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

Molecular & Cellular Proteomics : MCP
Diliana Dancheva SimeonovaMichael Przybylski

Abstract

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 masses 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 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...Continue Reading

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Mar 11, 2021·Proceedings of the National Academy of Sciences of the United States of America·Sophia D EwensJohn D Coates

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