Comparison of methods for simultaneous identification of bacterial species and determination of metabolic activity by protein-based stable isotope probing (Protein-SIP) experiments. Academic Article uri icon

Overview

abstract

  • 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.

publication date

  • June 1, 2009

Research

keywords

  • Bacterial Proteins
  • Carbon Isotopes
  • Nitrogen Isotopes
  • Pseudomonas putida
  • Spectrometry, Mass, Electrospray Ionization

Identity

Scopus Document Identifier

  • 67149131864

Digital Object Identifier (DOI)

  • 10.1002/rcm.4084

PubMed ID

  • 19449321

Additional Document Info

volume

  • 23

issue

  • 12