Related Experiment Video
Updated: May 2, 2026
14:48
Proteomic Profile of EPS-Urine through FASP Digestion and Data-Independent Analysis
Published on: May 8, 2021
7.8K
Enhanced FASP (eFASP) to increase proteome coverage and sample recovery for quantitative proteomic experiments
Jonathan Erde1, Rachel R Ogorzalek Loo, Joseph A Loo
1Department of Chemistry and Biochemistry and ‡Department of Biological Chemistry, University of California-Los Angeles , Los Angeles, California 90095, United States.
Journal of Proteome Research
|February 21, 2014
Summary
Enhanced filter-aided sample preparation (eFASP) improves proteomic analysis by increasing protein recovery and sensitivity. This method uses alternative reagents to reduce sample loss and enhance digestion efficiency for better proteomic coverage.
Area of Science:
- Proteomics
- Biochemistry
- Analytical Chemistry
Background:
- Quantitative proteomic experiments require robust protein extraction and purification.
- Traditional methods and filter-aided sample preparation (FASP) face challenges with sample loss and contaminant removal.
- Existing methods can lead to incomplete contaminant removal and significant sample loss.
Purpose of the Study:
- To present an enhanced filter-aided sample preparation (eFASP) approach.
- To improve sensitivity, recovery, and proteomic coverage compared to traditional FASP.
- To address limitations of current proteomic sample preparation techniques.
Main Methods:
- Developed eFASP by substituting reagents in traditional FASP.
- Utilized 0.2% deoxycholic acid for improved tryptic digestion efficiency.
- Incorporated prepassivation of Microcon filter surfaces with 5% TWEEN-20 to reduce peptide loss.
- Introduced an express eFASP method with tris(2-carboxyethyl)phosphine and 4-vinylpyridine for protein alkylation.
Main Results:
- eFASP demonstrated improved sensitivity, recovery, and proteomic coverage.
- Substitution of deoxycholic acid enhanced tryptic digestion for cytosolic and membrane proteins without additional cleanup.
- Prepassivation with TWEEN-20 reduced peptide loss by 300% in classic FASP.
- Express eFASP increased alkylation specificity and processing speed.
Conclusions:
- The eFASP approach offers significant improvements for proteomic sample preparation.
- Alternative reagents and methods enhance efficiency and reduce sample loss in FASP.
- eFASP provides a more sensitive and comprehensive proteomic analysis.
