Identifying the major proteome components of Haemophilus influenzae type‐strain NCTC 8143

作者：Andrew J. Link, Lara G. Hays, Edwin Carmack, John R. Yates · 发表于：Electrophoresis · 年份：1997 · DOI：10.1002/elps.1150180808 · 被引用次数：168 · 研究领域：Advanced Proteomics Techniques and Applications、Genomics and Phylogenetic Studies、Mass Spectrometry Techniques and Applications

With the completion of the Haemophilus influenzae Rd genomic sequence, we know the identity of most of the theoretical proteins in the proteome of this bacterium. However, the most abundant components of the actual proteome are unknown. Using mass spectrometry and two-dimensional gel electrophoresis (2-DE), we sequenced and analyzed the most abundant proteins observed in the ATCC reference strain of H. influenzae, NCTC 8143 (303 of approximately 400 Coomassie-stained 2-DE spots). To automate the identification of 2-DE spots, we coupled a liquid autosampler to a microcolumn liquid chromatography electrospray ionization tandem mass spectrometer capable of identifying 22 spots per day. From the 303 sequenced spots, we identified 263 unique proteins. Most of the abundant proteins lie in an isoelectric point range of pH 4-7 and a molecular mass range of 10-100 kDa. Of the observed proteins, the most abundant is the outer membrane protein P2. Based on variety and abundance, proteins involved in energy metabolism and macromolecular synthesis are the dominant classes of proteins. Unexpectedly, tryptophanase was identified as a highly abundant protein in the strain NCTC 8143 whose sequence is not present in the genome of the Rd strain. By searching the tandem mass spectra against the translated genomic sequence, we identified several proteins which were not annotated in the genomic sequence. Surprisingly, 22% of the identified 2-DE spots represent isoforms in which gene products with ...