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

As novel liquid chromatography-mass spectrometry(LC-MS) technologies for proteomics offer a substantial increase inLC-MS runs per day, robust and reproducible sample preparationemerges as a new bottleneck for throughput. We introduce a novelstrategy for positive-pressure 96-wellfilter-aided sample preparation(PF96) on a commercial positive-pressure solid-phase extractiondevice. PF96 allows for afive-fold increase in throughput inconjunction with extraordinaryreproducibility with Pearsonproduct-moment correlations on the protein level ofr= 0.9993,as demonstrated for mouse heart tissue lysate in 40 technicalreplicates. The targeted quantification of 16 peptides in the presence of stable-isotope-labeled reference peptides confirms that PF96variance is barely assessable against technical variation from nanoLC-MS instrumentation. We further demonstrate that protein loadsof 36-60 mu g result in optimal peptide recovery, but lower amounts >= 3 mu g can also be processed reproducibly. In summary, thereproducibility, simplicity, and economy of time provide PF96 a promising future in biomedical and clinical research.

Automated summary

This study introduces a novel strategy for positive-pressure 96-well filter-aided sample preparation (PF96), which significantly increases throughput and demonstrates high reproducibility. The study confirms the excellent reproducibility and stability of PF96 at the protein level, as well as its compatibility with nanoLC-MS instrumentation. This method has promising applications in biomedical and clinical research.