Positive pressure filter-aided sample preparation (FASP) in 96-well format

Cysteines were reduced using 10 mM dithiothreitol (Roche) at 56 °C for 30 min and alkylated in the presence of 25 mM iodoacetamide (Sigma) for 30 min at RT in the dark. Samples were diluted at least 1:4 in 8 M urea (Sigma-Aldrich) dissolved in 100 mM Tris–HCl, pH 8.5 (Applichem), and transferred to a 30-kDa AcroPrep Omega filter membrane plate (PALL, New York, USA, purchased via VWR, Hannover, Germany, REF 8035/518–0028) in a blocked randomized order, which was also kept for subsequent LC–MS measurements. The filter plate was placed on top of a 2.2-ml MegaBlock collection plate (Sarstedt, Nümbrecht, Germany) and the liquid of the protein solution was forced through the filter using a Resolvex A200 (Tecan, Männedorf, Switzerland) connected to nitrogen gas (N₂, 5.5 bar, purity 4.8 or higher, Linde, Düsseldorf, Germany) using a relative pressure of 20% of the low profile setting. Subsequently, the dispensing function of the A200 was used to wash the filter twice with 200 µl 8 M urea buffer in Tris–HCl pH 8.5 and twice with 200 µl 50 mM ammonium bicarbonate (ABC, Fluka). After each washing step, the liquid was forced through the filter using the same pressure profile as for loading. Afterwards, the plate was centrifuged for 2 min at RT and 1000 g to remove residual drops under the membrane. For digestion, 100 µl digestion buffer were added comprising 100 mM urea, 50 mM ABC, and 2 mM CaCl₂ (Merck) including sequencing-grade trypsin (Promega, sequencing-grade modified trypsin) in a concentration to meet a 1:3 (w/w) enzyme-to-sample ratio. After incubation for 16 h at 37 °C, the digested protein fraction was forced through the filter and collected in a 500-µl LoBind plate (Eppendorf, Hamburg, Germany). The filter was further washed with 100 µl 50 mM ABC followed by 100 µl H₂O, both wash steps were also collected in the LoBind plate. Then, tryptic digestion was stopped by reducing the pH < 2 through the addition of trifluoroacetic acid (TFA; Biosolve, Valkenswaard, Netherlands) to a final concentration of 1% (v/v). Aliquots were transferred to 700 µl glass-vial plates (Waters, Eschborn, Germany) for injection on a monolithic column HPLC (for tryptic digestion quality control) [74]. Based on the results, 4–5 replicates per condition were further analyzed by LC–MS.