“Unknown-genome” proteomics- based identification of a new NADP-epimerase/dehydratase from Desulf. phosphitoxidans by inverted-PCR, Edman-sequencing and high resolution mass spectrometry

Abstract

Protein identification in proteomics is generally based on the availability of genomic data. Using amenable databases, identifi cation in "bottom–up" proteomics is often straightforward but is highly complex in the absence of genome data, which typically requires "de novo"- identification approaches. We present here a new approach for identification of proteins from a bacterial strain, Desulfotignum phosphitoxidans, with unknown genomic background, using a combination of (i), inverted PCR of degenerate primers derived from N-terminal Edman sequencing, and (ii) high resolution MALDI-FTICR mass spectrometric peptide mass fingerprint-proteomics of expressed proteins. Desulfotignum phosphitoxidans is an anaerobic sulfatereducing bacterium from marine sediment in which phosphite oxidation was found crucial for energy metabolism. Culturing under different growth conditions provided 4 specifically expressed proteins with molecular masses of ca. 40 kDa in the presence of phosphate, which were subjected to 2D-gel electrophoretical separation for soluble and membrane fractions using PDQuest comparative analysis. N-Terminal sequences of the proteins, determined by Edman analysis were used for inverted PCR of degenerate primers, and provided a series of ORF candidates, one of which coded for a putative NAD-dependent dehydratase. In a complementary approach, protein spots from the 2D-gels were excised, digested with Lys-C protease, and digestion mixtures analysed by MALDI-FTICR-MS. The accurate peptide masses unequivocally matched to identify a new NAD-dependant epimerase/dehydratase. The detailed functional characterization of the new protein, and further development and application of this approach to proteome analysis with unknown genomic background, are currently in progress.