Proteomic Profile of EPS-Urine through FASP Digestion and Data-Independent Analysis
May 8th, 2021
Here, we present an optimized on-filter digestion protocol with detailed information about the following: protein digestion, peptide purification and data independent acquisition analysis. This strategy is applied to the analysis of expressed prostatic secretions-urine samples and allows high proteome coverage and low missing value label-free profiling of the urinary proteome.
Overview
This article details a comprehensive workflow for urinary proteome mapping, combining the filter-aided sample preparation (FASP) protocol, double StageTip purification, and LC-MS/MS in data-independent acquisition (DIA) mode. The protocol is demonstrated using expressed prostatic secretions (EPS)-urine, a sample type relevant for prostate cancer biomarker discovery. The workflow emphasizes deep proteome coverage, efficient sample concentration, and compatibility with various detergents, making it suitable for diluted protein samples.
Key Study Components
Area of Science
- Proteomics
- Analytical Chemistry
- Biomarker Discovery
Background
- FASP is widely used for proteomics sample preparation due to its compatibility with denaturing buffers and ability to concentrate diluted samples.
- Bottom-up proteomics workflows increasingly utilize LC-MS/MS in DIA mode for comprehensive proteome analysis.
- EPS-urine, collected after digital rectal exam, is a valuable sample for prostate cancer biomarker studies.
- Combining FASP with advanced purification and DIA enhances sensitivity and proteome coverage.
Purpose of Study
- To present a detailed workflow integrating FASP, double StageTip purification, and DIA LC-MS/MS for urinary proteome analysis.
- To demonstrate the protocol using EPS-urine as a model for prostate cancer biomarker discovery.
- To provide practical guidance for achieving deep urinary proteome coverage with minimal missing values.
Methods Used
- Sample centrifugation and storage of EPS-urine.
- Protein denaturation, reduction, and alkylation using SDS, DTT, and iodoacetamide.
- Concentration and buffer exchange via centrifugal filters (FASP protocol).
- Enzymatic digestion with trypsin.
- Peptide purification using strong cation exchange and reversed-phase StageTips.
- LC-MS/MS analysis in DIA mode with custom-packed analytical columns.
- Spectral library generation via data-dependent acquisition and sample fractionation.
Main Results
- The workflow enables deep coverage of the urinary proteome, identifying and quantifying proteins across a dynamic range spanning five orders of magnitude.
- Combining FASP with DIA scanning mode provides a rich and reproducible proteomic map.
- The protocol is effective for EPS-urine and can be extended to general urinary proteomics.
- StageTip purification efficiently removes detergents and concentrates peptides for LC-MS/MS analysis.
Conclusions
- The integrated FASP-DIA workflow is robust, sensitive, and suitable for biomarker discovery in urinary proteomics.
- It offers flexibility for different LC-MS setups, with or without trapping columns.
- The method is recommended for EPS-urine analysis and adaptable to other urinary proteome studies.
Frequently Asked Questions
Executive Industry Relevance
Comprehensive urinary proteome mapping using FASP digestion and DIA LC-MS/MS enables deep, quantitative profiling of low-abundance proteins in highly diluted clinical samples. This workflow supports biomarker discovery efforts, particularly in prostate cancer, by providing reproducible, high-coverage datasets critical for early-stage target validation and translational research. The protocol's compatibility with diverse sample matrices and detergents enhances its utility across biopharma discovery pipelines.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables robust interrogation of disease-relevant proteomic changes in EPS-urine for biomarker hypothesis testing.
- Supports functional target validation by quantifying protein abundance across a wide dynamic range.
- Facilitates biological de-risking through reproducible detection of low-abundance proteins in complex matrices.
Screening & Assay Development
- Prepares highly purified peptide samples suitable for downstream LC-MS/MS screening workflows.
- Delivers standardized, quantitative proteomic outputs for assay development and optimization.
- Reduces missing values, supporting reliable compound evaluation and screening scalability.
Translational & Preclinical Research
- Aligns proteomic outputs with translational biomarker strategies in prostate cancer research.
- Provides continuity from discovery-stage proteomics to preclinical validation of candidate biomarkers.
- Enables risk-adjusted advancement decisions based on quantitative, reproducible proteomic data.
Pipeline & Workflow Integration
This FASP-DIA workflow integrates into the discovery continuum from early biomarker identification through preclinical validation, supporting both hypothesis-driven and unbiased proteomic analyses.
- Discovery Biology: Facilitates null hypothesis testing and pathway clarification in disease-relevant samples.
- Screening: Provides reproducible, quantitative peptide readouts for assay readiness and platform reuse.
- Analytics: Enables robust statistical comparison of protein abundance across experimental conditions.
- Translational Research: Supports biomarker alignment and continuity into preclinical models.
- Enterprise Reuse: Offers a standardized, adaptable protocol for diverse urinary proteomics applications.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in biomarker discovery.
- Operational Value: Delivers standardized, scalable, and reproducible sample preparation and analysis.
- Strategic Value: Improves go/no-go decision quality and capital efficiency in early discovery.
- Portfolio Impact: Enables risk-adjusted prioritization of biomarker candidates for advancement.
Implementation Considerations
- Requires expertise in proteomics sample preparation and LC-MS/MS operation.
- Demands access to high-resolution mass spectrometry and robust data analysis infrastructure.
- Necessitates cross-team standardization of sample handling and purification steps.
- Adaptable to various urinary sample types and compatible with different LC-MS setups.
- Sample throughput and spectral library generation may limit scalability in large cohorts.
Biopharma FAQs
Chapters in this video
0:00
Introduction
0:55
Protocol
1:25
Protocol: FASP Digestion
5:29
Protocol: SCX Purification
8:08
Protocol: Protein Quantifcation by External Standard
9:23
Protocol: NanoLC-MS/MS and DIA Analysis
11:31
Protocol: High-pH C18 Fractionation for Spectral Library
13:09
Protocol: Data Analysis
13:35
Expected Results
13:53
Conclusion
