Scientists at Utrecht University have significantly expanded the number of human leukocyte antigen peptides detectable via mass spec.
In a study published last week in Proceedings of the National Academy of Sciences, the researchers identified more than 12,000 HLA class I-presented peptides, generating what Albert Heck, chair of the Biomolecular Mass Spectrometry and Proteomics group at Utrecht University and author on the study, said was by far the largest profile of such molecules to date.
In addition to providing new insights into the composition of HLA class I-presented peptide repertoire, the study also demonstrated an application of electron-transfer/higher-energy collision dissociation fragmentation (EThcD), Heck told ProteoMonitor.
Introduced by Heck's lab in a paper published in Analytical Chemistry in 2012, EThcD combines the two fragmentation methods to improve coverage and identification of fragment ions compared to conventional fragmentation techniques. In particular, Heck noted, the technique is useful for analysis of non-trypic peptides, such as the endogenous peptides his team targeted in its HLA work.
HLA class I molecules are a key part of the immune system, binding to short peptides generated by the degradation of cellular proteins, which they then present at the cell surface for detection by immune actors like T lymphocytes. Presentation of specific peptides marks cancerous or infected cells for destruction.
Given their key role in this process, researchers have sought to generate profiles of these peptides, and shotgun mass spec has emerged as the leading approach for such efforts.
Identifying large numbers of these peptides, however, has proven challenging using conventional mass spec techniques, Heck said, noting that thus far most efforts to identify them have maxed out at around 500 peptides. This, the PNAS authors noted, is likely due to the non-tryptic nature of these endogenously processed peptides and the fact that they may contain "certain amino acid residues that are known to hamper efficient backbone dissociation" by traditional fragmentation methods.
Heck and his colleagues hoped to achieve more complete coverage using the EThcD approach, which generates both ETD and HCD fragment ions, offering more informative mass spectra compared to traditional methods and potentially improving identifications.
Using the technique to analyze a human B-cell line on a customized Thermo Fisher Scientific Orbitrap Elite instrument, the Utrecht team identified more than 12,000 HLA class I-associated peptides, dramatically expanding the known repertoire of these molecules. Indeed, the researchers noted, "comparison of the complete dataset with recent large-scale studies revealed that 81 percent of the peptides have not been reported before."
In addition to the EThcD analysis, the researchers also performed the experiment using conventional fragmentation techniques including collision induced dissociation, HCD, and ETD alone and CID combined with HCD. EThcD analysis resulted in a 39 percent rate of high-confidence peptide identifications, roughly twice as high a rate as ETD alone and more than three times the rate of CID and HCD alone and CID combined with HCD.
The EThcD method does result in "a somewhat slower duty cycle" due to the two forms of fragmentation used, Heck said, but, he noted, this is more than made up for by the increase in peptide IDs.
The method could prove useful for analysis of a variety of analytes beyond HLA-associated peptides, Heck said. In particular, he noted, it could provide improved coverage of glycopeptides, with ETD fragmentation offering good information on the peptide backbone and HCD offering information on the attached glycans.
For the PNAS work, the Utrecht team used a specially modified Orbitrap Elite instrument that they customized in collaboration with Thermo Fisher scientists. Thermo Fisher's recently released Orbitrap Fusion, however, has a built-in EThcD, Heck said, "so [the method] will be available to anyone."
Perhaps more significant than the demonstration of the EThcD approach, though, Heck said, were the insights it offered into the composition of the HLA class I-associated peptide population.
He cited, for example, the technique's success at localizing phosphorylation sites on these peptides, a feature that could prove important in understanding HLA class I-associated peptides in cancerous cells. The notion, Heck said, is that dysregulation of cell signaling in cancer cells could be reflected in phosphorylation of their HLA-associated peptides. In total, the Utrecht researchers identified roughly 0.5 percent of all peptides in the study as being phosphorylated.
More generally, Heck said, the EThcD method's ability to generate deep profiles of HLA class I-associated peptides could enable identification of cell-specific differences in these peptide populations that could improve the ability to select specific cells for therapy.
"For instance, if you are able to identify that a certain peptide is presented on cancer cells but not on other [healthy] cells, you could use that for targeting those cells," he said.
Similar work has been done by previous researchers, Heck said, but these efforts have been limited by the relatively small number of peptide identifications generated by conventional workflows.
He and his colleagues are currently using the EThcD approach to compare the HLA-associated peptide profiles across a variety of cell types, including cancer cell lines, he said.