engleski

Rapid identification of microorganisms by protein reading concept using CAF-/CAF+ reagent

Currently used LC-MS/(MS) technique for protein identification is based on database matching of non-derivatized peptide signals recorded in the positive ion mode. Although widely used, such an approach does not always provide satisfactory sequence coverage to unambiguously identify a peptide/protein sample. We have introduced a peptide derivatization method that allows for spectra recording in both positive and negative ion mode in order to increase peptide sequence coverage. Peptides identified as overlapped amino acid sequences read from both ion modes assign peptides unambiguously after de novo sequencing and protein database identification. The protein-reading concept using CAF-/CAF+ reagent (chemically activated fragmentation negative/chemically activated fragmentation positive) enables fast, highly accurate, reliable and easy to use identification of microorganisms down to the species level. CAF-/CAF+ (5-formylbenzene-1, 3-disulfonic acid*) is a chemical reagent for the derivatization of peptide samples prior to analysis by MALDI (Matrix Assisted Laser Desorption Ionization) Tandem Mass Spectrometry (MS/MS). For the first time, mass spectrometry can exploit both positive and negative ion mode, either for species or proteins identification. The CAF-/CAF+ method enables de novo sequencing of derivatized peptides with negative and positive ion mode tandem mass spectrometry (MS/MS–and MS/MS+). Peptide sequences are read from MS/MS spectra and matched against the NCBInr database by developed software named ProteinReader and confirmed by the mass spectrometry data of elucidated peptide mass sequences derived from the annotated genome.

identification of microorganisms ; mass spectrometry ; chemically activated fragmentation (CAF) ; de novo sequencing

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