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Organic Solvent-Based Protein Precipitation for Robust Proteome Purification Ahead of Mass Spectrometry
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Ultrasonic-Based Filter Aided Sample Preparation as the General Method to Sample Preparation in Proteomics.

Luís B Carvalho1,2, José-Luis Capelo-Martínez1,2, Carlos Lodeiro1,2

  • 1BIOSCOPE Research Group, LAQV-REQUIMTE, Department of Chemistry, Faculty of Science and Technology, Universidade NOVA de Lisboa, Campus de Caparica, Caparica 2829-516, Portugal.

Analytical Chemistry
|June 3, 2020
PubMed
Summary

We developed a rapid ultrasonic-assisted filter-aided sample preparation (US-FASP) method, reducing proteomics sample processing time from overnight to 2.5 hours. This ultrafast, scalable technique maintains high protein identification rates and minimizes resource use.

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Area of Science:

  • Proteomics
  • Biochemistry
  • Analytical Chemistry

Background:

  • Traditional filter-aided sample preparation (FASP) is time-consuming, limiting high-throughput proteomics.
  • Optimizing sample preparation protocols is crucial for efficient large-scale proteomic analyses.

Purpose of the Study:

  • To develop and validate a significantly faster, high-throughput version of the FASP protocol.
  • To assess the impact of a novel ultrasonic-assisted approach on protein identification and quantification.

Main Methods:

  • Modified FASP protocol incorporating ultrasonic fields for protein reduction, alkylation, and digestion.
  • Rapid processing of 36 samples in 2.5 hours, scalable to 96-well plates.
  • Comparative analysis of US-FASP against standard overnight FASP using E. coli, mouse brain, and mouse liver proteomes.

Main Results:

  • Achieved protein reduction, alkylation, and digestion in 5.25 minutes using ultrasonic assistance.
  • No significant statistical differences in protein identification (>92%) compared to standard FASP across tested proteomes.
  • Successful relative label-free quantification of spiked proteins in E. coli samples.

Conclusions:

  • The US-FASP method offers a robust, rapid, and scalable alternative to conventional FASP protocols.
  • This approach minimizes time, cost, and waste while preserving analytical performance.
  • US-FASP enhances efficiency for large-scale proteomics studies.