High Sensitivity Proteomics: Immunopeptidomics

A comprehensive understanding of the immunopeptides that are presented to the immune system is vital for designing novel innovative therapeutics against cancer and other diseases.

The isolation and characterization of immunopeptides is being enabled by advances in mass spectrometry (MS)-based proteomics. Technology Networks recently spoke with Gary Kruppa, PhD, Vice President of Proteomics, Bruker Daltonics Inc., to learn more about how Bruker's MS portfolio is advancing research in this area, and to discuss the current status of single cell proteomics.

Molly Campbell (MC): What recent advances have been made in the high sensitivity applications of proteomics? How is mass spectrometry continuing to advance such applications?

Gary Kruppa (GK): The advances made in improving the sensitivity of mass spectrometry (MS) in the last two to three years have really been stunning, to the point where we can even talk about working towards proteomics at the single cell level. We can see (already) in the order of 750 to 1000 proteins, and that's not even digging that deep into the proteome. I think eventually single cell proteomics will arrive and we will be able to identify even larger numbers of proteins from a single cell.

In the meantime, even analyzing a tissue biopsy where there is, say, just a few micrograms of tissue or less to work with, we are able to see large numbers of proteins which can be used to really characterize cancer tissue. That's something we're very excited about, and combining this with spatially resolved proteomics, we hope to be able to spatially look at the distribution of proteins and small molecules in cancer cells/ tissues.

There's lots of other areas where you can't access much sample. For example, when looking at peptides that are presented to the immune system by the body.

The amount of immunopeptides that you can get from a reasonable sample from a human being is very, very small; you have to do a lot of careful sample workup and enrichment. Even then, you get very small amounts of immunopeptides. Characterizing those peptides is another important application of high sensitivity proteomics.

The advances are enabling work that, just a few years ago, wasn't possible; or at least possible at a sensitivity that then permits characterization of the immunopeptidome.

MC: For our readers that might be unfamiliar, please can you provide a bit of background on what immunopeptidomics is?

GK: Immunopeptides are proteins that are cut up from your own proteins and presented to the immune system on the surface of specific molecules and antigen-presenting molecules. Your immune system looks at the peptides that are presented and tries to make sure that there's nothing unusual.

Neoantigens are amino peptides that are from, say cancer cells, proteins that are mutated in cancer cells or onco viruses, and these are a way for your body to recognize that there are foreign molecules in your body. When these are presented to the immune system, it essentially tells your body that it needs to do something about it.

Studying such molecules is important because we can learn a lot about cancer cells from the type of neoantigens that are presented. It's very important in pharmaceutical drug development that biopharmaceutical products (which are proteins themselves) don't cause an immunogenic response. We can find out if they will produce an immunogenic response if we can detect any immunopeptides that are produced from them by the immune system. This requires really high sensitivity equipment because they are produced in very small quantities in the human cell culture; they're hard to work up.

MC: Previously, you stated that a "CCS signature could be advantageously used by adding a level of intelligence in immunopeptidomics". Can you please expand on this, firstly by discussing how the CCS signature is obtained?

GK: The timsTOF Pro has a trapped ion mobility spectrometer, and ion mobility spectrometry is a way that you measure collision cross section signatures. In an ion mobility spectrometer, you trap an ion in the presence of a stream of flowing gas, and the gas pushing on the ion, combined with an electric field opposing it, allows the ion to be trapped at a position which is determined by how big it is and its charge. We can scan those out, measure their ion mobility and calculate the CCS. The process of separating them according to ion mobility allows you to focus on ions that would have the right size to be immunopeptides. There are two classes of immunopeptides, some that are eight to nine amino acids long, and some that are 12 to 14 amino acids long; they're called class one and class two immunopeptides. They have very specific CCS ranges, and so you can use intelligent data acquisition to focus in on the ion mobility ranges where these peptides appear. Another exciting thing that we're working on is being able to predict the CCS of those peptides; we hope to be able to look at the sequence of a peptide predicted from the mass spectrometry data and tell how likely it is to be right by looking at its CCS. That is still a work in progress but it's something that we're very excited about.

MC: Are you able to talk about any of the interesting work Bruker's customers are undertaking in this space?

GK: There's a group working in the area of drug development, specifically pharmaceutical immunogenicity. There are biologics that are typically large proteins and antibody-type drugs in development that it's important to ensure the immune system doesn't react against and trigger an allergic reaction. Of course, you don't have an immunogenic sort of allergic.

Drug companies have developed workflows where, rather than waiting to study what the drug physiologically does in human clinical trials, they want to look at cell cultures. From the human cell cultures, they can apply the drug and work up the immunopeptides.

When you work up the immunopeptides from a cell culture, you'd like to see only native peptides. But if you see peptides from the drug sequence, then you know that the drug has the potential to cause an immunogenic response. Discovering this at an early stage saves a lot of money, but it does require very high sensitivity.

MC: What remains the biggest challenge in single cell proteomics and high sensitivity proteomics?

GK: You can always use more sensitivity. Until we get to the point where we can detect a single molecule, higher sensitivity is critical. Certainly, in cancer neoantigen studies, we really need high sensitivity. Being able to detect 700-1000 proteins from a single cell is nice but we expect in the order of 10,000 proteins to be present in a single cell, so we're still far away from characterizing the full proteome from a single cell. 

Sample preparation is also critical both in single cell proteomics and when working with small numbers of cells from tissues biopsies. When you're working with a small amount of material you can't afford to conduct lots of sample prep steps, which in a normal proteomics workflow would entail isolating the proteins, denaturing them, then reducing and digesting them before doing clean up, because all of the other steps involve processes that are not compatible with MS. So, sample prep for these methods is a big challenge: how do you process this sample in a way that will allow you to run it with minimum sample handling and preferably minimal clean up? Advances that are being made in these areas are very exciting. 

Gary Kruppa, PhD, Vice President of Proteomics, Bruker Daltonics Inc., was speaking to Molly Campbell, Science Writer, Technology Networks.