Proteoforms, which are distinct protein molecules arising from a single gene through various modifications, play an essential role in the physiological functions of living organisms. These proteoforms are generated through a complex series of post-translational modifications (PTMs) and alternative splicing of mRNA, offering a mechanism by which a single gene can encode multiple proteins with different functions. This vast diversity of proteoforms enables the intricate regulatory processes that sustain life, but understanding their full range and significance has been a major challenge in molecular biology.
The Mystery of Proteoforms
While the human genome has been mapped, revealing approximately 22,000 genes, the total number of proteoforms in humans remains an elusive figure. The complexity of proteoforms arises from the fact that proteins, once translated from their respective genes, undergo a series of transformations—such as phosphorylation, glycosylation, acetylation, and cleavage—that can alter their structure and function. These modifications are critical in regulating many biological processes, including signal transduction, cell division, and immune response. Despite extensive research, the true diversity of human proteoforms remains unknown due to technical limitations in detecting and analyzing these molecules.
Proteoform Analysis Using LC-MS
Liquid chromatography-mass spectrometry (LC-MS) has emerged as a powerful tool for the analysis of biomolecules, particularly proteins. This method allows for the identification and quantification of complex mixtures of proteins and their various forms (proteoforms). Traditional proteomics approaches, such as bottom-up proteomics, involve breaking down proteins into smaller peptides, which are then analyzed. However, this approach can often fail to capture the full complexity of the proteoform landscape, as it may overlook critical modifications that affect protein function.
A more advanced method, known as top-down proteomics, directly analyzes intact proteoforms without the need for digestion into smaller peptides. This technique enables the study of the full range of protein modifications and isoforms, providing a more comprehensive view of the proteome. Top-down proteomics offers significant advantages over traditional methods, particularly in its ability to preserve and study intact proteoforms, which may reveal novel aspects of protein function and regulation.
However, despite its promise, top-down proteomics faces several challenges. Biological samples, such as blood, tissue, or cultured cells, contain a highly complex mixture of proteins, which can make it difficult to detect low-abundance proteoforms and resolve closely related isoforms. As a result, scientists have sought ways to improve the sensitivity and resolution of proteoform detection in top-down proteomics.
The Development of PEPPI-MS
In 2020, the Takemori group at Ehime University introduced a groundbreaking method called PEPPI-MS (Proteoform Enrichment through Polyacrylamide Gel Electrophoresis and Ionization Mass Spectrometry), which aims to address the challenges of proteoform analysis in complex biological samples. This innovative technique combines the high-resolution fractionation capabilities of SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) with the sensitivity of LC-MS.
PEPPI-MS fractionation significantly improves the detectability of proteoforms by separating proteins based on their size and charge before mass spectrometry analysis. The simplicity and low cost of the SDS-PAGE system make this method particularly accessible, allowing laboratories without specialized equipment to perform high-quality proteoform analysis. The fractionation step enriches specific proteoforms, thereby improving the sensitivity of subsequent LC-MS analysis and increasing the overall number of proteoforms detected.
Since its introduction, PEPPI-MS has become widely adopted as a standard pre-fractionation technique in many top-down proteomics studies. The ability to enhance proteoform detection in complex biological samples has proven invaluable in expanding the knowledge of protein diversity, particularly in high-throughput proteomics applications.
Advancements in Proteoform Analysis with FAIMS-LC-MS
Building on the success of PEPPI-MS, the Takemori group collaborated with Prof. Andreas Tholey’s group at the University of Kiel, Germany, to further enhance the sensitivity and resolution of proteoform analysis. In 2022, they developed an ultra-sensitive proteoform measurement system by combining PEPPI-MS fractionation with FAIMS (Field Asymmetric Ion Mobility Spectrometry) and LC-MS.
FAIMS is a technique that separates ions in the gas phase based on their mobility in an electric field, providing an additional layer of separation beyond traditional LC-MS. By integrating FAIMS with LC-MS, the team was able to achieve unprecedented separation of proteoforms, even those with very similar sizes and charge properties. The FAIMS-LC-MS system enhances the ability to distinguish between closely related proteoforms, thereby enabling more detailed and accurate proteoform profiling.
This enhanced system allowed the group to conduct detailed top-down proteomic analysis of human cultured cells in 2023, resulting in a significant step forward in the study of proteoforms. They also developed a new methodology for middle-down proteomics, in which proteoforms are partially digested using Glu-C protease to produce intermediate-sized fragments. This approach strikes a balance between the sensitivity of top-down proteomics and the depth of information provided by bottom-up methods, offering a promising strategy for analyzing proteoforms in a more manageable size range.
The Promise of PEPPI-MS and FAIMS-LC-MS in Proteoform Research
The combination of PEPPI-MS fractionation and FAIMS-LC-MS ion mobility spectrometry represents a major leap forward in the analysis of proteoforms. These innovations have not only improved the sensitivity and resolution of proteoform detection but have also made high-resolution proteoform analysis more accessible to researchers worldwide. PEPPI-MS, in particular, has gained widespread adoption due to its simplicity, cost-effectiveness, and compatibility with standard laboratory equipment.
In 2024, the Takemori group published a comprehensive protocol in Nature Protocols, outlining a streamlined approach for applying PEPPI-MS and FAIMS-LC-MS in top-down and middle-down proteomics. This protocol provides researchers with a clear and reproducible method for fractionating biological samples and analyzing proteoforms at high resolution, contributing to the broader goal of creating proteoform atlases for various species.
Proteoform atlases aim to catalog the full spectrum of proteoforms present in an organism, similar to how the Human Genome Project mapped the human genome. Such atlases are expected to provide valuable insights into protein function, regulation, and disease mechanisms, ultimately facilitating the development of precision medicine and new diagnostic tools. For example, precise proteoform analysis could lead to more accurate biomarkers for disease detection or provide targets for drug development.
Future Directions and Applications
The ability to detect and characterize a broader range of proteoforms has profound implications for many areas of biological research, from neuroscience to cancer biology. As proteoform analysis techniques continue to improve, it is likely that researchers will uncover new protein functions, regulatory networks, and disease mechanisms that were previously hidden due to the limitations of older analytical methods.
One promising direction is the application of proteoform analysis to disease diagnostics. Many diseases, including cancer, neurodegenerative disorders, and cardiovascular diseases, are associated with specific changes in protein expression and modification patterns. By creating detailed proteoform profiles of healthy and diseased tissues, researchers may be able to identify new biomarkers for early disease detection or monitor disease progression.
Moreover, the insights gained from proteoform atlases could lead to the development of targeted therapies that address the specific molecular alterations in diseased cells. For instance, drugs could be designed to selectively interact with a particular proteoform or modulate its function, offering a more personalized approach to treatment.
Conclusion
The development of PEPPI-MS and the integration of FAIMS-LC-MS ion mobility spectrometry have revolutionized the field of proteomics by providing new ways to fractionate, analyze, and characterize proteoforms. These advances have made it possible to obtain a more comprehensive view of the proteome, revealing the complexity and diversity of proteins within living organisms.
As the techniques continue to evolve, the dream of constructing complete proteoform atlases for various species becomes increasingly achievable. Such efforts will not only enhance our understanding of basic biology but also pave the way for new diagnostic and therapeutic strategies that can improve human health. Through continued innovation in proteomics, we are on the brink of uncovering the full potential of the proteoform world and its impact on medicine and disease.
Reference: Ayako Takemori et al, PEPPI-MS: gel-based sample pre-fractionation for deep top-down and middle-down proteomics, Nature Protocols (2025). DOI: 10.1038/s41596-024-01100-0