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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

• 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 \(\it r\) = 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 \(\mu\)g result in optimal peptide recovery, but lower amounts \(\geq\)3 \(\mu\)g can also be processed reproducibly. In summary, the reproducibility, simplicity, and economy of time provide PF96 a promising future in biomedical and clinical research.