# Comparison of the Trapping Efficiency for Tryptic Peptides on Particle-Packed and Micro-Pillar Trap Columns for Proteomics Analyses

## TL;DR

This study compares two types of trapping columns for proteomics, finding that their design affects peptide recovery and proteome coverage.

## Contribution

The study provides a direct comparison of packed and micropillar monolithic trap columns for peptide trapping efficiency in proteomics.

## Key findings

- Packed trap columns recovered more total peptides, especially those with post-translational modifications and higher hydrophilicity.
- Monolithic columns favored peptides with intermediate hydrophobicity and showed reduced trapping efficiency for hydrophilic peptides.
- Both trap types identified over 2500 proteins, but with differences in peptide recovery profiles.

## Abstract

Background: Low-volume trapping columns are essential for sample enrichment, desalting, and injection profile focusing on nano-LC–MS-based proteomics. They enable higher sample loading, improve chromatographic performance, and protect the analytical column by removing salts and contaminants. Recently, monolithic trap columns with micropillar architecture have emerged as alternatives to conventionally packed traps. This study compares the performance of a packed and a micropillar monolithic trap column for the analysis of tryptic peptides. Methods: A tryptic digest of HeLa cell lysate was analyzed under identical LC–MS conditions using both trap types. Peptides were detected at 214 nm and analyzed by nano-ESI on a Q Exactive Plus Orbitrap. Data were searched against the human UniProt database (February 2023) using FragPipe v20.0, and statistical evaluation of MaxLFQ intensities was performed in Perseus using Welch’s t-test and clustering analysis. Results: Over 2500 proteins were identified with both setups. The packed trap column yielded more total peptides, particularly those with post-translational modifications and higher hydrophilicity, whereas the monolithic column favored peptides of intermediate hydrophobicity. Chromatographic profiles confirmed a slight reduction in the trapping efficiency of hydrophilic peptides by the monolithic trap. Conclusions: Trap column design significantly influences peptide recovery and proteome coverage.