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Advances in Mass Spectrometry Drive Immunopeptidomics

An image depicting single-cell sequencing.
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At the 71st Annual Society for Mass Spectrometry (ASMS) meeting, Technology Networks had the pleasure of speaking with Rohan Thakur, president of Life Sciences Mass Spectrometry at Bruker Daltonics. We discussed the new product launches from Bruker and focused specifically on the applications of the company’s newest mass spectrometer, the timsTOF Ultra, for immunopeptidomics.

Robustness and high coverage are critical alongside high sensitivity  

For many years, a key focus in mass spectrometry (MS) development has been increasing the technology’s sensitivity – a feature that is important for scientists studying low abundant proteins in small amounts of sample. The new timsTOF Ultra delivers a “breakthrough” level of sensitivity for single-cell analysis, but beyond this it also provides robustness and high sequence coverage – fundamental attributes that enable scientists to see more proteins and their associated pathways, Thakur explains.

Technical details: the timsTOF Ultra

The timsTOF Ultra mass spectrometer incorporates a new captive spray ionization (CSI) ultra ion source, which has a larger capillary and optimized gas flow. It also has a novel fourth-generation trapped ion mobility separation (TIMS) XR cell and a 14bit digitizer. Collectively, these features mean that the mass spectrometer can identify over 55,000 peptides, which map onto over 5,000 protein groups at 1% false detection rate from 0.125 ng of protein loading.

Scientists may encounter challenges when working to identify proteins in their sample due to high levels of sequence conservation across protein families. The presence of one sequence might not enable the identification of a protein with absolute confidence, as that sequence may also be found in another protein family. The high sequence coverage of the timsTOF Ultra is incredibly useful in this context, Thakur says, “particularly when studying protein isoforms.” Protein isoforms – also known as protein variants – occur when proteins are generated from the same DNA code but carry distinct amino acid sequences and biological functions due to mechanisms such as alternative splicing.

Driving immunopeptidomics

These features are also why the new mass spectrometer is helping to drive the field of immunopeptidomics, Thakur emphasizes. Immunopeptidomics is a growing research field that utilizes MS for antigen-associated peptide identification. Excitement surrounds its potential for cancer research, where identifying tumor-specific antigens can support the development of personalized medicines that harness the body’s immune system to fight cancer, known as immunotherapies.

As its name suggests, immunopeptidomics involves the study of peptides, not proteins – this distinction is important. Human leukocyte antigen (HLA)-bound peptides present on an infected cell or tumor tissue effectively inform the immune system that there are foreign substances present in the body, stimulating an immune response. Their presence therefore influences how capable the body is in fighting disease or invasion from a pathogen – a mechanism that can be harnessed for personalized medicine approaches. To achieve this, we need to know which immunopeptides are present. Researchers such as Dr. Nicola Ternette from the Jenner Institute at the University of Oxford are using the timsTOF Ultra to study the exact sequence of HLA-bound peptides extracted from patients’ tumors.

The first step in this process is to find the immunopeptides, which can be 8 to 9 amino acids long, or 12 to 14 amino acids long, depending on whether they are class 1 or class 2, respectively. “They exist in very low copy numbers – requiring very high sensitivity,” explains Thakur. “Contextually that becomes very important, because you’re sifting through enormous amounts of noise, and you’ve got to hit that peptide,” he adds, noting that the timsTOF Ultra system is having such a big impact in immunopeptidomics because it is “so sensitive” and “because of the high number of peptides it can see.”

Rohan Thakur, President of Life Sciences Mass Spectrometry at Bruker Daltonics, was speaking to Molly Campbell, Senior Science Writer for Technology Networks, at ASMS 2023.