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A Plasma Sample Preparation for Mass Spectrometry using an Automated Workstation
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Published on: April 24, 2020

A high-throughput sample preparation method for cellular proteomics using 96-well filter plates.

Linda Switzar1, Jordy van Angeren, Martijn Pinkse

  • 1AIMMS Division of BioMolecular Analysis, VU University, Amsterdam, The Netherlands.

Proteomics
|August 15, 2013
PubMed
Summary
This summary is machine-generated.

This study presents a new 96-well plate method for shotgun proteomics sample preparation. This high-throughput protocol enhances protein identification sensitivity and is ideal for cellular proteomics research.

Keywords:
Bottom-up proteomicsCellular proteomicsFilter plateHigh-throughput sample preparationLC-MSTechnology

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Area of Science:

  • Proteomics
  • Cellular Biology
  • Biochemistry

Background:

  • Shotgun proteomics requires efficient sample preparation for analyzing complex cell lysates.
  • Existing methods can be time-consuming and may lead to sample loss.
  • High-throughput methods are crucial for large-scale cellular proteomics studies.

Purpose of the Study:

  • To develop a high-throughput, 96-well plate-based sample preparation protocol for shotgun proteomics.
  • To improve the sensitivity and efficiency of protein identification from cell lysates.
  • To create a platform compatible with functional assays in cellular proteomics.

Main Methods:

  • A novel protocol utilizing 96-well molecular weight cutoff (MWCO) filter plates was developed.
  • All steps, including lysis, buffer exchange, denaturation, reduction, alkylation, and digestion, were performed in the 96-well format.
  • The protocol integrated sample concentration and washing steps using organic solvents.

Main Results:

  • The optimized protocol demonstrated high throughput and compatibility with automation.
  • The MWCO filter enabled sample concentration, leading to increased sensitivity.
  • The method identified more cellular proteins compared to a standard gel-filtration column protocol.

Conclusions:

  • The developed 96-well MWCO filter plate protocol is an efficient and sensitive method for shotgun proteomics.
  • This platform facilitates high-throughput analysis and is suitable for large-scale cellular proteomics.
  • The protocol's integration with functional assays offers a comprehensive approach to cellular proteomics.