Proteomic profiling of culture filtrates from M.tuberculosis H37Rv

In this report, a novel tandem cascade of biomimetic affinity chromatography (BiAC) system was used to fractionate the culture filtrate proteins (CFPs) of M. tuberculosis (MTB) prior to LC-MS/MS analysis. Consequently, in fractionations by 6 BiAC columns, 541 proteins from CFPs were identified using stringent database research by Mascot and scaffold software with ƿ < 0.05, each protein was determined with two distinct peptide sequences. And 248 proteins were identified in un-fractionated culture filtrates. Compared with un-fractionated sample, both proteomic coverage and accuracy of MS analysis were remarkably improved using BiAC fractionations. In 242 identifications by both BiAC fractionations and un-fractionated samples, the average number of matched peptide sequence was increased from 14 hits to 44 hits after BiAC fractionation. The average coverage of peptide sequence of these 242 proteins was also increased from 19.6% (un-fractionation) to 26.1% (fractionation). Compared with previous identifications in CFPs, 11.15% (61/547) identifications in this study have not been reported. The majority of these newly identifications possess extremely biochemical characteristics that usually are not suitable be fractionated by gel-based proteomic approach, of which 12 proteins with Mw<10kDa or Mw>140kDa, 10 with pI<4.0 or pI>10. And 20 membrane proteins, of which have 9 with 2-14 transmembrane domains. Taken together, the BiAC system can enhance the proteomic detection coverage especially by reducing the complexity of sample, improving the low copy and extremely biochemical characteristic proteins.