title: Methionine to isothreonine conversion as a source of false discovery identifications of genetically encoded variants in proteogenomics.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Institute of Biomedical Chemistry, Moscow 119121, Russia; Karolinska Institutet, Stockholm SE-171 77, Sweden.
authors.forename: Alexey L
authors.initials: AL
authors.lastname: Chernobrovkin
authors.affiliation: Institute of Biomedical Chemistry, Moscow 119121, Russia.
authors.forename: Arthur T
authors.initials: AT
authors.lastname: Kopylov
authors.affiliation: Institute of Biomedical Chemistry, Moscow 119121, Russia.
authors.forename: Victor G
authors.initials: VG
authors.lastname: Zgoda
authors.affiliation: Institute of Biomedical Chemistry, Moscow 119121, Russia.
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Moysa
authors.affiliation: Institute of Biomedical Chemistry, Moscow 119121, Russia.
authors.forename: Mikhail A
authors.initials: MA
authors.lastname: Pyatnitskiy
authors.affiliation: Institute of Biomedical Chemistry, Moscow 119121, Russia.
authors.forename: Ksenia G
authors.initials: KG
authors.lastname: Kuznetsova
authors.affiliation: Institute of Biomedical Chemistry, Moscow 119121, Russia.
authors.forename: Irina Y
authors.initials: IY
authors.lastname: Ilina
authors.affiliation: Institute of Biomedical Chemistry, Moscow 119121, Russia.
authors.forename: Maria A
authors.initials: MA
authors.lastname: Karpova
authors.affiliation: Institute of Biomedical Chemistry, Moscow 119121, Russia; Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow 119991, Russia.
authors.forename: Dmitry S
authors.initials: DS
authors.lastname: Karpov
authors.affiliation: Institute of Biomedical Chemistry, Moscow 119121, Russia.
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Veselovsky
authors.affiliation: Institute for Energy Problems of Chemical Physics, Russian Academy of Sciences, Moscow 119334, Russia; Moscow Institute of Physics and Technology (State University), Moscow region, Dolgoprudny 141700, Russia.
authors.forename: Mark V
authors.initials: MV
authors.lastname: Ivanov
authors.affiliation: Institute for Energy Problems of Chemical Physics, Russian Academy of Sciences, Moscow 119334, Russia; Moscow Institute of Physics and Technology (State University), Moscow region, Dolgoprudny 141700, Russia.
authors.forename: Mikhail V
authors.initials: MV
authors.lastname: Gorshkov
authors.affiliation: Institute of Biomedical Chemistry, Moscow 119121, Russia.
authors.forename: Alexander I
authors.initials: AI
authors.lastname: Archakov
authors.affiliation: Institute of Biomedical Chemistry, Moscow 119121, Russia; Pirogov Russian National Research Medical University, Moscow 117997, Russia. Electronic address: smosh@mail.ru.
authors.forename: Sergei A
authors.initials: SA
authors.lastname: Moshkovskii
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics*; D016477:Artifacts; D045744:Cell Line, Tumor; D005189:False Positive Reactions; D005819:Genetic Markers / Q000235:genetics; D014644:Genetic Variation / Q000235:genetics; D006801:Humans; D008715:Methionine / Q000235:genetics*; D009369:Neoplasms / Q000235:genetics*; D020543:Proteome / Q000235:genetics*; D040901:Proteomics / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013912:Threonine / Q000235:genetics*
chemical_list: D005819:Genetic Markers; D020543:Proteome; D013912:Threonine; D008715:Methionine
keywords: Cancer cell line; Iodoacetamide; Isothreonine; Methionine; Proteogenomics; Tandem mass spectrometry