Identification by HPLC and mass spectrometry of HPA-1a–derived peptides eluted from HLA-DR molecules. (A) RP-HPLC profile of the HLA-DR–associated peptides derived from the HLA-DRB3*0101 homozygous B lymphoblastoid cell line, HHKB. Cells were pulsed with glutathione S-transferase–HPA-1a (PSI) domain, and the associated peptides were acid eluted and separated by HPLC using an OD-300, aquapore, C-18, 3-cm × 2.1-mm column. The following gradient conditions were used: solvent A, 0.1% trifluoroacetic acid in water; solvent B, 70% acetonitrile in water containing 0.085% trifluoroacetic acid. The gradient was 0% to 100% B for 50 minutes with a flow rate of 0.5 mL/min. The UV absorbance of the eluates was monitored at 214 nm. (B) Fractions were introduced into the electrospray ionization source of a quadrupole tandem mass spectrometer. The total ion chromatogram of fraction 23 is shown. (C) The survey scan (+MS) for a compound eluting at 16.2 minutes within the m/z range of 300-1500 is shown. The most abundant ions were automatically subjected to collision-activated dissociation to yield peptide sequence-specific fragment ions (+MS[2]). (D) The fragmentation spectrum for the doubly protonated precursor ion [M + 2H]⁺ with an m/z of 934.15 was identified as the HPA-1a peptide MCAWCSDEALPLGSPR. The sequence of the peptide was acquired by comparing the observed mass of the type y (amino-terminal cleavage) and type b (carboxyl-terminal cleavage) ions with those of the corresponding theoretical ions, using the Matrix Science web server (www.matrixscience.com).