The plasma samples from 171 patients who were enrolled in the participating centers in Bochum and Bonn were digested according to the SP3 protocol with slight modifications. Briefly, 100 µg protein was purified using paramagnetic beads (Cytiva Sera-Mag Carboxyl-Magnet-Beads, GE Healthcare, Chicago, IL) and digested overnight using trypsin (SERVA Electrophoresis, Heidelberg, Germany). Subsequently, 300 ng tryptic peptides per sample were analyzed using an Ultimate 3000 RSLCnano HPLC coupled online to either an Orbitrap QExactive, Orbitrap QExactive HF, or Orbitrap Fusion Lumos mass spectrometer (all Thermo Scientific, Bremen, Germany). In total, 306 samples were analyzed and distributed over five batches and separated by either a 96-min (Batch 1) or 38-min (Batches 2–5) LC gradient. The mass spectrometers were operated in data-independent acquisition mode. Spectral libraries were generated with FragPipe (v.17.1) and protein quantification was conducted using DIA-NN (v.1.8) [30 (link)]. The Uniprot/SwissProt database restricted to homo-sapiens (release 01_2022; 20,386 entries) was used for protein identification. The resulting protein intensities were first normalized using the LOESS method [31 (link)]. The subsequent cross-batch normalization was based on linear regression models. A detailed description of the applied methods can be found in the Additional file.
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