Characterizing MHC-Associated Peptides by Mass Spectrometry

This issue’s Pillars of Immunology article (1) describes the characterization by mass spectrometry of endogenous peptides eluted from the class I MHC molecule HLA-A2.1 (A*0201). This was the first contribution from the collaboration between Don Hunt and Vic Engelhard on the identification of MHC-bound peptides, which soon would yield seminal work on tumor-specific CTL recognition (2, 3, 4), nonclassical MHC proteins (5), posttranslational modification of MHC-bound Ags (6, 7, 8), and minor histocompatibility Ags and graft-vs-host disease (9, 10).

The work was a tour de force in microchemical analysis and set the standard for ultrasensitive detection and characterization of biological peptides. The method used relied on a combination of several recent technical advances in mass spectrometry. First and most important was the development of tandem mass spectrometry (11). In this technique, two mass analyzers are used in series, the first to select particular molecules from a mixture by virtue of their exact masses, and the second to analyze the pattern of ions resulting from fragmentation of the selected molecule. Don Hunt had recently adapted this technique for peptide sequencing by building a triple quadrupole instrument that interposed a middle chamber filled with argon atoms to fragment peptide ions selected in the first chamber that could then be analyzed in the third chamber (12). Fragmentation occurred mostly at amide bonds, as in earlier chemical fragmentation approaches used for mass spectrometry sequencing, so that the sequence could be read from the pattern of fragment ions detected in the third chamber. The high sensitivity of mass spectrometric detection facilitated the analysis of minute quantities of molecules such as low abundance peptides eluted from MHC proteins. The second development was that of electrospray ionization, for which John B. Fenn would later be awarded the 2002 Nobel Prize in Chemistry (13). This technique allowed samples to be introduced continuously into the mass spectrometer. In earlier ionization methods, samples were individually mixed with volatilization-promoting compounds and introduced one at a time into the spectrometer. The volatile acidic solvents typically used for reverse phase HPLC proved to be a perfect match for electrospray ionization. Complex mixtures of peptides separated by HPLC could be fed directly into the tandem electrospray ionization mass spectrometer, allowing mixtures of unprecedented complexity to be analyzed. To fractionate the small quantities of peptide eluted from MHC molecules with minimal loss, Hunt et al. developed a microcapillary reverse phase HPLC column (75-μm diameter) for use in-line with the mass spectrometer. Immunologists who heard Don Hunt’s lectures from that time might recall marveling at his descriptions of these hair-thin columns and their resolving power. The same group would later develop a microcapillary effluent splitter in which a fraction of the HPLC eluent was diverted in-line to a microtiter plate for CTL assay (3). This approach allowed the same fraction to be analyzed for biological activity and molecular mass, and led to the identification of the peptides recognized by CTL specific to tumor Ags, alloantigens, and viral and bacterial epitopes (reviewed in Ref. 14). In this article, it was not T cell epitopes that were of interest but the set of naturally processed peptides presented by HLA-A2.1 on a normal, noninfected cell. Endogenous protein sources for these peptides were identified by matching the experimentally determined peptide sequences to protein and nucleic acid sequence databases, the third development on which this Pillar of Immunology rests. Only four of the 19 fully or partially sequenced peptides identified by Hunt et al. corresponded to peptides in the then-available database. At the time, GenBank had <0.1% of its current entries (15), basic local alignment search tool (BLAST) had just been developed (16), and the Human Genome Project was just getting off the ground (17). The use of database analysis in the identification of proteins from partial sequence data was to explode in a few years with increased sequence coverage in databases and with the development of two-dimensional gel tryptic peptide analysis (18).