Initial proteome analysis of model microorganism Haemophilus influenzae strain Rd KW20.
The proteome of Haemophilus influenzae strain Rd KW20 was analyzed by liquid chromatography (LC) coupled with ion trap tandem mass spectrometry (MS/MS). This approach does not require a gel electrophoresis step and provides a rapidly developed snapshot of the proteome. In order to gain insight into the central metabolism of H. influenzae, cells were grown microaerobically and anaerobically in a rich medium and soluble and membrane proteins of strain Rd KW20 were proteolyzed with trypsin and directly examined by LC-MS/MS. Several different experimental and computational approaches were utilized to optimize the proteome coverage and to ensure statistically valid protein identification. Approximately 25% of all predicted proteins (open reading frames) of H. influenzae strain Rd KW20 were identified with high confidence, as their component peptides were unambiguously assigned to tandem mass spectra. Approximately 80% of the predicted ribosomal proteins were identified with high confidence, compared to the 33% of the predicted ribosomal proteins detected by previous two-dimensional gel electrophoresis studies. The results obtained in this study are generally consistent with those obtained from computational genome analysis, two-dimensional gel electrophoresis, and whole-genome transposon mutagenesis studies. At least 15 genes originally annotated as conserved hypothetical were found to encode expressed proteins. Two more proteins, previously annotated as predicted coding regions, were detected with high confidence; these proteins also have close homologs in related bacteria. The direct proteomics approach to studying protein expression in vivo reported here is a powerful method that is applicable to proteome analysis of any (micro)organism.
Kolker
Eugene
E
BIATECH, Bothell, Washington 98011, USA. ekolker@biatech.org
Purvine
Samuel
S
Galperin
Michael Y
MY
Stolyar
Serg
S
Goodlett
David R
DR
Nesvizhskii
Alexey I
AI
Keller
Andrew
A
Xie
Tao
T
Eng
Jimmy K
JK
Yi
Eugene
E
Hood
Leroy
L
Picone
Alex F
AF
Cherny
Tim
T
Tjaden
Brian C
BC
Siegel
Andrew F
AF
Reilly
Thomas J
TJ
Makarova
Kira S
KS
Palsson
Bernhard O
BO
Smith
Arnold L
AL
D000332:Aerobiosis; D000693:Anaerobiosis; D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D002417:Cattle; D002853:Chromatography, Liquid; D015964:Gene Expression Regulation, Bacterial; D006193:Haemophilus influenzae / Q000737:chemistry* / Q000235:genetics / Q000254:growth & development; D006801:Humans; D020543:Proteome; D011817:Rabbits; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization; D014357:Trypsin
D001426:Bacterial Proteins; D020543:Proteome; D014357:Trypsin