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Searching deep proteome data for 9 NCI-60 cancer cell lines obtained earlier by Moghaddas Gholami et al. (Cell Reports, 2013) against a database from cancer genomes returned a variant tryptic peptide fragment 57-72 of molecular chaperone HSC70, in which methionine residue at 61 position is replaced by threonine, or isothreonine (homoserine), residue. However, no traces of the corresponding genetic alteration were found in the cell line genomes reported by Abaan et al. (Cancer Research, 2013). Studying on the background of this modification led us to conclude that a conversion of methionine into isothreonine resulted from iodoacetamide treatment of the probe during a sample preparation step. We found that up to 10% of methionine containing peptides experienced the above conversion for the datasets under study. The artifact was confirmed by model experiment with bovine albumin, where three of four methionine residues were partly converted to isothreonine by conventional iodoacetamide treatment. This experimental side reaction has to be taken into account when searching for genetically encoded peptide variants in the proteogenomics studies., Biological Significance: A lot of effort is currently put into proteogenomics of cancer. Studies detect non-synonymous cancer mutations at protein level by search of high-throughput LC-MS/MS data against customized genomic databases. In such studies, much attention is paid to potential false positive identifications. Here we describe one possible cause of such false identifications, an artifact of sample preparation which mimics methionine to threonine nucleic acid-encoded variant. The methionine to isothreonine conversion should be taken into consideration for correct interpretation of proteogenomic data., (Copyright © 2015 Elsevier B.V. All rights reserved.)