Identification of fur, aconitase, and other proteins expressed by Mycobacterium tuberculosis under conditions of low and high concentrations of iron by combined two-dimensional gel electrophoresis and mass spectrometry
Dk. Wong et al., Identification of fur, aconitase, and other proteins expressed by Mycobacterium tuberculosis under conditions of low and high concentrations of iron by combined two-dimensional gel electrophoresis and mass spectrometry, INFEC IMMUN, 67(1), 1999, pp. 327-336
Iron plays a critical role in the pathophysiology of Mycobacterium tubercul
osis. To gain a better understanding of iron regulation by this organism, w
e have used two-dimensional (2-D) gel electrophoresis, mass spectrometry, a
nd database searching to study protein expression in hi. tuberculosis under
conditions of high and low iron concentration. Proteins in cellular extrac
ts from M. tuberculosis Erdman strain grown under low-iron (1 mu M) and hig
h-iron (70 mu M) conditions were separated by 2-D polyacrylamide gel electr
ophoresis, which allowed high-resolution separation of several hundred prot
eins, as visualized by Coomassie staining, The expression of at least 15 pr
oteins was induced, and the expression of at least 12 proteins was decrease
d under low-iron conditions, In-gel trypsin digestion was performed on thes
e differentially expressed proteins, and the digestion mixtures were analyz
ed by matrix-assisted laser desorption ionization time-of-flight mass spect
rometry to determine the molecular masses of the resulting tryptic peptides
, Partial sequence data on some of the peptides were obtained by using afte
r source decay and/or collision-induced dissociation. The fragmentation dat
a were used to search computerized peptide mass and protein sequence databa
ses for known proteins. Ten iron-regulated proteins were identified, includ
ing Fur and aconitase proteins, both of which are known to be regulated by
iron in other bacterial systems. Our study shows that, where large protein
sequence databases are available from genomic studies, the combined use of
2-D gel electrophoresis, mass spectrometry, and database searching to analy
ze proteins expressed under defined environmental conditions is a powerful
tool for identifying expressed proteins and their physiologic relevance.