Tuberculosis(TB)has been a major global health threat,there is an urgent need to develop rapid,non-culture-required biomarker for rapidly and accurately detecting TB.Mycobacterium tuberculosis(MTB)antigens in patient can provide direct evidence of TB,while common applied antigens hadnt listed as available method owing to their homology with antigens of many nontuberculous mycobacteria(NTM).MTB-specific antigen will be make up for that deficit.Here,we refined H37Rv annotation by proteogenomics based on two public and our H37Rv MS datasets considering different growing period,protein resource,sample separation,digestion strategy,and so on.Total 50,820 peptides were identified,which mapping to 3,201(79.67%)proteins among 4,018 annotated protein-coding genes in H37Rv(TubercuList,2016).After novel peptides strict filtering by spectrum quality and peptide synthesis,we found 28 novel gene-coding N-terminus regions and verified 22 confirmed novel genes.We found half of novel genes behaved Mycobacterium tuberculosis complex(MTBC)specific,which were verified by PCR amplification and comparative genomics.This study describes a rational approach to searching and verifying H37Rv novel genes,and highlights the importance of accurate diagnosis of MTBC in clinic and selecting MTB-specific antigens.