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Advancements in Proteoform Analysis Through PEPPI-MS

Proteoforms, which are diversified protein molecules arising from a single gene, play crucial roles in various physiological processes within living organisms. Although humans possess around 22,000 genes, the exact number of distinct human proteoforms remains a mystery.

Liquid chromatography-mass spectrometry (LC-MS) is currently the primary technique utilized for proteoform analysis due to its sensitivity. Traditionally, proteoforms have been analyzed by breaking them down into smaller peptides. However, the innovative top-down proteomics approach allows for the examination of intact proteoforms, heralding a significant advancement in the field.

Efforts akin to the Human Genome Project have been made to create a comprehensive atlas of human proteoforms using top-down proteomics. However, the inherent complexity of proteoform components in biological samples poses challenges, as LC-MS alone cannot adequately identify all existing proteoforms.

In a notable development in 2020, researchers from Ehime University, led by the Takemori group, introduced PEPPI-MS. This method utilizes a cost-effective and straightforward SDS-PAGE system for high-resolution fractionation of proteoforms. PEPPI-MS has proven to effectively increase the number of detectable proteoforms in LC-MS analyses, leading to its widespread adaptation in pre-fractionation processes for top-down proteomics studies.

Building on this foundation, a collaboration in 2022 with Professor Andreas Tholey’s team at the University of Kiel in Germany resulted in the creation of an ultra-sensitive measurement system. This system merges PEPPI-MS fractionation with FAIMS ion mobility mass spectrometry, allowing for the separation of proteoforms in the gas phase.

In 2023, this advanced system facilitated a detailed analysis of human cultured cells through top-down proteomics and led to the establishment of middle-down proteomics, focusing on the analysis of Glu-C digestion products derived from proteoforms.

One of the significant benefits of PEPPI-MS is that it does not necessitate specialized equipment, making it accessible to standard biochemical laboratories. Its simplicity has made it a go-to method for sample fractionation in deep top-down proteomics.

To further enhance proteoform analysis capabilities, the Takemori group has established comprehensive experimental protocols for high-resolution proteoform fractionation using PEPPI-MS in conjunction with top-down and middle-down proteomics within the FAIMS-LC-MS framework. Recently, they published an integrated protocol in Nature Protocols, compiling their findings from 2020 to 2024.

The complete suite of PEPPI-MS protocols allows for high-resolution fractionation of trace biological samples, paving the way for extensive proteoform analysis through LC-MS. This work is anticipated to significantly contribute to the development of proteoform atlases across various species and enhance disease diagnostic methods grounded in precise proteoform profiling.

More information: 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

Citation: Streamlined protocol reveals chemical structure of cellular proteins on a large scale (2025, January 27) retrieved 27 January 2025 from https://phys.org/news/2025-01-protocol-reveals-chemical-cellular-proteins.html

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