I would like to ask a question. TMT labeled peptides by reacting with amino groups (primary amine groups), but the sulfhydryl-acetamide structure (carbamidomethyl of Cystein) formed after IAM (iodoacetamide) blocking Cystein also contains a primary amine group. Why does TMT not react with alkylated sulfhydryl groups? After all, there are primary amine groups in the structure（Blocked Cystein）, right? (It seems that this reaction is not considered when searching the library in TMT-proteomics, only the amino group of the N-terminal and K side chain are considered react with TMT).
The product of the reaction of iodoacetamide with the side chain of cysteine is not a primary amino group. It is an acetamide. The reactivity of an amide is not the same as the reactivity of an amine.
It may only be possible when free N-terminal cysteines exist which is not very frequent. Primary amines which can react with TMT reagent can only be presented by cysteine even after alkylation if they constitute the N terminal of the protein sequence and are not joined to peptide bonds through this side chain. This is the only primary amine at the carbamidomethyled cysteine structure. Otherwise, as previously indicated alkylated side is an acetamide that has no affinity due to the adjacent carbonyl moiety...
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