Updated project metadata. Protein glycation is a type of post-translational modification (PTM) involving complex non-enzymatic reactions of reducing sugars or reactive dicarbonyls with proteins, generating a heterogeneous group of advanced glycation end-products (AGEs) such as carboxymethyllysine (CML). In bottom-up proteomics, 2-iodoacetamide (IAA) is the most commonly used reagent for cysteine alkylation before trypsin digestion, which exogenously gives a carbamidomethylation (CAM) group on the side chain of cysteine residue. However, offisite alkylation of IAA can occur at the protein N-terminus and other amino acid residues such as lysine residue. Here, for the first time, we provided evidence that IAA alkylation can result in false-positive identifications of CML, and N-isopropylacrylamide (NIPAM) can be used as an IAA alternative to avoid these pitfalls.