"Unknown genome" proteomics: a new NADP-dependent epimerase/dehydratase revealed by N-terminal sequencing, inverted PCR, and high resolution mass spectrometry.
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 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 35,400 Da, which is consistent with an average degree of three phosphorylations.
Simeonova
Diliana Dancheva
DD
Laboratory of Microbial Ecology, Department of Biology and paragraph signLaboratory of Analytical Chemistry, University of Konstanz, 78457 Konstanz, Germany.
Susnea
Iuliana
I
Moise
Adrian
A
Schink
Bernhard
B
Przybylski
Michael
M
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D017931:DNA Primers / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D005798:Genes, Bacterial; D016680:Genome, Bacterial / Q000235:genetics*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism*; D016366:Open Reading Frames / Q000235:genetics; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D040901:Proteomics / Q000379:methods*; D019998:Racemases and Epimerases / Q000235:genetics / Q000378:metabolism*; D015203:Reproducibility of Results; D020539:Sequence Analysis, Protein / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001426:Bacterial Proteins; D017931:DNA Primers; D010455:Peptides; D009249:NADP; D019998:Racemases and Epimerases