Acclaim pepmap c18

After resuspended with 30 µL of buffer A (0.1% formic acid in water), the peptides were separated by nano-LC. And this process is analyzed by on-line electrospray tandem mass spectrometry. All experiments were performed on a Nano ACQUITY UPLC system (Waters Corporation, Milford, Massachusetts), including a Q-Exactive mass spectrometer (Thermo Fisher Scientific) equipped with an online nano-electrospray ion source. A total of 10 µL of peptide sample, with a flow of 10 µL/min for 3 minutes, was loaded onto the trap column (Thermo Scientific Acclaim PepMap C18, 100 µm × 2 cm). And then, the sample, from 3% B (B: 0.1% formic acid in ACN) to 32% B in 120 minutes, was separated on the analytical column (Acclaim PepMap C18, 75 µm × 15 cm) with a linear gradient. The column was reequilibrated for 10 minutes under initial conditions (flow rate = 300 nL/min). The electrospray voltage used was 2 kV, corresponding to the inlet of the mass spectrometer. Within data-dependent acquisition mode, the mass spectrometer was automatically switched under MS and MS/MS mode. The MS/MS resolution was maintained at 17.5K under HCD mode. MS1 mass resolution was maintained at 70K with m/z 300 to 1800 (dynamic exclusion time =10 seconds).

Afterwards, the peptides were diluted with 10 µL solvent A (water with 0.1% formic acid) and further analyzed using on-line nanospray LC-MS/MS on an Orbitrap Fusion lumos connected to an EASY-nLC system (Thermo Scientific, MA, USA). For LC-MS/MS analysis, 5 µL peptide sample was loaded onto the trap column (Thermo Scientific Acclaim PepMap C18, 100 µm x 2 cm) at a flow rate of 5 µL/min and subsequently separated on the analytical column (Acclaim PepMap C18, 75 µm x 15 cm) using a 120-min non-linear gradient, ranging from 3% solvent B (ACN with 0.1% formic acid) to 32% solvent B. The column flow rate was 300 nL/min. The electrospray voltage of 2.1 kV relative to the inlet of the mass spectrometer was used. The mass spectrometer was operated under data-dependent acquisition mode, and automatically switched between MS and MS/MS mode in 3.5 s cycle. MS1 mass resolution was set as 120 K with m/z 350–1550, MS/MS isolation window was set 1.6 Da, and MS/MS resolution was set as 30 K under HCD mode with a collision energy was 32%. The dynamic exclusion was set as n=1, and the dynamic exclusion time was 30 s, AGC target was 5e4, max injection time was 120 ms.

The proteolytic digests were loaded using a nano-HPLC (Dionex RSLCnano) on reverse-phase columns (trapping column: Acclaim PepMap c18, particle size 2 µm, L = 20 mm; analytical column: Acclaim PepMap c18, particle size 2 µm, L = 25 cm; Thermo Fisher Scientific) and eluted in organic phase gradients (Buffer A: 95% H₂O, 5% DMSO, and 0.1% formic acid; Buffer B: 80% acetonitrile, 15% H₂O, 5% DMSO, and 0.1% formic acid). Typically, gradients were ramped from 4 to 48% buffer B in 80 min at flowrates of 300 nl/min. Peptides eluting from the column were ionized online using a Nanospray Flex Ion source and analyzed in an Orbitrap Elite mass spectrometer. Mass spectra were acquired over the m/z range 350–1,600 at a resolution of 120,000, and sequence information was acquired by computer-controlled, data-dependent automated switching to tandem mass spectrometry mode using collision energies based on mass and charge state of the candidate ions (FTIT and TOP15).