PNNL

Abstract

Bottom-up proteomic workflows rely on sequential pre-processing 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. 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.