Analysis of the cytosolic proteome of Halobacterium salinarum and its implication for genome annotation

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Abstract
The halophilic archaeon Halobacterium salinarum (strain R1, DSM 671) contains 2784 protein‐coding genes as derived from the genome sequence. The cytosolic proteome containing 2042 proteins was separated by two‐dimensional gel electrophoresis (2‐DE) and systematically analyzed by a semi‐automatic procedure. A reference map was established taking into account the narrow isoelectric point (pI) distribution of halophilic proteins between 3.5 and 5.5. Proteins were separated on overlapping gels covering the essential areas of pI and molecular weight. Every silver‐stained spot was analyzed resulting in 661 identified proteins out of about 1800 different protein spots using matrix‐assisted laser desorption/ionization time of flight mass spectrometry (MALDI‐TOF MS) peptide mass fingerprinting (PMF). There were 94 proteins that were found in multiple spots, indicating post‐translational modification. An additional 141 soluble proteins were identified on 2‐D gels not corresponding to the reference map. Thus about 40% of the cytosolic proteome was identified. In addition to the 2784 protein‐coding genes, the H. salinarum genome contains more than 6000 spurious open reading frames longer than 100 codons. Proteomic information permitted an improvement in genome annotation by validating and correcting gene assignments. The correlation between theoretical pI and gel position is exceedingly good and was used as a tool to improve start codon assignments. The fraction of identified chromosomal proteins was much higher than that of those encoded on the plasmids. In combination with analysis of the GC content this observation permitted an unambiguous identification of an episomal insert of 60 kbp (“AT‐rich island”) in the chromosome, as well as a 70 kbp region from the chromosome that has integrated into one of the megaplasmids and carries a series of essential genes. About 63% of the chromosomally encoded proteins larger than 25 kDa were identified, proving the efficacy of 2‐DE MALDI‐TOF MS PMF technology. The analysis of the integral membrane proteome by tandem mass spectrometric techniques added another 141 identified proteins not identified by the 2‐DE approach (see following paper).