by Asim Siddiqui, SVP, Research & Tech Development, Seer
Today, scientists are looking to mass spectrometry-based proteomics to help them uncover novel insights into health and disease with new approaches enabling depth of protein coverage not previously possible. Advanced technologies are becoming more automated, cost-effective, and widely available to the broader life sciences community. With this progress, mass spec-based proteomics has evolved, allowing researchers to perform population-scale studies with deep molecular profiling, better throughput and flexible workflows.
Mass spec is considered the gold standard in proteomics, so it is essential to understand how it performs compared with other approaches, the evidence demonstrating its utility in definitively identifying peptides, challenges inherent to the methods, and what the future holds.
Basics of mass spec-based proteomics
Mass spectrometry allows researchers to accurately and reproducibly measure the mass of ions. A fundamental innovation that enabled proteomics in mass spec was the invention of electrospray ionization, enabling efficient ionization of peptides and the subsequent measurement of their mass as they fly through the mass spec.
In general, a majority of mass spec-based proteomics are performed using a method referred to as “bottom-up proteomics”, where the proteins are digested by an enzyme, typically trypsin, that leads to cleavage at particular sites along the peptide backbone. This approach leads to defined shorter peptides that are more generally compatible with currently available technologies. In the mass spec, each peptide is fragmented further. Analyzing a sub-fragment of the parent peptide chain allows scientists to gather more information about sub-components of that chain, reconstructing peptide sequences more accurately. Top-down proteomics analyzes intact proteins, which enables the identification of the complete protein sequences. However, its experimental challenges currently make it unsuitable for deep coverage, large-scale population studies.
In addition to the mass spec instrument itself, another key component of mass spec-based proteomics is the liquid chromatography machine and associated column which are used to separate peptides along a physicochemical gradient, such as hydrophilicity-hydrophobicity, prior to their introduction into the mass spec. The combination of liquid chromatography and mass spec is almost ubiquitously used for proteomics and is termed LC-MS. However, a challenge with LC-MS is that the separation of peptides is insufficient to deal with the wide range of concentrations of proteins within complex samples such as plasma. This wide range of concentrations overcomes the dynamic range of detection of the MS preventing low-abundance proteins of interest from being detected. This can be somewhat alleviated through a process of peptide fractionation to further separate peptides into distinct fractions and depletion of high abundant proteins, but these processes are challenging to run at scale.
Seer has pioneered the use of proprietary engineered nanoparticles to overcome the challenge of dynamic range in complex samples. Depending on the physicochemical properties engineered into the nanoparticle, each one enriches for specific subsets of proteoforms compressing the dynamic range of proteins present in the original sample and enabling the peptides to be resolved by the mass spec. This allows scientists to achieve deeper coverage of proteins and drive novel insights around proteoforms and low-abundance proteins that have biological impact.
Advantages of mass spec-based proteomics
The main advantage of evaluating proteomics through mass spec is that it allows for the direct measurement of the peptide, detecting its physical properties and counting ions by the amount of electrical current flowing. Electrical current leads to the quantitation of the peptide amount.
By comparison, other proteomic techniques, like affinity-based methods, use a reagent, which can be an aptamer or something more complex such as an antibody. A sensory fluorescent assay, like an ELISA, or a tagged DNA-based approach, is used for readout. While there are use cases for affinity-based approaches, multiplexing different reagents and minimizing cross-reactivity can be challenging. More specifically, generating reagents for each molecule of interest necessitates the creation of a specific affinity reagent, which can be costly and time-consuming.
Another downside of developing an antibody as a reagent is the specificity may be tied to a particular proteoform of the protein in question. Consequently, affinity-based approaches may have a limited view of the variations that will be present in the sample.
Mass spec provides a significant advantage over affinity-based approaches since the technique measures the mass of peptides in a sample rather than a single epitope. Since there is no need to utilize a specific reagent, researchers can identify an extensive range of proteins within the sample more cost-effectively with shorter timelines. The assay can work across species and in mixed-species samples such as those arising in biologic manufacturing, xenograft or cell culture applications.
Research applications of mass spec-based proteomics
Mass spec-based proteomics allows the life sciences community to better understand health and disease by identifying biomarkers and pathways of disease action. Mass spec-based proteomics enables us to uncover the link between proteins and disease progression or identify the mechanisms that differentiate treatment responders from non-responders.
Recently, scientists from Seer and Memorial Sloan Kettering Cancer Center used mass spec-based proteomics to analyze 188 plasma proteomes from non-small cell lung cancer subjects (NSCLC) and controls. They found specific protein isoforms of four proteins were associated with NSCLC progression. In one such example, they discovered that the short protein isoform of bone morphogenetic protein 1 (BMP1) occurred more frequently in lung cancer patients than in healthy individuals. Similarly, researchers at Seer and Massachusetts General Hospital conducted large-scale, deep plasma proteomics in 1,800 Alzheimer’s disease (AD) patients and found several proteins of interest for use in the identification of AD and disease progression. These two studies are examples of how mass spec-based proteomics is shedding new light into disease biology that will hopefully allow us to identify novel biomarkers.
Other researchers use mass spec-based proteomics for drug discovery and design by leveraging proteomic datasets to understand mechanisms of action and identify how drug interactions affect the proteome. By utilizing mass spec-based proteomics, drug developers can rationally design and develop targeted, safer and more effective therapeutics.
Challenges of mass spec-based proteomics
While mass spec-based proteomics has demonstrated its utility for biomarker development and drug discovery, a significant challenge is the standardization of methods, ensuring techniques are reproducible. It is important to have standardized protocols and workflows to ensure accurate and reproducible results across labs. Fortunately, there are automated lab robotics for sample prep that can help curb some of these problems with standardization. Additionally, spike-in controls are used to monitor performance and ensure consistent results. These approaches allow consistency within labs as well as across institutions worldwide.
Running deep unbiased proteomics at scale has been challenging, specifically large studies using plasma samples. This barrier is likely directly linked to the limited access of mass spec to the “everyday” biologist. Partnerships across institutions and borderlines are necessary if we want to ensure that scientists can access this technology.
Additional challenges exist around the concept of mass spectrometry instruments being difficult to run. While in some cases, running mass spec-based proteomics experiments requires highly qualified scientists, recent advances in automation coupled with standardized protocols from sample prep to mass spec workflow, can enable more junior technicians to run proteomics experiments provided they are supervised by a more skilled practitioner. This is another area where partnerships can help bridge any gaps in expertise.
A final challenge in mass spec-based proteomics is ensuring that low-abundant proteins are readily identifiable. As evident with numerous scientific studies, it is often those low-abundant proteins that are more difficult to identify when they are often the most relevant to the disease. We need to continue advancing proteomic approaches that will allow us to find those “needles in a haystack.”
To address some of these challenges, Seer formed a collaboration with Thermo Fisher Scientific. Under the partnership, Seer has established the Seer Technology Access Center, which will utilize the company’s Proteograph XT Assay Kit, along with Thermo Fisher’s new mass spectrometer, the Thermo Scientific Orbitrap Astral mass spectrometer. The Center will help researchers gain access to mass spec technology for deep unbiased proteomics at scale. Additionally, the collaboration will develop proteomics and proteogenomics workflows.
It is important to continue these efforts such that we can collectively as a life sciences community continue to advance the field, discovering novel drug and biomarker targets that are clinically actionable.
The future of proteomics
The future is bright for mass spec-based proteomics, and there are new and exciting technologies that will enable us to obtain deeper insights into the proteome.
Artificial intelligence will have a dramatic impact in the field of proteomics. As larger and larger amounts of proteomic datasets become available, we will need to build tools employing machine learning and large language models to make critical connections between those datasets and other clinically relevant patient information, such as imaging data, drug responses, comorbidities and outcomes.
By merging all of this information and having AI help us glean novel insights into disease and care management, we can significantly impact the healthcare industry. Further, extending the scale of mass spec methods to provide more data quickly will provide algorithms with sufficient amounts of data to learn from, evaluate and predict outcomes more accurately.
In parallel, advances in the fundamental technologies will continue as existing companies such as Thermo Fisher launch new, more advanced mass spec instruments and companies such as Seer advance technologies for deep proteomics at scale. As these and other technologies develop, they provide an ever-deepening view of the proteomic landscape. While it remains a long way off, eventually, the complete proteome of a sample will be measurable with all of its known constituent proteforms, such as protein isoforms, amino acid variants and post-translational modifications.
While this journey will be long, advances along the way will enable us to take steps in unraveling the processes that lie at the protein level that will allow us to move the field of biology forward. From cancer to neurodegenerative disorders to immunology, we have made significant progress in understanding how protein function and dysfunction lead to disease. As advancements in mass spec and proteomics technology progress and fundamental tools continue to improve, we will uncover new insights to drive our understanding of biology and disease processes.