T cells recognizing a peptide contaminant undetectable by mass spectrometry

• Published in: PloS one 2011-12, Vol.6 (12), p.e28866-e28866
• Main Authors: Brezar, Vedran, Culina, Slobodan, Østerbye, Thomas, Guillonneau, François, Chiappetta, Giovanni, Verdier, Yann, Vinh, Joelle, Wong, F Susan, Buus, Søren, Mallone, Roberto
• Format: Article
• Language: English
• Subjects: Adaptive immunology; Amino Acid Sequence; Amino acids; Animals; Antigenic determinants; Antigens; Biology; Catalysis; CD8 antigen; CD8-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes - immunology; Chromatography; Clone Cells; Cloning; Contaminants; Cytotoxicity; Dendritic cells; Diabetes; Diabetes mellitus; Epitopes; Epitopes, T-Lymphocyte; Epitopes, T-Lymphocyte - immunology; Glucose-6-Phosphatase; Glucose-6-Phosphatase - immunology; Health sciences; Histocompatibility Antigens Class I; Histocompatibility Antigens Class I - immunology; Humans; Identification; Immunization; Immunodominance; Immunology; Impurities; Interferon-gamma; Interferon-gamma - immunology; Islets of Langerhans; Islets of Langerhans - immunology; Life Sciences; Liquid chromatography; Lymphocytes; Lymphocytes T; Major histocompatibility complex; Mass Spectrometry; Mass Spectrometry - methods; Mass spectroscopy; Mice; Mice, Inbred NOD; Molecular Sequence Data; Obesity; Peptides; Peptides - chemistry; Peptides - immunology; Proteins; Proteins - immunology; Quality control; Reproducibility of Results; Rodents; Scientific imaging; Sequence Analysis, Protein; Spectroscopy; Studies; Synthetic peptides; T cell receptors; T cells; Trace contaminants
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0028866

Synthetic peptides are widely used in immunological research as epitopes to stimulate their cognate T cells. These preparations are never completely pure, but trace contaminants are commonly revealed by mass spectrometry quality controls. In an effort to characterize novel major histocompatibility complex (MHC) Class I-restricted β-cell epitopes in non-obese diabetic (NOD) mice, we identified islet-infiltrating CD8+ T cells recognizing a contaminating peptide. The amount of this contaminant was so small to be undetectable by direct mass spectrometry. Only after concentration by liquid chromatography, we observed a mass peak corresponding to an immunodominant islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)(206-214) epitope described in the literature. Generation of CD8+ T-cell clones recognizing IGRP(206-214) using a novel method confirmed the identity of the contaminant, further underlining the immunodominance of IGRP(206-214). If left undetected, minute impurities in synthetic peptide preparations may thus give spurious results.