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Author Spotlight: Advancing Tear Fluid Analysis Using a Standardized Protocol for Proteomics Research

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Published on: December 1, 2023

Optimized Suspension Trapping Method for Phosphoproteomics Sample Preparation.

Fujia Wang¹, Tim Veth¹, Marije Kuipers²

¹Biomolecular Mass Spectrometry and Proteomics, Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, the Netherlands.

Analytical Chemistry

|June 15, 2023

Summary

This summary is machine-generated.

The standard Suspension Trapping (S-Trap) method hinders phosphoproteomics. Optimizing S-Trap by replacing phosphoric acid with trifluoroacetic acid improves phosphopeptide enrichment for mass spectrometry.

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A Streamlined Approach for Mass Spectrometry-Based Proteomics Using Selected Tissue Regions

A Streamlined Approach for Mass Spectrometry-Based Proteomics Using Selected Tissue Regions

Published on: April 18, 2025

Area of Science:

Proteomics
Mass Spectrometry
Biochemistry

Background:

Effective sample preparation is crucial for mass spectrometry-based phosphoproteomics.
Suspension trapping (S-Trap) is a widely used method for bottom-up proteomics sample preparation.
The suitability of the standard S-Trap protocol for phosphoproteomics remains unclear.

Purpose of the Study:

To evaluate the performance of the S-Trap protocol for both proteomics and phosphoproteomics.
To identify limitations of the standard S-Trap protocol in phosphoproteomics analysis.
To develop an optimized S-Trap method for improved phosphopeptide enrichment.

Main Methods:

Systematic evaluation of S-Trap digestion for proteomics and phosphoproteomics using large- and small-scale samples.
Comparison of the standard S-Trap protocol (using phosphoric acid) with an optimized protocol (using trifluoroacetic acid).
Application of the optimized S-Trap protocol to extracellular vesicle samples.

Main Results:

The standard S-Trap protocol, particularly the use of phosphoric acid, is detrimental to downstream phosphopeptide enrichment.
An optimized S-Trap protocol, substituting trifluoroacetic acid for phosphoric acid, significantly enhances phosphoproteomics analysis.
The optimized S-Trap method demonstrates superior performance for low-abundance, membrane-rich samples like extracellular vesicles.

Conclusions:

The standard S-Trap protocol is suboptimal for phosphoproteomics due to the detrimental effects of phosphoric acid.
An optimized S-Trap protocol using trifluoroacetic acid offers a simple and effective solution for phosphoproteomics sample preparation.
This optimized workflow is particularly beneficial for analyzing challenging samples such as extracellular vesicles.