# Theoretical Investigation of the Material Usage During On-Bead Enrichment of Post-Translationally Modified Peptides in Suspension Systems

## TL;DR

This paper presents a theoretical model to optimize the selective enrichment of low-abundance protein modifications, validated with experiments on glycosylated and phosphorylated peptides.

## Contribution

A novel thermodynamic model linking material dosage to analyte recovery in PTM enrichment is introduced and experimentally validated.

## Key findings

- Enrichment efficiency follows a non-linear trend, peaking at an optimal dosage before declining.
- Experimental validation confirmed the model's predictions for both glycosylated and phosphorylated peptides.
- Optimal dosage varies among peptides with the same modification, emphasizing the need for tailored strategies.

## Abstract

Over the past decade, the number and diversity of identified protein post-translational modifications (PTMs) have grown significantly. However, most PTMs occur at relatively low abundance, making selective enrichment of modified peptides essential. To address this, we developed a thermodynamic model describing the free beads enrichment in suspension enrichment process and derived a theoretical relationship between material dosage and analyte recovery. The model predicts a non-linear trend, with enrichment efficiency increasing up to an optimal dosage and declining thereafter—a pattern confirmed by experimental data. We validated the model using centrifugation-based enrichment for glycosylated peptides and magnetic-based enrichment for phosphorylated peptides. In both cases, the results aligned with theoretical predictions. Additionally, the optimal dosage varied among peptides with the same modification type, highlighting the importance of tailoring enrichment strategies. This study provides a solid theoretical and experimental basis for optimizing PTMs enrichment and advancing more sensitive, accurate, and efficient mass spectrometry-based proteomic workflows.