Development, validation, and application of a method to characterize major histocompatibility complex associated peptides from equivalent to 1x10(8) cells

The class I major histocompatibility complex (MHC) presents peptides on almost all cells within the human body. Intracellular proteins are degraded into peptides of 8–11 amino acids allowing them to fit in the groove of an empty MHC class I molecule. Characterization of these MHC associated peptides can be challenging, a major difficulty being the need to obtain peptides in an adequate concentration for detection. Published protocols require a sample size that is impractically large to be clinically realistic. The objective of this research was to establish a method able to characterize MHC associated peptides from a sample of reasonable clinical availability. Based on extrapolations, it should be possible to characterize these peptides from 1 × 10 8 cells (or 20–30 ml of whole blood). Key to making this work, was a citric acid wash of whole cells release the peptides followed by sample clean-up using RP-HPLC and a peptide trap. Peptides were analyzed by LC-MS/MS. MHC associated peptides from several cellular samples were characterized to validate this method. Peptides from proteins with general cellular function (ubiquitin, talin, actin, and unnamed protein), platelet specific function (thrombospondin, platelet factor 4, and GPIb), and an immune response (MRP-14), were all identified. The most remarkable finding, when the method was applied to a clinical problem, was the purification of GPIb peptide from individuals with Idiopathic Thrombocytopenic Purpura (ITP). ITP is characterized by the premature immune destruction of platelets, and is associated with the production of antiplatelet autoantibodies, most often targeting platelet membrane GPIIb/IIIa and/or GPIb/IX. The GPIb peptide was identified in 4 of the 5 ITP patients (who coincidently all had HLA-B7) and in none of the random controls. To determine if the peptide was specific for the ITP patients or HLA-B7, 3 non-ITP individuals with HLA-B7 were selected as controls. GPIb (4–12) was isolated from 2 of these 3 controls. In conclusion, platelets can present GPIb peptides associated with MHC class I. Since both HLA-B7 ITP and controls present GPIb peptides, development of autoimmunity may not reside with peptide presentation, but with other mechanisms, such as loss of self-tolerance.