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Rapid, One-Step Sample Processing for Label-Free Single-Cell Proteomics.

S Madisyn Johnston1, Kei G I Webber1, Xiaofeng Xie1

  • 1Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, United States.

Journal of the American Society for Mass Spectrometry
|July 6, 2023
PubMed
Summary
This summary is machine-generated.

A new one-step method simplifies single-cell proteomics sample preparation, reducing hands-on time and increasing protein identification. This faster, more accessible approach enhances proteome coverage without compromising results.

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Area of Science:

  • Proteomics
  • Biochemistry
  • Analytical Chemistry

Background:

  • Single-cell proteomics sample preparation is typically a multi-step, time-consuming process.
  • Existing workflows can be labor-intensive and introduce errors, leading to long sample-to-answer times.
  • There is a need for streamlined methods to increase accessibility and efficiency in single-cell proteomics.

Purpose of the Study:

  • To develop and validate a rapid, one-step sample preparation method for single-cell proteomics.
  • To compare the efficiency and proteome coverage of the new method against traditional multi-step workflows.
  • To evaluate the impact of different substrate materials on sample recovery and proteome coverage.

Main Methods:

  • A novel one-step protocol combining cell lysis, protein denaturation, and digestion in one hour.
  • Evaluation of four different reagent compositions for the one-step method.
  • Comparison of microfluidic chip materials (glass vs. polypropylene) for sample recovery.
  • Analysis of proteome coverage using Orbitrap mass spectrometry with a data-dependent acquisition workflow.

Main Results:

  • The optimized one-step method achieved cell lysis, denaturation, and digestion within 1 hour.
  • The one-step preparation demonstrated increased proteome coverage compared to the multi-step workflow.
  • Injection-molded polypropylene chips yielded improved proteome coverage over glass nanowell chips.
  • An average of nearly 2400 proteins per cell were identified using the combined one-step method and polypropylene substrates.

Conclusions:

  • The developed one-step sample preparation method significantly simplifies single-cell proteomics workflows.
  • This approach enhances proteome coverage and reduces labor and potential errors.
  • Utilizing polypropylene substrates further improves results, making single-cell proteomics more accessible without sacrificing data quality.