Mapping and identification of Mycobacterium tuberculosis proteins by two-dimensional gel electrophoresis, microsequencing and immunodetection

Mycobacterium tuberculosis is the infectious agent giving rise to human tub erculosis. The entire genome of M. tuberculosis, comprising approximately 4 000 open reading frames, has been sequenced. The huge amount of information released from this project has facilitated proteome analysis of M. tubercu losis. Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was ap plied to fractions derived from M. tuberculosis culture filtrate, cell wall , and cytosol, resulting in the resolution of 376, 413, and 395 spots, resp ectively, in silver-stained gels. By microsequencing and immunodetection, 3 8 culture filtrate proteins were identified and mapped, of which 12 were id entified for the first time. In the same manner, 23 cell wall proteins and 19 cytosol proteins were identified and mapped, with 9 and 10, respectively , being novel proteins. One of the novel proteins was not predicted in the genome project, and for four of the identified proteins alternative start c odons were suggested. Fourteen of the culture filtrate proteins were propos ed to possess signal sequences. Seven of these proteins were microsequenced and the N-terminal sequences obtained confirmed the prediction. The data p resented here are an important complement to the genetic information, and t he established 2-D PAGE maps (also available at: www.ssi.dk/publichealth/tb immun) provide a basis for comparative studies of protein expression.