NHS-SS-Biotin for Protein Labeling[1]
This experimental protocol is compiled based on the research content and for reference only. Please adjust according to actual needs.
(1) Reagent preparation: Dissolve NHS-SS-Biotin in dimethyl sulfoxide (DMSO) to prepare a 100mM stock solution. Store in a desiccator at -30℃ to maintain anhydrous conditions. Prepare the target protein in phosphate-buffered saline (PBS) at a concentration of 2mg/mL. Prepare 10% hydroxylamine solution. Prepare 20mM tris(2-carboxyethyl)phosphine (TCEP) solution. Prepare 50mM iodoacetamide (IAA) solution. Prepare 0.1% Rapigest solution. Prepare 50mM Tris-HCl buffer (pH=7.5). Prepare 30mM NaCl solution. Prepare 1% formic acid (FA) solution. Prepare 10% trifluoroacetic acid (TFA) solution.
(2) Protein chemical labeling: Incubate the target protein with NHS-SS-Biotin reagent at 25℃ for 30 minutes. The molar ratio of protein to NHS-SS-Biotin is 1:50. Add 10% hydroxylamine and incubate at 25℃ for 15 minutes to quench the labeling reaction. Store the labeled sample at -80℃.
(3) Streptavidin magnetic bead enrichment: Take streptavidin magnetic beads and incubate with the labeled protein sample at 4℃ for 16 hours. Remove the supernatant. Wash sequentially with buffer A (2% SDS), buffer B (50mM Tris-HCl, 500mM NaCl, 2% Triton-X), buffer C (50mM Tris-HCl, 250mM NaCl, 0.5% SDS, 0.5% Triton-X), and buffer D (2M Urea, 50mM Tris-HCl) twice each.
(4) Disulfide bond reduction and protein elution: Add elution buffer (20mM TCEP, 0.1% Rapigest, 30mM NaCl, 50mM Tris-HCl) to the magnetic beads and incubate at 37℃ for 60 minutes. Then add 1% FA (dissolved in 50mM Tris-HCl) and incubate at 37℃ for 30 minutes. Combine the two eluates. Add IAA for alkylation in the dark for 30 minutes. Then add 20mM TCEP and treat for 10 minutes to remove excess IAA.
(5) Protein digestion and peptide desalting: Add Trypsin/Lys-C mixed enzyme (enzyme/protein=1:30, w/w) to the eluate and incubate at 37℃ for 16 hours. Add 10% TFA until pH<3 to terminate digestion. Incubate at 37℃ for 45 minutes. Centrifuge at 13000rpm for 10 minutes to remove Rapigest. Desalt peptides using a C18 96-well μElution plate. Dry the peptides. Store at -30℃ for LC-MS/MS analysis.
Precautions:
(1) NHS-SS-Biotin mainly modifies lysine side chains and N-termini of proteins, but also modifies serine, threonine, histidine, and other amino acids. The modification efficiency is approximately 10-14%.
(2) Biotinylation at lysine sites reduces trypsin digestion efficiency, resulting in over 90% of modified peptides having at least one missed cleavage site. However, SS-biotinylated peptides partially alleviate this problem compared to traditional biotinylated peptides.
(3) For your safety and health, please wear a lab coat and disposable gloves during the operation.
References:
[1] Li H, Frankenfield AM, Houston R, et al. Thiol-Cleavable Biotin for Chemical and Enzymatic Biotinylation and Its Application to Mitochondrial TurboID Proteomics. J Am Soc Mass Spectrom. 2021 Sep 1;32(9):2358-2365.
