Development of an MHC-class I peptide selection assay combining nanoparticle technology and matrix-assisted laser desorption/ionisation mass spectrometry.
Human leukocyte antigen (HLA)-bound peptides are central for recognition of infected/transformed cells by T cells, and have formed the basis for many immunotherapy strategies. Epitopes from a given protein sequence (e.g. from viral proteins or oncoproteins) can be predicted by algorithms, as individual HLA receptors bind peptides through defined binding motifs. Peptides with the highest predicted binding score are then normally tested for their binding ability in binding assays. However, with the assays already established, only one peptide can be tested for binding per assay. This is certainly not a reflection of the in vivo situation, where several peptides generated via the major histocompatability complex (MHC)-class I processing pathway compete for HLA-receptor binding. Here, we describe the development of a method that can mimic the competition between multiple peptides for binding to a single HLA receptor molecule. We used silica nanoparticles with immobilised HLA-A2 complexes to screen HLA-A2 binder-peptides out of a known peptide mixture. The washed beads were analysed for selectively bound peptides by matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry. The advantage of the system is that the bound peptides can be unambiguously identified without any prior modification (e.g. radioactive or fluorescence labelling), even from complex peptide mixtures.
Flad
Thomas
T
Section for Transplantation Immunology and Immunohematology, University of Tuebingen, Germany. thomas.flad@uni-tuebingen.de
Schiestel
Thomas
T
Brunner
Herwig
H
Tolson
Jonathan
J
Ouyang
Qin
Q
Pawelec
Graham
G
Mueller
Gerhard A
GA
Mueller
Claudia A
CA
Tovar
Guenter E M
GE
Beck
Hermann
H
D019921:Biotinylation; D015789:HLA-A2 Antigen / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D036103:Nanotechnology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D015789:HLA-A2 Antigen