Rational engineering of a virulence gene from Mycobacterium tuberculosis facilitates proteomic analysis of a natural protein N-terminus.
Mass spectrometry (MS) for the detection of proteins is an indispensable tool for evaluating the biological processes of the proteome. Proteomics frequently requires proteolysis of proteins into peptide fragments. Proteins can be refractory to ideal proteolysis at the sequence level rendering them difficult to analyze by routine proteomics methods. EsxA (ESAT-6, Early Secreted Antigen, 6kDa) is a major virulence determinant of Mycobacterium tuberculosis, the cause of human tuberculosis. EsxA is routinely used to evaluate mycobacterial virulence in the laboratory and as a biomarker for tuberculosis in humans. The sequence of EsxA hinders deeper MS analysis beyond routine detection. Here we engineer the sequence of EsxA to add desirable tryptic properties aimed at improving complex MS analysis. We demonstrate that EsxA variants are amenable to MS analysis and remain functional in established in vitro and ex vivo assays of Esx-1-function. We provide the first demonstration of molecular engineering to specifically improve MS analysis of individual microbial proteins.
Department of Biological Sciences, Eck Institute for Global Health, Center for Rare and Neglected Diseases, University of Notre Dame, Notre Dame IN, USA.
Cristal
C
Reyna
Department of Biological Sciences, Eck Institute for Global Health, Center for Rare and Neglected Diseases, University of Notre Dame, Notre Dame IN, USA.
Felix
F
Mba Medie
Department of Chemistry and Biochemistry, Eck Institute for Global Health, Center for Rare and Neglected Diseases, University of Notre Dame, Notre Dame IN, USA.
Matthew M
MM
Champion
Department of Biological Sciences, Eck Institute for Global Health, Center for Rare and Neglected Diseases, University of Notre Dame, Notre Dame IN, USA.
Patricia A
PA
Champion
D000942:Antigens, Bacterial / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods; D006801:Humans; D013058:Mass Spectrometry; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000472:pathogenicity; D040901:Proteomics; D014376:Tuberculosis / Q000175:diagnosis / Q000235:genetics* / Q000382:microbiology; D037521:Virulence Factors / Q000235:genetics*
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C093276:ESAT-6 protein, Mycobacterium tuberculosis; D037521:Virulence Factors