 90% of the predicted methanogenesis proteins were detected. In contrast, only 27−37% of predicted hypothetical proteins, proteins involved in transport and binding, and proteins with regulatory functions were identified. High peptide number, spectrum count, and sequence coverage have been used as indicators of high expression levels and are in good agreement with codon bias analysis. Predicted intein peptides were detected in MJ1043 (DNA-directed RNA polymerase, subunit A‘ ‘), MJ0542 (phosphoenolpyruvate synthase), MJ0782 (transcription initiation factor IIB), and MJ1422 (putative replication factor C subunit). New peptides created by protein splicing were detected in MJ0885 (DNA dependent DNA polymerase), MJ0542, and MJ0782. The methanogenesis pathway and the enzymes involved are also discussed. Keywords: proteomics • MudPIT • LC/LC/MS/MS • <i>Methanococcus jannaschii</i> • mass spectrometry • SEQUEST • methanogenesis • archaea • intein