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Title (Primary) Protein-based stable isotope probing
Author Jehmlich, N.; Schmidt, F.; Taubert, M.; Seifert, J.; Bastida, F.; von Bergen, M.; Richnow, H.-H.; Vogt, C.;
Journal Nature Protocols
Year 2010
Department ISOBIO; PROTEOM;
Volume 5
Issue 12
Language englisch;
Keywords Protein-SIP; Stable isotope probing; Microbiology; Proteomics
Abstract We describe a stable isotope probing (SIP) technique that was developed to link microbe-specific metabolic function to phylogenetic information. Carbon (13C)- or nitrogen (15N)-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.
ID 10124
Persistent UFZ Identifier https://www.ufz.de/index.php?en=20939&ufzPublicationIdentifier=10124
Jehmlich, N., Schmidt, F., Taubert, M., Seifert, J., Bastida, F., von Bergen, M., Richnow, H.-H., Vogt, C. (2010):
Protein-based stable isotope probing
Nat. Protoc. 5 (12), 1957 - 1966