The cytoplasmic phosphoproteome of the Gram-negative bacterium Campylobacter jejuni: evidence for modification by unidentified protein kinases.
We have undertaken a comprehensive analysis of cytoplasmic protein phosphorylation in Campylobacter jejuni by mass spectrometric identification of phosphoproteins and localization of the sites of modification by phosphopeptide analyses. Cell extracts, enriched for phosphoproteins using Fe(III) IMAC or commercial phosphoprotein purification kits, were analyzed by 1-D and 2-D SDS-PAGE and subjected to mass fingerprinting by in-gel tryptic digestion and MALDI-TOF MS. Fifty-eight phosphopeptides were identified from 1-D gel bands by nano-LC-MS/MS and automated searching in a C. jejuni ORF database resulting in the unequivocal identification of 36 phosphoproteins of diverse function. In addition to elongation factors and chaperonins, which have been reported to be phosphorylated in other bacteria, the major phosphoproteins included bacterioferritin and superoxide dismutase. The sequences around the phosphorylated Ser and Thr residues are indicative of specific kinases being responsible for some of the modifications. However, many of the other identified proteins are enzymes that have phosphorylated substrates, including ATP, hence other modifications may arise from autophosphorylation. Comparative analyses of IMAC extracts from the Escherichia coli strain AD202 and Helicobacter pylori resulted in the identification of homologs of six of the C. jejuni phosphoproteins, though their overall phosphoproteome maps were distinctly different.
Voisin
Sébastien
S
Institute for Biological Sciences, National Research Council of Canada, Ottawa, Canada.
Watson
David C
DC
Tessier
Luc
L
Ding
Wen
W
Foote
Simon
S
Bhatia
Smita
S
Kelly
John F
JF
Young
N Martin
NM
D000446:Aldehyde-Lyases / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D016123:Campylobacter jejuni / Q000737:chemistry* / Q000378:metabolism; D003573:Cytochrome b Group / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D005293:Ferritins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D016480:Helicobacter pylori / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D010748:Phosphopeptides / Q000032:analysis / Q000378:metabolism; D010750:Phosphoproteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D020543:Proteome / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D053719:Tandem Mass Spectrometry
D001426:Bacterial Proteins; D003573:Cytochrome b Group; D010748:Phosphopeptides; D010750:Phosphoproteins; D020543:Proteome; D005293:Ferritins; C018032:bacterioferritin; C020011:2-dehydro-3-deoxyphosphooctonate aldolase; D011494:Protein Kinases; D000446:Aldehyde-Lyases