RESUMEN
Electrospray ionisation (ESI) mass spectrometry (MS) has been used extensively for the detection of peptides presented by major histocompatibility complex (MHC) molecules. This review focuses on the optimisation of electrospray mass spectrometry and the use of tandem mass spectrometry to sequence MHC class I peptides. We review the isolation of MHC class I peptides from the surface of cells with particular reference to tumour cells. In addition, we also discuss the advantages and disadvantages of the methods available to concentrate and fractionate the peptides prior to analysis by electrospray mass spectrometry.
Asunto(s)
Antígenos de Histocompatibilidad Clase I/análisis , Neoplasias/química , Neoplasias/inmunología , Péptidos/análisis , Animales , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Péptidos/química , Péptidos/inmunología , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
The BCR-ABL oncogene is central in the pathogenesis of chronic myeloid leukemia (CML). Here, tandem nanospray mass spectrometry was used to demonstrate cell surface HLA-associated expression of the BCR-ABL peptide KQSSKALQR on class I-negative CML cells transfected with HLA-A*0301, and on primary CML cells from HLA-A3-positive patients. These patients mounted a cytotoxic T-lymphocyte response to KQSSKALQR that also killed autologous CML cells, and tetramer staining demonstrated the presence of circulating KQSSKALQR-specific T cells. The findings are the first demonstration that CML cells express HLA-associated leukemia-specific immunogenic peptides and provide a sound basis for immunization studies against BCR-ABL.
Asunto(s)
Presentación de Antígeno , Antígenos de Neoplasias/inmunología , Antígenos de Superficie/inmunología , Proteínas de Fusión bcr-abl/inmunología , Antígeno HLA-A3/inmunología , Leucemia Mielógena Crónica BCR-ABL Positiva/inmunología , Proteínas de Neoplasias/inmunología , Células Madre Neoplásicas/inmunología , Fragmentos de Péptidos/inmunología , Adulto , Secuencia de Aminoácidos , Antígenos de Neoplasias/química , Antígenos de Superficie/química , Femenino , Proteínas de Fusión bcr-abl/química , Antígeno HLA-A3/genética , Humanos , Células K562/inmunología , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/química , Fragmentos de Péptidos/química , Proteínas Recombinantes de Fusión/inmunología , Espectrometría de Masa por Ionización de Electrospray , Linfocitos T Citotóxicos/inmunología , TransfecciónRESUMEN
The formation of copper/peptide complex ions by nano-electrospray and microbore HPLC-electrospray mass spectrometry has been investigated for major histocompatibility complex (MHC) class I and class II restricted peptides. Post-column addition of copper(II) acetate following microbore HPLC-MS separation was carried out using a mixing T-piece or via the sheath flow inlet of the electrospray source. Optimal analytical conditions for copper complex ion formation were determined by variation of copper concentration, pH, nebulization gas supply and spray voltage. Tandem mass spectrometry of copper/peptide complex ions provides peptide sequence information and insight into the peptide chelation sites. Copper associated y fragment ions dominate the product ion spectrum for non-histidine containing peptides, but both b and y copper complex ions were observed for the histidine containing MHC class I associated peptide gp70.