# Comparative Analysis of Digestion Methods for Bile Proteomics: The Key to Unlocking Biliary Biomarker Potential

## TL;DR

This study compares different digestion methods for bile proteomics to find the best approach for identifying biliary biomarkers.

## Contribution

The study provides a systematic comparison of trypsin digestion methods for bile proteomics, highlighting optimal approaches for protein identification.

## Key findings

- Methods involving precipitation identified more proteins than crude methods, except for in-gel digestion.
- In-gel, in-solution, and EasyPep methods showed the least intermethod variability.
- Age-related differences in protein expression were observed, with younger donors showing metabolic processes and older donors showing immune and cell cycle processes.

## Abstract

Background: Bile’s
potential to reflect the health of the
biliary system has led to increased attention, with proteomic analysis
offering deeper understanding of biliary diseases and potential biomarkers.
With the emergence of normothermic machine perfusion (NMP), bile can
be easily collected and analyzed. However, the composition of bile
can make the application of proteomics challenging. This study systematically
evaluated various trypsin digestion methods to optimize proteomics
of bile from human NMP livers. Methods: Bile was collected from 12
human donor livers that were accepted for transplantation after the
NMP viability assessment. We performed tryptic digestion using six
different methods: in-gel, in-solution, S-Trap, SMART, EasyPep, and
filter-aided sample purification, with or without additional precipitation
before digestion. Proteins were analyzed using untargeted proteomics.
Methods were assessed for total protein IDs, variation, and protein
characteristics to determine the most optimal method. Results: Methods
involving precipitation surpassed crude methods in protein identifications
(4500 vs 3815) except for in-gel digestion. Filtered data (40%) resulted
in 3192 versus 2469 for precipitated and crude methods, respectively.
We found minimal differences in mass, cellular components, or hydrophobicity
of proteins between methods. Intermethod variability was notably diverse,
with in-gel, in-solution, and EasyPep outperforming others. Age-related
biological comparisons revealed upregulation of metabolic-related
processes in younger donors and immune response and cell cycle-related
processes in older donors. Conclusions: Variability between methods
emphasizes the importance of cross-validation across multiple analytical
approaches to ensure robust analysis. We recommend the in-gel crude
method for its simplicity and efficiency, avoiding additional precipitation
steps. Sample processing speed, cost, cleanliness, and reproducibility
should be considered when a digestion method is selected for bile
proteomics.