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

We present an effective, fast and user-friendly method to reduce co-digestion of bead-bound ligands, such as antibodies or streptavidin, in affinity purification-mass spectrometry experiments. A short pre-incubation 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 non-toxic 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.