Update publication information. Update information. Here we employed a non-enzymatic chemical labeling approach to introduce lactylation on lysine residues of model peptides and proteins. Tandem MS acquisition of lactylated peptide carriers on different MS platforms unequivocally led us to note an intense peak corresponding to the “cyclic” immonium ion of lactyllysine that is generated from the “linear” immonium ion upon the loss of NH3. The cycIm ion provides superior sensitivity and specificity as a lactylation marker than the linIm ion. Together, the ability to confidently identify protein lactylation empowers mapping the landscape of human lactylation proteome and fosters ensuing investigation into the regulatory mechanisms of lactylation when our understanding to this newly discovered PTM is still during its infancy.