Analysis of recombinant protein expression by MALDI-TOF mass spectrometry of bacterial colonies

E. coli expressing soluble recombinant HIV antigens were analyzed directly by MALDI-TOF mass spectrometry (MS) from bacterial colonies picked from aga r plates. An HIV envelope (ENV) antigen construct, penvA, was expressed in E. coli by transformation of the plasmid pPL/penvA-M. The plasmid was co-tr ansformed into E. coli DH5 alpha cells with an equal quantity of the plasmi d pKRR826, the parent vector without the penvA insert, and plated at medium density on L-agar plus ampicillin plates. A total of 24 colonies from four agar plates (six colonies per plate) were picked and transferred into 50% acetonitrile-0.1% trifluoroacetic acid aliquots for analysis by MALDI-TOF M S. The MS analysis detected 10 of 24 colonies expressing the recombinant pr otein; one colony expressed a mutant penvA protein; eleven of 24 colonies s howed ions only from E. coli; and two of 24 colonies showed no detectable p roteins. When E. coli transformed only with plasmid pPL/penvA-M were examin ed all (10 of 10) colonies showed the penv insert by the MALDI-TOF MS metho d. The method is fast (less than 1.5 h for 24 colonies) and allows identifi cation of colonies expressing intact or mutant proteins directly from cultu re plates without sample purification.