Analysis of protein species differentiation among mycobacterial low-Mr-secreted proteins by narrow pH range Immobiline gel 2-DE-MALDI-MS

Abstract

Secreted proteins of bacteria are preferentially capable of interacting with host cells and are therefore of special biological and medical interest. Narrow pH range 2-DE and MALDI-TOFTOF-MS combine high-resolution protein separation with highly sensitive identification of proteins. Secreted proteins of Mycobacterium tuberculosis were separated at the protein species level, distinguishing different protein species of one protein. We focused on the pI range 4.0-4.7 and the Mr range 6-20kDa of the 2-DE pattern. Out of 128 analyzed spots, 121 were identified resulting in 33 different proteins with 277 different protein species, accumulating in a mean of 8.4 protein species per protein. Overrepresentation was found for the protein classes "virulence, detoxification, adaption", "information pathways", "cell wall and cell processes" and "intermediary metabolism and respiration". Thus far, 15 protein species of the ESX-1 family are characterized with 100% sequence coverage. More automated 2-DE procedures and more sensitive identification techniques are required for complete characterization of all of the protein species even in highly enriched samples, such as culture filtrates. Only then the functional level of proteomics will be achieved and potential biomarkers can be postulated at the molecular level. Proteomics is dominated by bottom-up approaches largely ignoring protein speciation. A prerequisite to reach the protein species level is to obtain 100% sequence coverage, which is a major challenge in proteomics. Here we show complete sequence information with a 2-DE-MS approach for 15 protein species. Acetylation of the N-terminus of ESAT-6 inhibits interaction with CFP-10, with direct consequences for pathogen-host interaction. This article is part of a Special Issue entitled: Trends in Microbial Proteomics.