RESUMEN
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.
Asunto(s)
Sustitución de Aminoácidos/genética , Artefactos , Metionina/genética , Neoplasias/genética , Proteoma/genética , Treonina/genética , Línea Celular Tumoral , Reacciones Falso Positivas , Marcadores Genéticos/genética , Variación Genética/genética , Humanos , Proteómica/métodos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Cancer genome deviates significantly from the reference human genome, and thus a search against standard genome databases in cancer cell proteomics fails to identify cancer-specific protein variants. The goal of this Article is to combine high-throughput exome data [Abaan et al. Cancer Res. 2013] and shotgun proteomics analysis [Modhaddas Gholami et al. Cell Rep. 2013] for cancer cell lines from NCI-60 panel to demonstrate further that the cell lines can be effectively recognized using identified variant peptides. To achieve this goal, we generated a database containing mutant protein sequences of NCI-60 panel of cell lines. The proteome data were searched using Mascot and X!Tandem search engines against databases of both reference and mutant protein sequences. The identification quality was further controlled by calculating a fraction of variant peptides encoded by the own exome sequence for each cell line. We found that up to 92.2% peptides identified by both search engines are encoded by the own exome. Further, we used the identified variant peptides for cell line recognition. The results of the study demonstrate that proteome data supported by exome sequence information can be effectively used for distinguishing between different types of cancer cell lines.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Exoma , Proteoma/metabolismo , Secuencia de Aminoácidos , Biomarcadores de Tumor/química , Biomarcadores de Tumor/genética , Línea Celular Tumoral , Humanos , Mutación Missense , Fragmentos de Péptidos/química , Polimorfismo de Nucleótido Simple , Proteoma/química , Proteoma/genéticaRESUMEN
PURPOSE: The aim of this study was to estimate a possibility of mycosis fungoides (MF) diagnostics based on protein profiling in blood serum. EXPERIMENTAL DESIGN: We obtained and analysed samples of blood serum from 23 patients with MF, and 29 psoriasis patients and 22 healthy donors as controls. Protein profiling was carried out using SELDI TOF MS SELDI-TOF and also profiling of 27 cytokines with multiplex immunoassay technology was implemented. RESULTS: MS data analysis of sera did not give satisfactory statistical discrimination between the groups. Antibody-based cytokine profiling revealed a number of cytokines with a change in their concentrations in both MF and psoriasis (IL-1Ra, IL-4, G-CSF). The C-X-C motif chemokine 10 (IP-10, CXCL10) cytokine had a significantly increased concentration (p<0,001) in samples from MF patients as compared with the other groups. CONCLUSIONS AND CLINICAL RELEVANCE: IP-10 may be considered as a promising biomarker for the differentiation between MF and other skin conditions.