Easy spray c18 analytical column

LC-ESI-MS/MS analysis was performed on an Easy nanoLC1200 (Thermo Fisher Scientific) coupled to a Q-Exactive HF-X mass spectrometer (Thermo Fisher Scientific). 2 µg of the pooled, labeled peptides were loaded onto a C18 pre-column connected to an easy spray C18 analytical column (2 mm, 75 mm × 50 cm, Thermo Fisher Scientific) and analyzed using positive mode by data dependent acquisition. The full scan range was set to 300 to 1650 m/z with an AGC of 3e⁶ followed by 15 MS/MS scans. The normalized collision energy was set to 32% with a dynamic exclusion window of 40 s. The mobile phase was 2% ACN with 0.1% FA (A) and 80% ACN with 0.1% FA (B). The gradient profile was 5% B at 0 min, 30% B at 90 min, 100% B from 99 to 116 min, and 2% B at 117 to 120 min. The column oven was set to 55°C.

The peptides obtained from a total of 30 fractions were subjected to LC-MS/MS analysis using Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) coupled to Easy-nLC1200 nano-flow UHPLC (Thermo Scientific, Odense, Denmark). The peptide digests were reconstituted in 0.1% formic acid and loaded onto nanoViper trap column 2 cm (3 μm C18 Aq) (Thermo Fisher Scientific). Peptide separation was carried out using EASY-Spray C18 analytical column (15 cm, 75 μm PepMap C18, 2 μm C18 Aq) (Thermo Fisher Scientific) at a flow rate of 300 nL/min. A linear gradient of 5-35% solvent B (80% acetonitrile in 0.1% formic acid) over 100 min was used to resolve the peptide mixture with a total run time of 120 min including sample loading and column reconditioning. Data dependent acquisition with full scans in 400–1,600 m/z range was carried out using an Orbitrap mass analyzer at a mass resolution of 120,000 at 200 m/z. The most intense precursor ions from a survey scan were selected for MS/MS and fragmented using HCD mode with 34% normalized collision energy and detected at a mass resolution of 30,000 at 200 m/z. Peptide charge state was set to 2–6, and dynamic exclusion was set to 30 s. Internal calibration was carried out using lock mass option (m/z 445.1200025) from ambient air.

Peptide samples were separated
on a Thermo Scientific Ultimate 3000 UHPLC (Thermo Fisher Scientific)
using 10 min loading at a 3 μL/min flow rate to a trap column
(Acclaim PepMap 100, 2 cm × 75 μm, 3 μm, 100 Å,
Thermo Fisher Scientific). The separation was performed on an EASY-Spray
C18 analytical column (25 cm × 75 μm, 1.9 μm, 300
Å, ES802A, Thermo Fisher Scientific). A constant flow rate of
100 nL/min was applied during sample separation achieved in a linear
gradient ramped from 5% B to 27% B over 120 min, with solvents A and
B being 2% ACN in 0.1% FA and 98% ACN in 0.1% FA, respectively. LC-MS/MS
data were acquired on an Orbitrap Fusion Lumos Tribrid mass spectrometer
(Thermo Fisher Scientific, San José CA), using nano-electrospray
ionization in positive ion mode at a spray voltage of 1.9 kV. Data-dependent
acquisition (DDA) mode parameters were set as follows: isolation of
top 20 precursors in full mass spectra at 120 000 mass resolution
in the m/z range of 375–1500, maximum allowed
injection time of 100 ms, dynamic exclusion of 10 ppm for 45 s, MS2
isolation width of 0.7 Th with higher-energy collision dissociation
(HCD) of 35% at a resolution of 50 000, and maximum injection
time of 150 ms in a single microscan. The mass spectrometry proteomics
data were deposited to the ProteomeXchange Consortium via the PRIDE
partner repository^{29 (link)} with the dataset identifier
PXD025481.