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Poster Presentations
Day 1, June 22（Sun.）
Room P (Maesato East, Foyer, Ocean Wing)
- 1P-PM-37
Native Digestion-Based Sample Preparation for Plasma Proteomics
(1Kyoto Univ., 2NIBIOHN)
oHiroto Kakiuchi1, Ayana Tomioka1, Kosuke Ogata1, Eisuke Kanao1,2, Yasushi Ishihama1,2
Plasma proteomics is highly challenging due to the presence of thousands of proteins and peptides spanning a dynamic range of over 1012. However, plasma is an essential source for biomarker and drug discovery because it is minimally invasive and rich in biological information. Despite global efforts, no standardized method for plasma proteome analysis has been established.
We developed a high-throughput plasma sample preparation strategy using tryptic digestion under non-denaturation condition (“native digestion"). Native digestion poorly digests highly abundant, folded plasma proteins such as albumin and immunoglobulins, while unfolded proteins and those with intrinsically disordered regions are more accessible to the protease, yielding more peptide fragments. A novel fractionation step utilizing pipette tips packed with reversed-phase sponge-like polymer (ChocoTip) removes folded proteins.
Using 100 µg of non-denatured HeLa cell protein extracts, native digestion identified 2,332 proteins, with a 98.0% overlap with proteins identified under denaturation digestion. Gene ontology analysis revealed significant enrichment of “RNA binding" proteins (FDR = 10-54), suggesting a preference for proteins with lower structural rigidity. In human plasma, our method increased protein identification from 232 to 394 (1.7-fold) and reduced the dominance of highly abundant proteins. This approach enhances the detection of low-abundance proteins in complex plasma samples.