Chemical acetylation of ligands and two-step digestion protocol for reducing codigestion in affinity purification–mass spectrometry

Abstract: 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

Object Details

• Location: Deutsche Nationalbibliothek Frankfurt am Main
• Extent: Online-Ressource
• Language: Englisch
• Notes: Journal of proteome research. - 22, 10 (2023) , 3383-3391, ISSN: 1535-3907

Contributors, Places and Time

• Event: Veröffentlichung
• (where): Freiburg
• (who): Universität
• (when): 2023
• Creator: Hollenstein, David M. ; Maurer-Granofszky, Margarita ; Reiter, Wolfgang ; Anrather, Dorothea ; Gossenreiter, Thomas ; Babic, Riccardo ; Hartl, Natascha ; Kraft, Claudine ; Hartl, Markus

Further information

• DOI: 10.1021/acs.jproteome.3c00424
• URN: urn:nbn:de:bsz:25-freidok-2393573
• Rights: Open Access; Der Zugriff auf das Objekt ist unbeschränkt möglich.
• Last update: 14.08.2025, 10:56 AM CEST