Protein-based stable isotope probing Academic Article Article uri icon

Overview

MeSH Major

  • Cell Transformation, Viral
  • Oncogenes
  • Receptors, Cell Surface

abstract

  • We describe a stable isotope probing (SIP) technique that was developed to link microbe-specific metabolic function to phylogenetic information. Carbon (13 C)- or nitrogen (15 N)-labeled substrates (typically with >98% heavy label) were used in cultivation experiments and the heavy isotope incorporation into proteins (protein-SIP) on growth was determined. The amount of incorporation provides a measure for assimilation of a substrate, and the sequence information from peptide analysis obtained by mass spectrometry delivers phylogenetic information about the microorganisms responsible for the metabolism of the particular substrate. In this article, we provide guidelines for incubating microbial cultures with labeled substrates and a protocol for protein-SIP. The protocol guides readers through the proteomics pipeline, including protein extraction, gel-free and gel-based protein separation, the subsequent mass spectrometric analysis of peptides and the calculation of the incorporation of stable isotopes into peptides. Extraction of proteins and the mass fingerprint measurements of unlabeled and labeled fractions can be performed in 2-3 d. © 2010 Nature America, Inc. All rights reserved.

publication date

  • November 2010

Research

keywords

  • Academic Article

Identity

Digital Object Identifier (DOI)

  • 10.1038/nprot.2010.166

PubMed ID

  • 21127489

Additional Document Info

start page

  • 1957

end page

  • 1966

volume

  • 5

number

  • 12