Mycobacterium tuberculosis (MTB) is a species of pathogenic bacteria and the causative agent of tuberculosis. The type strain H37Rv has been sequenced in 1998, while many previous studies found its predicted genes exhibit frequent errors, particularly in start codons. Here, we applied a TMPP [(N-Succinimidyloxycarbonylmethyl) tris (2,4,6-trimethoxyphenyl) phosphonium bromide] labeling combined with StageTip separating strategy to characterize the N-terminal peptides. We identified 2,598 annotated proteins, which 1,078 proteins were labeled by TMPP.