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Biotin Switch-Based Capture

Biotin switch methodology was first described for the study of 5-nitrosothiols, whereby nitrosylated cysteines are converted to biotinylated cysteines using N-hydroxysuccinimide (NHS) chemistry. The biotinylated peptides are purified by avidin affinity chromatography and analyzed by LC-MS [108]. The procedure has been modified for application to detection of endogenously lysine-acetylated peptides, termed the acetyl-biotin switch technique [94]. In this technique, NHS forms a stable amide bond with free (N-terminal, lysine-e) amines, resulting in a mixture of peptides with either NHS-blocked lysines or preexisting Kac. Lysine acetylation is removed in vitro by treatment with deacetylase enzyme. The resulting free lysines are derivatized with NHS-SS- biotin followed by affinity capture on streptavidin resin. The identity of the biotin-captured peptides is determined by LC-MS/MS. This approach has been applied to the identification of a range of potential substrates, including the 14-3-3Z, as a substrate for Sirtl deacetylase, a finding that links Sirtl and 14-3-3Z, acetylation to control of caspase-2-dependent apoptotic cell death mediated by chemotherapeutic agents [94]. In general, biotin switch techniques can be compromised by incomplete reaction and side reactions. Technical aspects of the acetyl-biotin switch technique, including protocol details, advice on how to reduce false-positive protein identification, and coupling to quantitative MS approaches, including label-free analysis, are available [109]. In principle, the biotin technique could be applied to methylation by blocking of unmodified lysine and arginine residues, in vitro demethylation, and capture followed by MS analysis.

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