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DOE OSTI · 3375676

Aggregation Methods for Quantifying PTM and Structural Changes in Bottom-Up Proteomics

VonKaenel, Erik D. [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)] (ORCID:0000000289337413)·Rozum, Jordan C. [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)] (ORCID:0000000243569809)·Zhang, Tong [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)] (ORCID:0000000325402017)·Stratton, Kelly G. [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)] (ORCID:0000000217219688)·Bramer, Lisa M. [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)] (ORCID:0000000283841926)·Wiley, Henry Steven [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)] (ORCID:0000000302326867)·Qian, Wei-Jun [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)] (ORCID:0000000253932827)·Sims, Amy C. [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)]·Melchior, John T. [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)] (ORCID:0000000337812566)·Feng, Song [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)] (ORCID:0000000339839009)
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Abstract

Bottom-up proteomic workflows rely on sequential preprocessing steps, commonly including peptide-to-protein aggregation (“roll-up”), to enhance data reliability and interpretability. While roll-up is effective for protein-centered analyses, it may be suboptimal for applications focused on post-translational modifications (PTMs) or protein structural changes, such as limited proteolysis–mass spectrometry (LiP-MS). Here, we investigate how different roll-up strategies influence site-level quantification in PTM differential analysis. Moreover, we introduce a novel site-centric roll-up approach tailored for LiP-MS, which quantifies proteolytic fragments rather than solely tryptic peptides. We benchmark these methods through simulation studies, comparing their sensitivity and specificity in detecting structural and PTM-driven changes. We found that the median and mean roll-up methods outperform the sum method in both PTM and LiP proteomics, and site-level quantification in LiP outperforms peptide-level quantification. Our findings offer the first systematic, data-driven guidance for selecting roll-up techniques in site-level proteomic analyses, with implications for both PTM-focused and structural proteomics studies.

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aggregationbottom-up proteomicslimited proteolysis mass spectrometry (LiP-MS)modificationpeptides and proteinspost-translational modifications (PTMs)protein structureproteomicsproteomics quantification

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VonKaenel, Erik D. [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)] (ORCID:0000000289337413), Rozum, Jordan C. [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)] (ORCID:0000000243569809), Zhang, Tong [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)] (ORCID:0000000325402017), Stratton, Kelly G. [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)] (ORCID:0000000217219688), Bramer, Lisa M. [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)] (ORCID:0000000283841926), Wiley, Henry Steven [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)] (ORCID:0000000302326867), Qian, Wei-Jun [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)] (ORCID:0000000253932827), Sims, Amy C. [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)], Melchior, John T. [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)] (ORCID:0000000337812566), Feng, Song [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)] (ORCID:0000000339839009). 2026-05-15. Aggregation Methods for Quantifying PTM and Structural Changes in Bottom-Up Proteomics. https://doi.org/10.1021/acs.jproteome.5c00782

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