Irt standard peptide

L-3000 HPLC system combined with Q Exactive HFX mass spectrometer (Thermo Fisher, Waltham, USA) and NanosprayFlex™ electrospray ionization (ESI) ion source was used for library generation in DDA mode. 24 dried fractions were resuspended respectively by 0.1% formic acid (FA) with and iRT standard peptide (Biognosys, Schlieren, Switzerland). In DDA mode, the full scan range was M/Z 350–1500, with resolution at 120,000, autogain control (AGC) at 3e6 and maximum injection time of 80 ms. MS/MS spectra were acquired at a resolution of 15000, with an AGC at 5e4, maximum injection time of 45 ms and a normalized collision energy of 27.
Samples of each group were resuspended respectively by 0.1% FA with iRT standard peptide (Biognosys, Schlieren, Switzerland). In DIA mode, the full scan range was m/z 350–1250, with resolution at 120,000, AGC at 3e6 and maximum injection time of 60 ms. MS/MS spectra were acquired at a resolution of 12000, with AGC at 3e6, maximum injection time of 60 ms and a normalized collision energy of 27, with 60 variable windows ranging from 349.5 to 1250.5 m/z.

The main reagents required for this experiment are as follows: iRT standard peptide was purchased from Biognosys; SDS lysis solution was purchased from Beyotime Biotechnology; bicinchoninic acid (BCA) kit, mass spectrometry grade water, and acetonitrile were purchased from ThermoScientific; performed gum was purchased from GenScript Inc. PMSF was purchased from Amresco; disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate monohydrate, NaCl, Tris-HCl (pH 6.8; pH 8.8), Tris, Dithiothreitol (DTT), glycerol, bromophenol blue, trifluoroacetic acid (TFA), indole-3-acetic acid (IAA), urea, and triethylammonium borate; triethylamine-boric acid (TEAB) were purchased from Biotech; glycine and SDS were purchased from Sinopharm; trypsin was purchased from Wallys; anhydrous ethanol and isopropanol were purchased from GENERAL-REAGENT; and de-high abundance kit was purchased from MilliPore.

UbiSite samples for data-independent acquisition (DIA) MS runs were prepared in the same way as for the large-scale DDA experiments, excluding the HpH fractionation. After digestion with trypsin, the resulting peptide mixtures from each sample were purified with Stage-Tips, supplemented with iRT standard peptides (Biognosys AG, Switzerland) and subjected to nanoLC-MS/MS analysis using an EASY-nLC 1000 ultra-high-pressure system (Thermo Fisher Scientific) coupled online with the Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific), using Xcalibur (version 4.3) and Tune (version 3.0). Peptide loading and nanoLC settings were the same as described above. Acquisition time of the samples was 120 min. We used DIA methodology with the following settings: MS1 scan with mass range 350–1400 m/z had a normalised AGC target set to 300% with resolution 120,000 and injection time 45 ms. For DIA scans acquisition AGC target value was set at 1000%, R = 30,000 and IT to 54 ms. 50 windows of 13 Da with an overlap of 1 Da were used. Normalised collision energy was set at 28%. Data were acquired in profile mode using positive polarity.