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Quick 96FASP for high throughput quantitative proteome analysis.

Yanbao Yu1, Shiferaw Bekele1, Rembert Pieper1

  • 1J. Craig Venter Institute, 9714 Medical Center Drive, Rockville, MD 20850, United States.

Journal of Proteomics
|July 4, 2017
PubMed
Summary
This summary is machine-generated.

Quick 96-well filter aided sample preparation (q96FASP) significantly reduces processing time for high-throughput proteomics. This method generates protein digests comparable to standard FASP, proving cost- and time-effective for large-scale biomarker discovery.

Keywords:
96-well filter plateApplicationsFASPHuman urineKlebsiella pneumoniaeQuantitative proteomics

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Area of Science:

  • Proteomics
  • Biomarker Discovery
  • Analytical Chemistry

Background:

  • Filter aided sample preparation (FASP) is a key method for proteomic sample cleanup and peptide generation.
  • Previous 96-well FASP (96FASP) offers high throughput but is limited by lengthy centrifugation times.
  • Reducing sample processing time is critical for large-scale quantitative proteomics.

Purpose of the Study:

  • To develop a faster, high-throughput 96-well FASP method (q96FASP).
  • To maintain sample quality comparable to conventional single-well FASP.
  • To assess the suitability of q96FASP for quantitative proteomics and biomarker discovery.

Main Methods:

  • Utilized filter membranes with a large molecular weight cut-off (MWCO) of ~30kDa.
  • Adapted the 96-well filter plate format for rapid sample preparation.
  • Performed side-by-side comparisons with single FASP using whole cell lysates and human urine.

Main Results:

  • q96FASP significantly reduced centrifugation times from ~1.5 hours to ~20 minutes per spin.
  • Protein digests generated by q96FASP were of similar quality to those from single FASP.
  • Label-free quantitation demonstrated excellent experimental repeatability in parallel sample processing.

Conclusions:

  • q96FASP offers a cost- and time-effective solution for shotgun proteomics.
  • The method is highly suitable for large-scale biomarker discovery studies.
  • q96FASP represents a significant advancement in multi-well plate-based proteomic sample preparation.