C18 pre column

Peptides were analyzed by nanoLC-MS/MS using an UltiMate 3000 RSLCnano system coupled to a Q-Exactive-Plus mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Five µL of each sample were loaded on a C-18 precolumn (300 µm ID × 5 mm, Dionex) in a solvent made of 5% acetonitrile and 0.05% TFA and at a flow rate of 20 µL/min. After 5 min of desalting, the precolumn was switched online with the analytical C-18 column (75 µm ID × 15 cm, Reprosil C18) equilibrated in 95% solvent A (5% acetonitrile, 0.2% formic acid) and 5% solvent B (80% acetonitrile, 0.2% formic acid). Peptides were eluted using a 5 to 50% gradient of solvent B over 105 min at a flow rate of 300 nL/min. The Q-Exactive-Plus was operated in a data-dependent acquisition mode with the XCalibur software. Survey scan MS were acquired in the Orbitrap on the 350–1500 m/z range with the resolution set to a value of 70,000. The 10 most intense ions per survey scan were selected for HCD fragmentation. Dynamic exclusion was employed within 30 s to prevent repetitive selection of the same peptide. At least 3 injections were performed for each sample.

Peptides were analyzed by nanoLC-MS/MS using an UltiMate 3000 system (Dionex) coupled to an LTQ Orbitrap Velos ETD mass spectrometer (Thermo Fisher Scientific)^{38 (link)}. Each sample (5 µl) was loaded onto a C18 precolumn (300 μm inner diameter × 5 mm; 5 µm particule size; 100 Å pore size; Dionex) at 20 μl/min in 5% acetonitrile, 0.05% trifluoroacetic acid. After 5 min of desalting, the precolumn was switched online with the analytical C18 column (75 μm inner diameter × 50 cm; 3 µm particule size; 120 Å pore size in-house; packed with Reprosil C18) and was equilibrated in 95% solvent A (5% acetonitrile, 0.2% formic acid) and 5% solvent B (80% acetonitrile, 0.2% formic acid). Peptides were eluted using a 5–50% gradient of solvent B over 110 min at a flow rate of 300 nl/min. The mass spectrometer was operated in a data-dependent acquisition mode with Xcalibur software. Survey MS scans were acquired in the Orbitrap on the 300–2000 m/z range with the resolution set at 60,000. The 20 most intense ions per survey scan were selected for CID fragmentation and the resulting fragments were analyzed in the linear ion trap (LTQ). A dynamic exclusion of 60 s was used to prevent repetitive selection of the same peptide. Each sample was injected once for MS analysis.

Peptide mixtures were analyzed by nanoLC-MS/MS using a nanoRS UHPLC system (Dionex, Amsterdam, Netherlands) coupled to an LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Each biochemical replicate (three per conditions) samples was analyzed twice. Five microliters of each sample were loaded on a C18 pre-column (5 mm × 300 μm; Dionex) at 20 μL/min in 2% acetonitrile and 0.05% trifluoroacetic acid. After 5 min of desalting, the pre-column was switched online with the analytical C18 column (50 cm × 75 μm inner diameter; in-house packed with Reprosil C18) equilibrated in 95% of solvent A (5% acetonitrile + 0.2% formic acid in water) and 5% of solvent B (80% acetonitrile + 0.2% formic acid in water). Peptides were then eluted using a 5–50% gradient of solvent B for 105 min at a 300 nL/min flow rate. The LTQ-Orbitrap was operated in data-dependent acquisition mode with Xcalibur software. Survey scan MS spectra were acquired in the Orbitrap on the 300–2000 m/z range with the resolution set to a value of 60,000. The 20 most intense ion survey scans were selected for CID (collision-induced dissociation) fragmentation and the resulting fragments were analyzed in the linear trap (LTQ). Dynamic exclusion was used within 60 s to prevent repetitive selection of the same peptide.

LC-MS/MS analysis for proteomics was performed using an EASY-nLC 1000 system (Thermo Fisher Scientific) coupled to a QExactive Plus mass spectrometer (Thermo Fischer Scientific) equipped with a nano-ESI source. Solvent A was 0.1% formic acid (FA) in H₂O and solvent B was 0.1% FA in acetonitrile (ACN). Peptides were reconstituted in solvent A to a nominal concentration of 0.2 μg/μL, and 5 μL (1 μg) from each sample was injected onto a C₁₈ pre-column (75 µm i.d., 2 cm length, 3 µm particle size, Thermo Fischer Scientific). Peptide separation was carried out using a linear 150 min gradient from 4 to 100% B with a flow rate of 250 nL/min through the pre-column and the following analytical C₁₈ column (75 µm i.d., 15 cm length, 2 µm particle size, Thermo Fischer Scientific). The mass spectrometer was operated in positive mode with an electrospray voltage of 2.2 kV. The survey MS spectra (400–1750 m/z) were generated at a resolution of 70,000 (FWHM) using an automatic gain control (AGC) target of 3 × 10⁶. Data were recorded in data-dependent mode, where the top ten most abundant peaks were selected for fragmentation using higher energy collision-induced fragmentation with nitrogen. A normalized collision energy of 25% was applied. The AGC target for MS/MS was set to 5 × 10⁵ at a resolution of 17,500. Dynamic exclusion was used with an exclusion period of 20 s.

Trypsin digests were analyzed by nanoLC-MS² using a nanoRS UHPLC system (Dionex) coupled to an ETD-enabled LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific), with fluoranthene as reagent anion. 5 μL of sample was loaded on a C18-precolumn (300 μm ID × 5 mm, Thermo Fisher Scientific) at 20 μL/min in 2% acetonitrile, 0.05% TFA. After 5 min desalting, the precolumn was switched online with the analytical C18 nanocolumn (75 μm ID × 15 cm, in-house packed with C18 Reprosil) equilibrated in 95% solvent A (0.2% formic acid) and 5% solvent B (80% acetonitrile, 0.2% formic acid). Peptides were eluted using the following gradient of solvent B at 300 nL/min flow rate: 5 to 25% gradient during 75 min; 25 to 50% during 30 min; 50 to 100% during 10 min. The LTQ-Orbitrap Velos was operated in data-dependent acquisition mode with the XCalibur software. Survey scan MS were acquired in the Orbitrap on the 300–2000 m/z range with the resolution set to a value of 60,000. The 20 most intense ions per survey scan were selected for ETD fragmentation and the resulting fragments were analyzed in the linear trap (LTQ). Activation time was dependent on the precursor charge state, and supplemental activation was enabled.
Both top-down and bottom-up data have been deposited to the MassIVE repository with the dataset identifier MSV000080396 (ftp://MSV000080396@massive.ucsd.edu).

The deglycosylated peptides were resuspended in 0.1% FA, then analyzed with LC−MS/MS. LC-MS/MS was performed using a Q-Exactive mass spectrometer coupled with an Easy nLC (Thermo Fisher Scientific, MA). Testing samples were loaded onto a 100 μm × 20 mm C18 precolumn (Thermo Fisher Scientific, CA) and chromatographic separation was performed on a 75 μm × 10 cm C18 nanocolumn. A high-performance liquid chromatography gradient was achieved using 0−55% buffer B (0.1% FA and 95% acetonitrile) and buffer A (0.1% FA) at a flow rate of 300 nL/min for more than 90 min. A Q-Exactive mass spectrometer was used for the analysis. The MS data was acquired with a precursor ion range of 300−1800 in positive ion mode. Resolution was set to 70 000 at m/z 200, the dynamic exclusion 25 s, maximum ion injection time 20 ms and automatic gain control target 3 × 10⁶. Higher-energy collisional dissociation at a normalized collision energy of 27 eV was used to acquire the 10 most intense ions for MS2 scans with a resolution of 17500 at m/z 200 and 60 ms maximum IT.