Towards zero variance in proteomics - positive pressure FASP in 96-well format (PF96) enables highly reproducible and time-efficient analysis of sample cohorts

As novel LC-MS technologies for proteomics offer 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 5-fold increase in throughput in conjunction with extraordinary reproducibility on protein level (Pearson Product-Moment correlations of r=0.9993), as demonstrated for mouse heart tissue lysate in 40 technical replicates. Subsequent targeted quantification of 16 endogenous peptides in presence of stable-isotope-labeled reference peptides confirmed that PF96 variance is barely accessible against the technical variation originating from nanoLC-MS instrumentation. We further demonstrate that protein loads of 36-60 µg result in optimal peptide recovery, but also that lower amounts ≥ 3 µg can be reproducibly processed. In summary, reproducibility, simplicity and economy of time render PF96 a promising future for various applications in the field of proteomics including clinical and biomedical research.