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Comparison of methods for simultaneous identification of bacterial species and determination of metabolic activity by protein‐based stable isotope probing (Protein‐SIP) experiments
Author(s) -
Jehmlich Nico,
Schmidt Frank,
Taubert Martin,
Seifert Jana,
von Bergen Martin,
Richnow HansHermann,
Vogt Carsten
Publication year - 2009
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.4084
Subject(s) - chemistry , stable isotope probing , isotopic labeling , stable isotope ratio , peptide , shotgun , mass spectrometry , shotgun proteomics , isotope , stable isotope labeling by amino acids in cell culture , metabolic pathway , isobaric labeling , isotopes of nitrogen , fragmentation (computing) , biochemistry , nitrogen , protein sequencing , chromatography , peptide sequence , metabolism , proteomics , tandem mass spectrometry , protein mass spectrometry , gene , bacteria , organic chemistry , microorganism , computer science , genetics , biology , operating system , quantum mechanics , physics
We developed a concept for analysing carbon and nitrogen fluxes in microbial communities by employing protein‐based stable isotope probing (Protein‐SIP) in metabolic labelling experiments with stable isotope labelled substrates. For identification of microbial species intact protein profiling (IPP) can be used, whereas the assessment of their metabolic activity is achieved by shotgun mass mapping (SMM). Microbial cultures were grown on substrates containing 13 C or 15 N. For identification of species we tested both the IPP and the SMM approaches. Mass spectra (MALDI‐MS) were taken from mixtures of either intact proteins or peptides from tryptic digestion for generating species‐specific peak patterns. In the case of SMM, the fragmentation of peptides was additionally used to obtain sequence information for species identification. Mass spectra of peptide sequences allow calculation of the amount of 13 C or 15 N incorporation within peptides for determining metabolic activity of the specific species. The comparison of IPP and SMM revealed a higher robustness of species identification by SMM. In addition, the assessment of incorporation levels of 13 C and 15 N into peptides by SMM revealed a lower uncertainty (0.5–0.8 atom %) compared to IPP (6.4–8.9 atom %). The determination of metabolic activity and function of individual species by Protein‐SIP can help to analyse carbon and nitrogen fluxes within microbial communities. Copyright © 2009 John Wiley & Sons, Ltd.