Easylc 1200

Tryptic peptides were desalted (Rappsilber et al., 2007 (link)) and separated by reverse phase nano-LC-MS/MS (column: 12 cm/75um C18 built-in-emitter column, Nikkyo Technos Co., Ltd. Japan, EasyLC 1200, Thermo Scientific) using a 70-min analytical gradient, increasing from 2% B/98% A to 38%B/62% A (A: 0.1% formic acid, B: 80% Acetonitrile/0.1% formic acid) at 300 nL/min. The mass spectrometer (Fusion Lumos, Thermo Scientific) was operated in high/high mode (120,000 and 30,000 for MS1 and MS2, respectively). Auto Gain Control was set at 50,000 for MS2. MS1 scan range was set to m/z 375–1500 and m/z 110 was set as lowest recorded mass in MS2. One-point lock mass calibration was used. All data were quantified and searched against a Uniprot mouse database using MaxQuant (v. v. 1.6.0.13) (Cox et al., 2014 (link)). Oxidation of methionine and protein N-terminal acetylation were allowed as variable modifications, cysteine carbamidomethyl was set as a fixed modification, and two missed cleavages were allowed. The ‘match between runs’ option was enabled, and false discovery rates for proteins and peptides were set to 1%. Protein abundances measured using label free quantitation (Tyanova et al., 2016 (link)).

LC–MS/MS instrumentation consisted of an Easy‐LC 1200 (Thermo Fisher Scientific) coupled via a nano‐electrospray ionization source to a QExactive HF‐x mass spectrometer (Thermo Fisher Scientific). For peptide separation, an in‐house packed column (inner diameter: 75 μm, length: 40 cm) was used. A binary buffer system (A: 0.1% formic acid and B: 0.1% formic acid in 80% acetonitrile) was applied as follows: Linear increase in buffer B from 4% to 32% within 33 min, followed by a linear increase to 55% within 5 min. The buffer B content was further ramped to 95% within 2 min. 95% buffer B was kept for further 5 min to wash the column. Prior to each sample, the column was washed using 6 μl buffer A and the sample was loaded using 7 μl buffer A.
The mass spectrometer operated in a data‐dependent mode and acquired MS1 spectra at a resolution of 60,000 (at 200 m/z) using a maximum injection time of 20 ms and an AGC target of 3e6. The scan range was defined from 350–1,650 m/z, and the data type was set to profile. MS2 spectra were acquired at a 15,000 resolution (at 200 m/z) using an isolation window of 1.4 m/z and a normalized collision energy of 32. The Top22 peaks were targeted for MS2 spectra acquisition. The first mass was set to 110 m/z. Dynamic exclusion was enabled and set to 20 s.

Samples were analyzed on Orbitrap Velos Pro, Q-Exactive, or Orbitrap Fusion mass spectrometers (all Thermo Scientific) using default settings. Mass spectrometers were coupled to UPLC systems (Waters nanoAcquity UPLC or Thermo EASY LC 1200). Peptides were loaded onto trap columns (Waters nanoAcquity Symmetry C₁₈, 5 μm, 180 μm × 20 mm [for Waters nanoAcquity UPLC] or Acclaim PepMap100 C18 Nano-Trap 2cm × 100 μm × 5 μm [for EASY nLC 1200]) with Buffer A (0.1% formic acid in water) and separated over 25 or 50 cm analytical columns (Acclaim PepMap RSLC, 75 μm × 2 μm) using 90, 145, or 240 min linear gradients from 3%–40% Buffer B (0.1% formic acid in Acetonitrile). Peptides were introduced into the mass spectrometer using a Pico-Tip Emitter (360 μm outer diameter × 20 μm inner diameter, 10 μm tip, New Objective). MS2 Fragmentation was set to HCD (Q-Exactive and Fusion) or CID (Orbitrap Velos Pro), and MSMS scans were acquired in the ion trap (Orbitrap Velos Pro and Fusion) or Orbitrap (Q-Exactive).

LC–MS/MS instrumentation consisted of an Easy‐LC 1200 (Thermo Fisher Scientific) coupled via a nano‐electrospray ionisation source to an Exploris 480 mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). An in‐house packed column (inner diameter: 75 μm, length: 40 cm) was used for peptide separation. A binary buffer system (A: 0.1% formic acid and B: 0.1% formic acid in 80% acetonitrile) was applied as follows: