"Unknown-genome" proteomics : a new NAD(P)-dependent epimerase/dehydratase revealed by N-terminal sequencing, inverted PCR and high resolution mass spectrometry

Abstract

We present here a new approach that enabled the identification of a newstrain 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 hosphorylations.