We present here a new approach that enabled the identification of a new protein from a bacterial strain with unknown genomic background, using a combination of inverted PCR with degenerate primers derived from N-terminal protein sequences, and high resolution peptide mass determination of proteolytic digests from two dimensional electrophoretic separation. Proteins of the sulfate-reducing bacterium Desulfotignum phosphitoxidans specifically induced in the presence of phosphite were separated by two dimensional gel electrophoresis as a series of apparent soluble and membrane-bound isoforms with molecular weights of approximately 35 kDa. Inverted PCR based on N-terminal sequences and high resolution peptide mass fingerprinting by Fourier transform-ion cyclotron resonance mass spectrometry provided the identification of a new NAD(P)-epimerase/dehydratase by specific assignment of peptide masses to a single open reading frame (ORF), excluding other possible ORF candidates. The protein identification was ascertained by chromatographic separation and sequencing of internal proteolytic peptides. Metal ion affinity-isolation of tryptic peptides and high resolution mass spectrometry provided the identification of five phosphorylations, identified in the domains (23 – 47) and (91 – 118) of the protein. In agreement with the phosphorylations identified, direct molecular weight determination of the soluble protein eluted from the two dimensional gels by mass spectrometry provided a molecular mass of 35400 Da, consistent with an average degree of three phosphorylations.