Toward Zero Variance in Proteomics Sample Preparation: Positive-Pressure FASP in 96-Well Format (PF96) Enables Highly Reproducible, Time- and Cost-Efficient Analysis of Sample Cohorts

Stefan Loroch*, Dominik Kopczynski, Adriana C Schneider, Cornelia Schumbrutzki, Ingo Feldmann, Eleftherios Panagiotidis, Yvonne Reinders, Roman Sakson, Fiorella A Solari, Alicia Vening, Frauke Swieringa, Johan W M Heemskerk, Maria Grandoch, Thomas Dandekar, Albert Sickmann*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

3 Citations (Web of Science)


As novel liquid chromatography-mass spectrometry (LC-MS) technologies for proteomics offer a substantial increase in LC-MS runs per day, robust and reproducible sample preparation emerges as a new bottleneck for throughput. We introduce a novel strategy for positive-pressure 96-well filter-aided sample preparation (PF96) on a commercial positive-pressure solid-phase extraction device. PF96 allows for a five-fold increase in throughput in conjunction with extraordinary reproducibility with Pearson product-moment correlations on the protein level of r = 0.9993, as demonstrated for mouse heart tissue lysate in 40 technical replicates. The targeted quantification of 16 peptides in the presence of stable-isotope-labeled reference peptides confirms that PF96 variance is barely assessable against technical variation from nanoLC-MS instrumentation. We further demonstrate that protein loads of 36-60 μg result in optimal peptide recovery, but lower amounts ≥3 μg can also be processed reproducibly. In summary, the reproducibility, simplicity, and economy of time provide PF96 a promising future in biomedical and clinical research.

Original languageEnglish
Pages (from-to)1181-1188
Number of pages8
JournalJournal of Proteome Research
Issue number4
Publication statusPublished - 1 Apr 2022


  • Animals
  • Chromatography, Liquid/methods
  • Humans
  • Mass Spectrometry/methods
  • Mice
  • Peptides/analysis
  • Proteomics/methods
  • Reproducibility of Results
  • proteomics
  • sample preparation
  • automation
  • PF96
  • FASP

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