Reprosil pur c18 resin

The Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific, USA) was used in combination with an Easy-nLC 1000 ultrahigh-pressure liquid chromatography pump (Thermo Fisher Scientific, USA) for the LC-MS/MS analysis. Separation was achieved using a trap column and an analytic column with a spray tip, both filled with 3 µm/120 Å ReproSil-Pur C18 resins (Dr. Maisch GmbH, DE). The separation buffers were comprised of 0.1% formic acid in both water and acetonitrile. A fraction of the collected samples was initially introduced into the trap column with a 2 µL/min flow rate, and then, it was separated via the analytical column at a flow rate of 300 nL/min. The separation gradient was established as starting with 3%–7% acetonitrile over 2 min, increasing to 22% acetonitrile over the next 50 min, then to 35% acetonitrile in 10 min, surging to 90% acetonitrile within 2 min, maintaining at 90% for 6 min, dropping back to 3% acetonitrile in 2 min, and finally stabilizing at 3% acetonitrile for a duration of 13 min. Full MS scans were performed in an Orbitrap mass analyzer over m/z range of 395–1 205 with a mass resolution of 60 000. Data was processed and analyzed for DIA-Based proteomics using Spectronaut version 14.9 (Biognosys, CH).^{81 (link)}

An Eksigent NanoLC system (nano2D ultra) coupled with a Q Exactive mass spectrometer (Thermo Fisher Scientific Inc.) was used for analysis. Tryptic peptides were reconstituted in 0.1% (vol/vol) formic acid to approximately 0.25 μg/μL, and 1 μg of peptides was loaded. The column used for peptide separation was of 75-μm inner diameter and was 15 cm long, packed with reverse-phase C₁₈ resin (1.9-μm/120-Å ReproSil-Pur C₁₈ resin; Dr. Maisch GmbH). A 90-min gradient with acetonitrile changing from 5% to 30% (vol/vol) was used, at a flow rate of 300 nL/min. Solvent A was composed of 0.1% (vol/vol) formic acid, and solvent B was composed of 0.1% (vol/vol) formic acid and 80% (vol/vol) acetonitrile. MS analysis was performed with a Q Exactive mass spectrometer (Thermo Fisher Scientific Inc.). Full MS scans were performed from m/z 300 to m/z 1,800, and data-dependent MS/MS scans were performed for the 12 most intense ions. MS and MS/MS scans were performed with resolutions of 70,000 and 17,500, respectively. Samples were loaded in a randomized order. In this study, 197 samples were analyzed over a period of 23 days. All raw data from LC-MS/MS have been deposited with the ProteomeXchange Consortium (http://www.proteomexchange.org) via the PRIDE (41 (link)) partner repository via the data set identifier PXD030458.

LC-MS/MS was performed on a Q Exactive Plus equipped with an ultra-high pressure liquid chromatography unit Easy-nLC1000 and a Nanospray Flex Ion-Source (all three from Thermo Fisher Scientific, Waltham, MA). Peptides were separated on an in-house packed column (100 µm inner diameter, 30 cm length, 2.4 µm Reprosil-Pur C18 resin [Dr. Maisch GmbH, Germany]) using a gradient from mobile phase A (4% acetonitrile, 0.1% formic acid) to 30% mobile phase B (80% acetonitrile, 0.1% formic acid) for 60 min followed by a second step to 60% B for 30 min, with a flow rate of 250 nl/min. MS data were recorded in data-dependent mode selecting the 10 most abundant precursor ions for HCD fragmentation. The full MS scan range was set from 200 to 2000 m/z with a resolution of 70,000. Ions with a charge ≥2 were selected for MS/MS scan with a resolution of 17,500 and an isolation window of 2 m/z. Dynamic exclusion of selected ions was set to 30 s. Data were acquired using the Xcalibur software (Thermo Fisher Scientific).

An Eksigent NanoLC system (nano2D ultra) coupled with a Q Exactive mass spectrometer (Thermo Fisher Scientific Inc.) was used for analysis. Tryptic peptides were reconstituted in 0.1% (vol/vol) formic acid to approximately 0.25 μg/μL, and 1 μg of peptides was loaded. The column used for peptide separation was of 75-μm inner diameter and was 15 cm long, packed with reverse-phase C₁₈ resin (1.9-μm/120-Å ReproSil-Pur C₁₈ resin; Dr. Maisch GmbH). A 90-min gradient with acetonitrile changing from 5% to 30% (vol/vol) was used, at a flow rate of 300 nL/min. Solvent A was composed of 0.1% (vol/vol) formic acid, and solvent B was composed of 0.1% (vol/vol) formic acid and 80% (vol/vol) acetonitrile. MS analysis was performed with a Q Exactive mass spectrometer (Thermo Fisher Scientific Inc.). Full MS scans were performed from m/z 300 to m/z 1,800, and data-dependent MS/MS scans were performed for the 12 most intense ions. MS and MS/MS scans were performed with resolutions of 70,000 and 17,500, respectively. Samples were loaded in a randomized order. In this study, 197 samples were analyzed over a period of 23 days. All raw data from LC-MS/MS have been deposited with the ProteomeXchange Consortium (http://www.proteomexchange.org) via the PRIDE (41 (link)) partner repository via the data set identifier PXD030458.

The LC-mass spectrometry (MS) analysis was performed on a Q Exactive instrument (Thermo Fisher Scientific Inc., USA) with the Easy-nLC 1200 system (Thermo Fisher Scientific Inc., USA). All 48 samples were run in a randomized order. We loaded 1 μg of peptides on a high-performance liquid chromatography (HPLC) column packed in an electrospray ionization (ESI) spraying tip (75-μm inner diameter by 10 cm) packed with reverse-phase beads (3-μm/120-Å ReproSil-Pur C₁₈ resin; Dr. Maisch GmbH, Ammerbuch, Germany) for separation. Formic acid (FA) (0.1%, vol/vol) in water was used as buffer A, and 0.1% FA in 80% acetonitrile was used as buffer B. The MS1-only method has full scans with a resolution of 7,000, a scan range from 200 to 1,400 m/z, an automatic gain control (AGC) target of 3E6, and a maximum injection time (IT) of 200ms. No dependent scans followed. A 15-min LC gradient was used: solvent B was linearly changed from 4% to 35% in 9 min, followed by another gradient from 35% to 80% in 2 min, and finally, the gradient was kept constant at 80% for 4 min. Example chromatograms are visualized in Fig. S1 in the supplemental material. Raw files were then used to create data set 1.