Easy nlc 1000 nano lc system

The peptides were dissolved in 0.1% (vol/vol) formic acid (FA) and analyzed with nanoscale LC-MS/MS using an Easy-nLC 1000 nano-LC system (Thermo Scientific) coupled with a quadrupole Orbitrap mass spectrometer (Q Exactive; ThermoElectron, Bremen, Germany) as previously reported (80 (link)). The obtained MS raw data were processed via MaxQuant software (version v.1.6.1.0) with the built-in search engine Andromeda (81 (link), 82 (link)), using a protein database comprising all 5,564 Mmr protein sequences (UniProt proteome up000257451, genome accession PEDF01000000), both forward and reverse. Carbamidomethyl (C) was set as fixed, and methionine oxidation was set as a variable modification. Tolerance was set to 20 ppm in the first search and 4.5 ppm in the main search. Trypsin without the proline restriction enzyme option and with two allowed miscleavages was used. The minimal unique plus+ razor peptide number was set to 1, the false-discovery rate (FDR) was set to 0.01 (1%) for peptide and protein identification, and LFQ with default settings was used. The mass spectrometry proteomics data were deposited in the ProteomeXchange Consortium via the PRIDE (83 (link)) partner repository with the data set identifier PXD02010.

Each sample pool was loaded on a ReproSil-Pur C18 precolumn (3 cm × 100 μm, 5 μm, 120 Å; Dr Maisch, Germany) at 5 μl/min using an Easy nLC-1000 nano-LC system (Thermo Scientific, San Jose, CA, USA). For separation, mobile phase A was 0.1% FA in 2% ACN, and mobile phase B was 0.1% FA in 98% ACN. A step gradient of 2–8% B, 0–5 min; 8–22% B, 5–85 min; 22–30% B, 85–105 min; 30–90% B, 105–110 min; and 90% B, 110–120 min was used at 300 nl/min. Data-dependent MS/MS was performed using an Orbitrap Fusion mass spectrometer in positive ion mode with the following parameters: 2.2 kV spray voltage, 275°C capillary temperature, 55% S-lens level, 350–1,550 mass acquisition range, and 120,000 resolution for MS analysis. Each precursor ion scan was followed by a 4-s top speed data-dependent HCD MS/MS at 35% normalized collision energy. The resolution for MS/MS analysis was 30,000. The quadrupole isolation width was 2 m/z. The dynamic exclusion time was 60 s with a ±10 ppm exclusion mass width. The raw data were processed using Proteome Discoverer version 1.4.0.28 and the UniProt database. The PhosphoRS 3.0 algorithm was used to evaluate the localization probabilities of phosphorylation sites.

The resulting peptide mixture was analyzed using an Orbitrap Fusion Lumos Tribrid mass spectrometer coupled with an EASY-nLC 1,000 nano-LC system (ThermoFisher Scientific, Waltham, MA, USA). Peptide separation was performed on a 15-cm length reverse phase C₁₈ column (150 nm id, 1.9 μm, 100 Å) using A and B buffers (buffer A:0.1% formic acid in water; buffer B:0.1% formic acid in acetonitrile) at a constant flow rate of 600 nl min⁻¹. The gradient was set as follows: 7–15% B for 7 min, 15–25% B for 37 min, 25–40% B for 20 min, and 40–100% B for 7 min. The dynamic exclusion duration of data-dependent MS2 acquisition (DDA) is 18 s. For MS1 scan, mass spectra were acquired in the positive-ion mode over the range of 300–1,400 m/z with a resolution of 120,000 and a maximum ion injection time of 50 ms. MS2 spectra were acquired with an automatic gain control target value of 5.e3 and a maximum injection time of 35 ms with higher-energy collision dissociation (HCD) with a normalized collision energy of 30%.