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.

Minimal and hybrid hydrogenases are active from archaea

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“…Labeled cells were lysed with RIPA buffer, and protein concentration was measured using Pierce Protein Assay Kit . Prior to affinity purification, streptavidin bead slurry (Thermo-Fisher Scientific, Cat #88817) was acetylated with 5 mM sulfo-NHS acetate in 50 mM ammonium bicarbonate with 0.2% Tween at RT for 1 h, as described [49]. In brief, beads were washed three times with 50 mM HEPES, pH 7.8, 0.2% Tween on a magnetic rack, incubated with sulfo-NHS acetate while rotating for 1 h at RT, subsequently washed in 50 mM ammonium bicarbonate, 0.2% Tween, and finally stored in PBS-T (0.2% Tween), 0.02% sodium azide at 4°C until further use.…”