Easy nlc 1000 uplc

All samples were analyzed on a Q-Exactive Plus (Thermo Scientific) mass spectrometer that was coupled to an EASY nLC 1000 UPLC (Thermo Scientific). Peptides were loaded with solvent A (0.1% formic acid in water) onto an in-house packed analytical column (50 cm × 75 µm I.D., filled with 2.7 µm Poroshell EC120 C18, Agilent). Peptides were chromatographically separated at a constant flow rate of 250 nL/min using the following gradient: 5–30% solvent B (0. 1% formic acid in 80% acetonitrile) within 119 min, 30–50% solvent B within 19 min, followed by washing and column equilibration. The mass spectrometer was operated in data-dependent acquisition mode. The MS1 survey scan was acquired from 300–1750 m/z at a resolution of 70,000. The top ten most abundant peptides were isolated within a 2 Da window and subjected to HCD fragmentation at a normalized collision energy of 27%. The AGC target was set to 5 × 10E5 charges, allowing a maximum injection time of 55 ms. Product ions were detected in the Orbitrap at a resolution of 17,500. Precursors were dynamically excluded for 20 s.

The interested bands were excised from the gel, followed by in-gel tryptic digestion. The resulting peptides were loaded onto a homemade reversed-phase analytical column on an EASY-nLC 1000 UPLC system followed by tandem mass spectrometry (MS/MS) in Q Exactive^TM Plus (Thermo). The electrospray voltage 2.0 kV was applied. The m/z scan range was between 350 and 1800 for a full scan, and intact peptides were detected in the Orbitrap at a resolution of 70,000. All MS/MS data were processed using Proteome Discoverer 1.3. Trypsin was specified as a cleavage enzyme allowing up to 2 missing cleavages. A mass error was set to 10 ppm for precursor ions and 0.02 Da for fragment ions. Carbamidomethyl on Cys was specified as fixed modification and oxidation on Met was specified as variable modification. Peptide confidence was set at high, and peptide ion score was set >20.

Peptides were eluted from SDB-RPS tips with 30 μl of 5 % (w/v) ammonium hydroxide in 80% acetonitrile (ACN) and dried a speed vac.
All samples were analyzed on a Q-Exactive Plus (Thermo Scientific) mass spectrometer that was coupled to an EASY nLC 1000 UPLC (Thermo Scientific). After resuspension in 10 μl 5% formic acid, 2% ACN the peptides were loaded with 8 μl solvent A (0.1% formic acid in water) onto an in-house packed analytical column (50 cm × 75 μm I.D., filled with 2.7 μm Poroshell EC120 C18, Agilent). Peptides were separated at a constant flow rate of 250 nL/min using the following gradient: 3 - 5% solvent B (0.1% formic acid in 80 % ACN) within 1 min, 5-30% solvent B within 40 min, 30-50% solvent B within 8 min, followed by washing with 95 % solvent B for 10 min. The mass spectrometer was operated in data-dependent acquisition mode.
The MS1 survey scan was acquired from 300-1750 m/z at a resolution of 70.000. The top 10 most abundant peptides were isolated within a 1.8 Th window and subjected to HCD fragmentation at normalized collision energy of 27%. The AGC target was set to 5e5 charges, allowing a maximum injection time of 110 ms. Product ions were detected in the Orbitrap at a resolution of 35.000. Precursors were dynamically excluded for 20 s.

Trypsin-based method was used to digest protein. Before digestion, protein solution was first reduced for 1 h at 37 °C with 10 mM DTT. Proteins then were alkylated at room temperature in darkness with 20 mM iodoacetamide (IAA) for 45 min. Afterward, a two-step trypsin digestion was performed as previously described [16 (link)].
To enrich succinylated peptides, tryptic peptides dissolved in NETN buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, 0.5% NP-40, pH 8.0) were incubated overnight with prewashed antibody beads (PTM Biolabs) at 4 °C. After washing four times with NETN buffer and twice with ddH₂O, the bound peptides were eluted from the beads with 0.1% TFA.
After cleaning with C18 ZipTips (Millipore), the enriched peptides were analyzed following the procedure as described, with minor modification [16 (link)]. In brief, the peptides dissolved in 0.1% formic acid (FA) were firstly separated using a reversed-phase analytical column (Acclaim PepMap RSLC, Thermo Scientific) at a constant flow rate of 700 nl/min on EASY-nLC 1000 UPLC system. Then, the resulting peptides were subjected to MS/MS by an Orbitrap Fusion mass spectrometer (ThermoFisher Scientific). Detection of intact peptides and ion fragments in the Orbitrap were carried out at a resolution of 60,000 and 15,000, with the NCE setting as 35. For MS scans, the m/z scan range was 350 to 1550.

Digested peptides were analyzed on a QExactive Plus mass spectrometer (Thermo Scientific) coupled to an EASY nLC 1000 UPLC (Thermo Scientific). Dried peptides were resolubilized in solvent A (0.1% formic acid), and loaded onto an in-house packed C18 column [50 cm × 75 μm I.D., filled with 2.7 μm Poroshell 120 (Agilent)]. Following loading, samples were eluted from the C18 column with solvent B (0.1% formic acid in 80% acetonitrile) using a 2.5 h gradient, comprising: linear increase from 4 to 27% B over 120 min, 27–50% B over 19 min, followed by column washing and equilibration. Flow rate was at 250 nL/min. Data-dependent acquisition was used to acquire MS/MS data, whereby the 10 most abundant ions (charges 2–5) in the survey spectrum were subjected to HCD fragmentation. MS scans were acquired from 300 to 1,750 m/z at a resolution of 70,000, while MS/MS scans were acquired at a resolution of 17,500. Following fragmentation, precursor ions were dynamically excluded for 25 s.