Mass spectrometry analysis and quantitation of peptides presented on the MHC II molecules of mouse spleen dendritic cells

Abstract

Major histocompatibility complex class II (MHC II) molecules are expressed on the surface of antigen-presenting cells and display short bound peptide fragments derived from self- and nonself antigens. These peptide-MHC complexes function to maintain immunological tolerance in the case of self-antigens and initiate the CD4(+) T cell response in the case of foreign proteins. Here we report the application of LC-MS/MS analysis to identify MHC II peptides derived from endogenous proteins expressed in freshly isolated murine splenic DCs. The cell number was enriched in vivo upon treatment with Flt3L-B16 melanoma cells. In a typical experiment, starting with about 5 × 10(8) splenic DCs, we were able to reliably identify a repertoire of over 100 MHC II peptides originating from about 55 proteins localized in membrane (23%), intracellular (26%), endolysosomal (12%), nuclear (14%), and extracellular (25%) compartments. Using synthetic isotopically labeled peptides corresponding to the sequences of representative bound MHC II peptides, we quantified by LC-MS relative peptide abundance. In a single experiment, peptides were detected in a wide concentration range spanning from 2.5 fmol/μL to 12 pmol/μL or from approximately 13 to 2 × 10(5) copies per DC. These peptides were found in similar amounts on B cells where we detected about 80 peptides originating from 55 proteins distributed homogenously within the same cellular compartments as in DCs. About 90 different binding motifs predicted by the epitope prediction algorithm were found within the sequences of the identified MHC II peptides. These results set a foundation for future studies to quantitatively investigate the MHC II repertoire on DCs generated under different immunization conditions.

Figure 1

A schematic diagram summarizing the steps performed to identify peptides presented by MHC II molecules on DCs and B cells, from sample preparation to peptide identification by LC-MS/MS and quantitation by LC-MS.

Figure 2

Identification of two representative MHC class II binding peptides derived from DCs and B cells. Comparison of MS/MS spectra of naturally eluted peptides SLAM7 (A) and APOE (C), identified using MASCOT software in B cell and DC samples respectively, with MS/MS spectra of the matching synthetic isotopically labeled peptides for SLAM7 (B) and APOE (D). The corresponding y and b series are marked.

Figure 3

Cellular localization of source proteins for endogenous MHC II peptides. A) Pie charts and percentages of source proteins identified as MHC II peptides endogenously presented by DCs and B cells in multiple biological replicates (n=7 for DCs and n=4 for B cells). B) Venn diagram representation of the overlap of endogenous MHC II peptides eluted from DCs and B cells in multiple replicates as indicates above.

Figure 4

Absolute Quantitation Analysis (AQUA) by LC-MS of selected MHC II endogenous peptides. Synthetic isotopically labeled peptides (0.8 ng) were spiked into DC and B cell samples. Quantitation of endogenous counterparts was obtained comparing peaks’ intensity of the selected peptide pair. Heavy isotopes peaks are indicated with a (*). Each peptide was analyzed separately after LC-MS run. A) MS profile of the SLAM 7 peptides pair identified in the MHC II peptide mixture eluted from one representative B cell sample. B) MS profile of the Apo E peptides pair identified in the MHC II peptide mixture eluted from one representative DC sample. Isotopic patterns of the ions were consistent with the predicted patterns based on the isotopic ratios. Mass shifts of the isotopically labeled peptides are consistent with the predicted values (see Table 1).

Figure 5

MHC II peptide repertoire in DCs and B cells reflects their distinct roles in the immune system. The functional analysis was generated through the use of Ingenuity Pathways Analysis Software (Ingenuity® Systems, www.ingenuity.com). Data sets of source proteins identified from MHC II peptide analysis of DCs and B cells in one representative experiment were uploaded into Ingenuity application. The biological functions that had the most significant (score ≥) are shown (see Materials and Methods).