Journal of Chromatography B, 782 (2002) 227–243 www.elsevier.com / locate / chromb Global analysis of a ‘‘simple’’ proteome: Methanococcus jannaschii a, b a a c * Carol S. Giometti , Claudia Reich , Sandra Tollaksen , Gyorgy Babnigg , Hanjo Lim , c c b Wenhong Zhu , John Yates III , Gary Olsen a Biosciences Division, Argonne National Laboratory, 9700 South Cass Avenue, Building 202, Room B117, Argonne, IL 60439, USA b Biochemistry Department, University of Illinois, Urbana, IL, USA c Mass Spectrometry Group, The Scripps Institute, La Jolla, CA, USA Abstract The completed genome of Methanococcus jannaschii, including the main chromosome and two extra-chromosomal elements, predicts a proteome comprised of 1783 proteins. How many of those proteins are expressed at any given time and the relative abundance of the expressed proteins, however, cannot be predicted solely from the genome sequence. Two-dimensional gel electrophoresis coupled with peptide mass spectrometry is being used to identify the proteins expressed by M. jannaschii cells grown under different conditions as part of an effort to correlate protein expression with regulatory mechanisms. Here we describe the identification of 170 of the most abundant proteins found in total lysates of M. jannaschii grown under optimal fermentation conditions. To optimize the number of proteins detected, two different protein specific stains (Coomassie Blue R250 or silver nitrate) and two different first dimension separation methods (isoelectric focusing or nonequilibrium pH gradient electrophoresis) were used. Thirty-two percent of the proteins identified are annotated as hypothetical (21% conserved hypothetical and 11% hypothetical), 21% are enzymes involved in energy metabolism, 12% are proteins required for protein synthesis, and the remainder include proteins necessary for intermediary metabolism, cell division, and cell structure. Evidence of post-translational modification of numerous M. jannaschii proteins has been found, as well as indications of incomplete dissociation of protein–protein complexes. These results demonstrate the complexity of proteome analysis even when dealing with a relatively simple genome.  2002 Elsevier Science B.V. All rights reserved. Keywords: Proteomics; Methanococcus jannaschii ; Two-dimensional gel electrophoresis 1. Introduction methane or the methane can escape into the atmos- phere as a significant greenhouse gas. Due to its M. jannaschii, an extreme thermophile (optimal infrared spectrum, on a molar basis, CH is a greater 4 growth temperature is 83 8C), was isolated from a contributor to the greenhouse effect than CO . In 2 deep sea hydrothermal vent. An obligate anaerobe, certain contexts, such as anaerobic sludge digesters the autotrophic M. jannaschii utilizes H and CO and landfills, methane gas produced is sometimes 2 2 for energy production, producing methane (CH ). captured and used as fuel, thereby generating useful 4 Methanotrophic bacteria can oxidize the released energy and protecting the atmosphere. Of all hydro- carbon fuels, CH yields the most energy per unit 4 CO produced, and burning CH is less harmful than 2 4 *Corresponding author. Tel.: 11-630-252-3839; fax: 11-630- releasing it. The biochemical mechanisms used by 252-5517. E-mail address: csgiometti@anl.gov (C.S. Giometti). methanogenic microorganisms in general, and more 1570-0232 / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S1570-0232(02)00568-8