Characterization of major histocompatibility complex-associated peptides from a small volume of whole blood.
Class I major histocompatibility complex (MHC) presents intracellular-derived peptides on the majority of cells within the human body. Intracellular proteins are degraded into peptides of 8-11 amino acids, allowing them to fit into the groove of an empty MHC class I molecule. Detection of MHC-associated peptides can be challenging with the major difficulty being the ability to obtain peptides in adequate concentration. Published protocols require a large sample size that is unrealistic for a clinically available sample. Based on calculations, it should be possible to characterize MHC-associated peptides from cells obtained from 30 ml of whole blood. A citric acid wash of whole platelets was implemented to release the peptides with sample cleanup by reversed-phase high-performance liquid chromatography on a peptide trap. Peptides were analyzed by liquid chromatography tandem mass spectrometry. Four peptides were identified from an individual's platelets. The binding motifs of the peptides were consistent with the published MHC binding motif of the individual. Since red blood cells do not express MHC, they were used as a negative control. Using citric acid wash of whole cells and a peptide trap, the more abundant MHC-associated peptides can be identified. This report demonstrates the identification of peptides from a sample volume compatible with reasonable clinical availability.
Hopkins
Leann M
LM
Department of Pathobiology and Diagnostic Investigation, Michigan State University, East Lansing, MI 48824, USA.
Schall
Michael
M
Leykam
Joseph F
JF
Gerlach
John A
JA
D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism; D001774:Blood Chemical Analysis; D001792:Blood Platelets / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D008285:Major Histocompatibility Complex; D013058:Mass Spectrometry; D010455:Peptides / Q000032:analysis / Q000737:chemistry
D000596:Amino Acids; D010455:Peptides