期刊
JOURNAL OF PROTEOME RESEARCH
卷 22, 期 10, 页码 3383-3391出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.jproteome.3c00424
关键词
mass spectrometry; proteomics; affinity purification; immunopurification; nanobody; GFP; streptavidin; Sulfo-NHS-Acetate; chemical acetylation; ligand
We present an effective method to reduce codigestion of bead-bound ligands in affinity purification-mass spectrometry experiments. The method utilizes nontoxic chemicals and mild chemical reaction conditions, leading to a significant reduction in contaminating ligand peptides while improving sensitivity and quantitative accuracy in analysis.
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.
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