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

[Image: see text] 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 ly...

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Detalles Bibliográficos
Autores principales: Hollenstein, David M., Maurer-Granofszky, Margarita, Reiter, Wolfgang, Anrather, Dorothea, Gossenreiter, Thomas, Babic, Riccardo, Hartl, Natascha, Kraft, Claudine, Hartl, Markus
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2023
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10563155/
https://www.ncbi.nlm.nih.gov/pubmed/37712406
http://dx.doi.org/10.1021/acs.jproteome.3c00424
Descripción
Sumario:[Image: see text] 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.