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This is followed by the second alkylation step, where newly formed sulfhydryl groups are chemically blocked with an alkylating reagent distinct from that used in the first step [e.g., NEM, IAM, 2-chloroacetamide, iodoacetic acid (IAA), 4-vinylpyridine, methanethiosulfonate (MMTS)] (McDonagh et al., 2014)

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