TY - JOUR
T1 - Chemical Acetylation of Ligands and Two-Step Digestion Protocol for Reducing Codigestion in Affinity Purification-Mass Spectrometry
AU - Hollenstein, David M
AU - Maurer-Granofszky, Margarita
AU - Reiter, Wolfgang
AU - Anrather, Dorothea
AU - Gossenreiter, Thomas
AU - Babic, Riccardo
AU - Hartl, Natascha
AU - Kraft, Claudine
AU - Hartl, Markus
PY - 2023/10/6
Y1 - 2023/10/6
N2 - 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.
AB - 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.
KW - affinity purification
KW - chemical acetylation
KW - GFP
KW - immunopurification
KW - ligand
KW - mass spectrometry
KW - nanobody
KW - proteomics
KW - streptavidin
KW - Sulfo-NHS-Acetate
UR - http://www.scopus.com/inward/record.url?scp=85174684678&partnerID=8YFLogxK
U2 - 10.1021/acs.jproteome.3c00424
DO - 10.1021/acs.jproteome.3c00424
M3 - Article
C2 - 37712406
SN - 1535-3893
VL - 22
SP - 3383
EP - 3391
JO - Journal of Proteome Research
JF - Journal of Proteome Research
IS - 10
ER -