Ultimate 3000 rslcnano hplc

Samples for global analysis were fractionated after digestion and subjected to LC-MS/MS analysis using an Ultimate 3000 RSLCnano HPLC coupled to a Q Exactive HF mass spectrometer, while samples dedicated to label-free quantification and PRM analysis were measured unfractionated on an Ultimate 3000 RSLCnano HPLC coupled to an Orbitrap Fusion Lumos mass spectrometer (all from Thermo Scientific). Detailed settings are described in Text S1 in the supplemental material.

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.

Concatenated fractions were vacuum concentrated once more until approximately 5 μL and then reconstituted to a total volume of 30 μL with 0.1% (v/v) formic acid (FA), sonicated in a water bath for 15 min and centrifuged at 16000 x g for 10 min to remove particulates. Using an Ultimate 3000 RSLCnano HPLC (Thermo Scientific), each sample was loaded via a trap column (100 μm x 2 cm nanoViper PepMap 100; Thermo Scientific) in 2% (v/v) ACN, 0.1% (v/v) FA at a flow rate of 15 μL/min onto an analytical nanocolumn (75 μm x 50 cm PepMap 100 C18 3 μm 100Å; Thermo Scientific) at a flow rate of 300 μL/min. Peptides were separated using increasing concentrations of 80% (v/v) ACN, 0.1% (v/v) FA (buffer B) (2.5% B to 42.5% B over 20 min) and analysed with a Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Scientific). Up to 12 MS/MS spectra were acquired per cycle with maximum accumulation time of 50 ms and 100 ms for MS1 and MS2, respectively. To prevent multiple sequencing of the same peptide (dynamic exclusion), MS1 masses were excluded for 10 secs.