Zorbax 300sb c18

Each peptide sample was reconstituted with 0.1% formic acid (FA) and analyzed on a nano-LC–LTQ-Orbitrap Hybrid Mass Spectrometer (Thermo Fisher, CA, USA) as described previously [22 (link)]. Briefly, samples were loaded across a trap column (Zorbax 300SB-C18, 0.3 × 5 mm; Agilent Technologies, Wilmington, DE, USA) at a flow rate of 0.2 μL/min in HPLC buffer (0.1% FA) and separated on a resolving 10 cm analytical C18 column (inner diameter, 75 μm) using a 15 μm tip (New Objective, Woburn, MA, USA). Peptides were eluted using a linear gradient of 0–10% HPLC elution buffer (99.9% ACN containing 0.1% FA) for 3 min, 10–30% buffer B for 35 min, 30–35% buffer B for 4 min, 35–50% buffer B for 1 min, 50–95% buffer B for 1 min, and 95% buffer B for 8 min, with a flow rate of 0.25 μL/min across the analytical column. The resolution of Orbitrap was 30,000, and the ion signal of (Si(CH3)2O)6H+ at 445.120025 (m/z) used as a lock mass for internal calibration. One MS scan alternating with six MS/MS scans for the 10 most abundant precursor ions was applied. The m/z values selected for MS/MS were dynamically excluded for 180 s. For MS scans, the m/z value of the scan range was 400–2000 Da. For MS/MS scans, >1 × 10⁴ ions accumulated in the ion trap to generate spectra. MS and MS/MS spectra were acquired using one scan with maximum fill times of 1000 and 100 ms, respectively.

The LC system (Ettan MDLC, GE Healthcare, USA) was used for desalting and separation of tryptic peptides mixtures. In this system, samples were desalted on reverse phase (RP) trap columns (Zorbax 300 SB C18, Agilent Technologies, USA), and then separated on a RP column (150 μm × 100 mm, Fremont Column Technology Inc., USA). The mobile phase A was 0.1% formic acid in HPLC-grade water and the mobile phase B was 0.1% formic acid in acetonitrile. 20 μg of tryptic peptide mixtures was separated at a flow rate of 2 μL/min with a linear gradient (4–50% solution B, 50 min; 50–100% solution B, 4 min; 100% solution B, 6 min) in the columns. A mass spectrometer (LTQ Velos, Thermo Scientific, USA) was equipped with a micro-spray interface and connected to the LC setup to detecte the eluted peptides. The MS/MS spectra were set so that one full scan mass spectrum (m/z 300–1800) was followed by twenty MS/MS events of the most intense ions using dynamic exclusion (repeat count 2, repeat duration 30 seconds, exclusion duration 90 seconds).

The purity of the peptides was assessed by analysing around 20 μg of the peptide by RP-HPLC (1260 HPLC system, Agilent) using a C18 column (ZORBAX 300SB-C18, 5 μm, 300 Å, 4.6 × 250 mm, Agilent). Peptides were run at a flow rate of 1 mL min⁻¹ with a linear gradient of 0–100% of solvent B over 15 min (A: 94.9% H₂O, 5% ACN, and 0.1% TFA; B: 99.9% ACN and 0.1% TFA). The mass was determined by electrospray ionization mass spectrometry (ESI-MS) in positive ion mode on a single quadrupole liquid chromatography-mass spectrometer (LC-MS-2020, Shimadzu) and the data were analyzed using the Shimadzu LabSolutions software.

Detail information about SDS PAGE and In-gel trypsin digestion of cell fractions proteins can be found in Supplemental information 2. LC-MS/MS analysis of tryptic peptides was carried out using Ultimate-3000 HPLC system (Thermo Scientific) coupled to a maXis qTOF after HDC-cell upgrade (Bruker) with a nano-electrospray source. Chromatographic separation of peptides was performed on a C-18 reversed phase column (Zorbax 300SB-C18, 150 mm x 75 um, particle diameter 3.5 um, Agilent). Gradient parameters were as follows: 5–35% acetonitrile in aqueous 0.1% (v/v) formic acid, duration 120 min, column flow 0.3 ul/min. Positive MS and MS/MS spectra were aquired using AutoMS/MS mode (capillary voltage 1700, curtain gas flow 4 l/min, curtain gas temperature 170 C, spectra rate 10 Hz, 4 precursors, m/z range 50–2200, active exclusion after 2 spectra, release after 0.5 min). Detail information about Search Database Creation and Proteins and Peptides Identification can be found in Supplemental information 2.