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Easy Proteomics Sample Preparation: Technical Repeatability and Workflow Optimization Across 8 Biological Matrices in

Paraskevi Karousi1,2, Maria Voumvouraki2,3, Panagiota Efstathia Nikolaou4,5

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Proteomics
|October 24, 2025
PubMed
Summary
This summary is machine-generated.

This study optimized the Sample Preparation by Easy Extraction and Digestion (SPEED) protocol for scalable, high-throughput proteomics. The adapted SPEED method enables accurate protein analysis from minimal cell numbers across diverse biological samples.

Keywords:
SPEED protocolbottom‐up proteomicsdiaPASEFtechnical repeatabilitytryptic peptides

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Area of Science:

  • Proteomics
  • Biochemistry
  • Analytical Chemistry

Background:

  • Bottom-up proteomics requires efficient sample preparation for accurate protein identification and quantification.
  • Existing protocols can be complex and may not suit diverse biological matrices, including lysis-resistant samples.

Purpose of the Study:

  • To evaluate and adapt the Sample Preparation by Easy Extraction and Digestion (SPEED) protocol for simplified, detergent-free proteomics.
  • To enable standardized, cost-effective, and scalable proteomics analysis across various biological samples.
  • To demonstrate the protocol's down-scalability for low cell input and its compatibility with high-throughput workflows.

Main Methods:

  • Refined protein extraction and denaturation steps for 8 biological matrices.
  • Adapted the SPEED protocol for 96-well plate compatibility and used a low-detergent RIPA buffer for tissue samples.
  • Implemented optimized data-independent acquisition (DIA) windows for diaPASEF analysis with short nanoLC-MS/MS runs.

Main Results:

  • Achieved robust proteomics measurements from as few as 3000 cells for preparation and 300 cells for LC-MS/MS analysis.
  • Enabled a throughput of 15-20 samples per day with 30-minute nanoLC-MS/MS runs.
  • Demonstrated enhanced proteome coverage and streamlined workflows for challenging biological matrices.

Conclusions:

  • The adapted SPEED protocol offers a robust, scalable, and cost-effective solution for high-throughput proteomics.
  • This method facilitates reproducible proteomic studies across diverse biological contexts, even with limited sample material.
  • Early-stage optimization and feasibility testing are crucial for successful proteomics pipeline development.