Chemical Acetylation of Ligands and Two-Step Digestion Protocol for Reducing Codigestion in Affinity Purification-Mass Spectrometry.

Autor:	Hollenstein DM; Department for Biochemistry and Cell Biology, University of Vienna, Center for Molecular Biology, Vienna Biocenter Campus (VBC), Dr. Bohr-Gasse 9, Vienna 1030, Austria.; Mass Spectrometry Facility, Max Perutz Laboratories, Vienna Biocenter Campus (VBC), Dr. Bohr-Gasse 7, Vienna 1030, Austria., Maurer-Granofszky M; St. Anna Children's Cancer Research Institute (CCRI), Zimmermannplatz 10, Vienna 1090, Austria., Reiter W; Mass Spectrometry Facility, Max Perutz Laboratories, Vienna Biocenter Campus (VBC), Dr. Bohr-Gasse 7, Vienna 1030, Austria., Anrather D; Mass Spectrometry Facility, Max Perutz Laboratories, Vienna Biocenter Campus (VBC), Dr. Bohr-Gasse 7, Vienna 1030, Austria., Gossenreiter T; Mass Spectrometry Facility, Max Perutz Laboratories, Vienna Biocenter Campus (VBC), Dr. Bohr-Gasse 7, Vienna 1030, Austria., Babic R; Institute of Biochemistry and Molecular Biology, ZBMZ, Faculty of Medicine, University of Freiburg, Freiburg 79104, Germany.; Faculty of Biology, University of Freiburg, Freiburg 79104, Germany.; Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, Freiburg 79104, Germany., Hartl N; Department for Biochemistry and Cell Biology, University of Vienna, Center for Molecular Biology, Vienna Biocenter Campus (VBC), Dr. Bohr-Gasse 9, Vienna 1030, Austria.; Mass Spectrometry Facility, Max Perutz Laboratories, Vienna Biocenter Campus (VBC), Dr. Bohr-Gasse 7, Vienna 1030, Austria., Kraft C; Institute of Biochemistry and Molecular Biology, ZBMZ, Faculty of Medicine, University of Freiburg, Freiburg 79104, Germany.; CIBSS - Centre for Integrative Biological Signalling Studies, University of Freiburg, Freiburg 79104, Germany., Hartl M; Department for Biochemistry and Cell Biology, University of Vienna, Center for Molecular Biology, Vienna Biocenter Campus (VBC), Dr. Bohr-Gasse 9, Vienna 1030, Austria.; Mass Spectrometry Facility, Max Perutz Laboratories, Vienna Biocenter Campus (VBC), Dr. Bohr-Gasse 7, Vienna 1030, Austria.
Jazyk:	angličtina
Zdroj:	Journal of proteome research [J Proteome Res] 2023 Oct 06; Vol. 22 (10), pp. 3383-3391. Date of Electronic Publication: 2023 Sep 15.
DOI:	10.1021/acs.jproteome.3c00424
Abstrakt:	We present an effective, fast, and user-friendly method to reduce codigestion of bead-bound ligands, such as antibodies or streptavidin, in affinity purification-mass spectrometry experiments. A short preincubation of beads with Sulfo-NHS-Acetate leads to chemical acetylation of lysine residues, making ligands insusceptible to Lys-C-mediated proteolysis. In contrast to similar approaches, our procedure offers the advantage of exclusively using nontoxic chemicals and employing mild chemical reaction conditions. After binding of bait proteins to Sulfo-NHS-Acetate treated beads, we employ a two-step digestion protocol with the sequential use of Lys-C protease for on-bead digestion followed by in-solution digestion of the released proteins with trypsin. The implementation of this protocol results in a strong reduction of contaminating ligand peptides, which allows significantly higher amounts of sample to be subjected to LC-MS analysis, improving sensitivity and quantitative accuracy.
Databáze:	MEDLINE
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