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.

For targeted LC-MS, 2.5 µl mouse heart digest with a theoretical concentration of 75 ng /uL were injected including 22 SIL peptides ranging from 0.5-50 fmol/uL. Samples were loaded in 0.1 % TFA at a flow rate of 100 µl/min and the pre-column (Acclaim PepMAP C18, 0.3 x 5mm, 5 µm, 100 Å) was switched in-line after 30 s. Peptides were separated at a flowrate of 500 nL/min (Acclaim PepMAP C18, 0.075 x 150 mm, 2 µm, 100 Å) using a 12 min linear ACN gradient from 4 - 25 % ACN in presence of 0.1 % FA. The Q Exactive HF was operated in parallel reaction monitoring mode (PRM) using a resolution of 60,000 (at 200 m/z) and maximum ion injection time of 120 ms to reach the AGC target value of 10^6. Corresponding heavy and light precursor ions were isolated for 1.5 min at their expected retention time (scheduled, maximum 10 of 32 precursors in parallel) using an 0.4 m/z window and an NCE of 27.

All animals were treated in accordance with federal guidelines according to the Committee on Animal Research of the regional government (Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen LANUV-NRW). Left ventricular tissue of a PBS-perfused wild type mouse heart was used for lysis. 300 µL lysis buffer (50 mM Tris-HCl pH 7.8, 150 mM NaCl, 1% SDS supplemented with complete mini-EDTA free protease inhibitor and PhosStop (Roche, Penzberg)) and approx. 20 glass beads were added to approx. 8 mg of punched tissue. Tissue was disrupted by sonication for 20 min (30 s on, 30 s off) using a Bioruptor Plus (Diagenode) and 10 µL were subjected to amino acid analysis.