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

Area of Science:

• Proteomics
• Mass Spectrometry
• Bioinformatics

Background:

• Bottom-up proteomics uses peptide-to-protein aggregation (roll-up) for data reliability.
• Standard roll-up may be suboptimal for post-translational modification (PTM) and limited proteolysis-mass spectrometry (LiP-MS) studies.

Purpose of the Study:

• Investigate the impact of different roll-up strategies on site-level quantification in PTM differential analysis.
• Introduce and evaluate a novel site-centric roll-up approach for LiP-MS.

Main Methods:

• Benchmarking various roll-up methods (sum, mean, median) using simulation studies.
• Comparing sensitivity and specificity for detecting structural and PTM-driven changes.
• Evaluating site-level vs. peptide-level quantification in LiP-MS.

Main Results:

• Median and mean roll-up methods demonstrate superior performance over the sum method for both PTM and LiP proteomics.
• Site-level quantification in LiP-MS proves more effective than peptide-level quantification.
• The novel site-centric approach enhances LiP-MS data interpretation.

Conclusions:

• Provides the first systematic, data-driven guidance for selecting roll-up techniques in site-level proteomic analyses.
• Highlights the importance of tailored roll-up strategies for PTM and structural proteomics.
• Site-centric roll-up offers significant advantages for LiP-MS applications.