Use of selected reaction monitoring mass spectrometry for the detection of specific MHC class I peptide antigens on A3 supertype family members

Abstract

The development of peptide-based vaccines that are useful in the therapeutic treatment of melanoma and other cancers ultimately requires the identification of a sufficient number of antigenic peptides so that most individuals, regardless of their major histocompatibility complex (MHC)-encoded class I molecule phenotype, can develop a cytotoxic T lymphocyte (CTL) response against one or more peptide components of the vaccine. While it is relatively easy to identify antigenic peptides that are presented by the most prevalent MHC class I molecules in the population, it is problematic to identify antigenic peptides that are presented by MHC class I molecules that have less frequent expression in the population. One manner in which this problem can be overcome is by taking advantage of known MHC class I supertypes, which are groupings of MHC class I molecules that bind peptides sharing a common motif. We have developed a mass spectrometric approach which can be used to determine if an antigenic peptide is naturally processed and presented by any given MHC class I molecule. This approach has been applied to the A3 supertype, and the results demonstrate that some, but not all, A3 supertype family-associated peptides can associate with all A3 supertype family members. The approach also demonstrates the shared nature of several newly identified peptide antigens. The use of this technology negates the need to test peptides for their ability to stimulate CTL responses in those cases where the peptide is not naturally processed and bound to the target MHC class I molecule of interest, thus allowing resources to be focused on the most promising vaccine candidates.

Fig. 1

Immunoaffinity purification of A3 supertype peptides. Cell lysates obtained from each of the indicated cell lines were sequentially run over immunoaffinity columns in the order indicated. The antibody used in each of the sequential columns is indicated, and is followed by the MHC class I molecule that was retained by that antibody. Peptides obtained from the MHC class I molecules in bold type were analyzed for the presence of specific peptides using SRM mass spectrometry.

Fig. 2

The CAD mass spectra of ALLAVGATK and ALL_D10AVG_D2ATK. The predicted masses for the product ions of types b_n and y_n are written above and below the peptide sequence, respectively. Ions of type b_n and y_n that are observed in the spectrum are underlined.

Fig. 3

SRM mass spectrometric detection of ALLAVGATK. Single ion chromatograms (SIC) for three product ions of ALLAVGATK are shown. The product ions observed for the synthetic peptide and each of the purified peptide samples have equal retention times and b₂/y₅/y₇ ratios, indicating that ALLAVGATK is present in each of the samples. *Indicates that the ratio is high due to ion contribution from the internal standard deuterium-labeled ALLAVGATK (the contribution of the internal standard is observed in the b₂ product ion because the mass of b₂ for both the deuterium-labeled and non-deuterium-labeled peptides is 185.4).

Fig. 4

Peptide quantification by comparison to the deuterium-labeled ALLAVGATK peptide. The average ion abundance was calculated from the three measured product ions. The ionization efficiency was determined from each peptide compared to the deuterium-labeled ALLAVGATK. The average ion abundance and ionization efficiencies were used to calculate the abundance of the peptides.