C18 cl 120

Mass spectrum analysis was conducted via a Triple Time of Flight (TOF) 5600 mass spectrometer (AB SCIEX, USA). A NanoLC pre-column (Chromxp C18-LC-3 μm, Eksigent) and an analytical column (C18-CL-120, Eksigent) were separately used to trap and elute peptides via gradient wash from 5 to 35% Buffer B (Buffer A: 2% ACN, 98% H₂O, Buffer B: 98% ACN, 2% H₂O, 0.1% formic acid) at a flow rate of 300 nL/min. Full-scan MS was performed in the positive ion mode with a nano-ion spray voltage of 2.5 kV from 350 to 1500 (m/z), with up to 30 precursors selected for MS/MS if the precursors exceeded a threshold of 125 counts per second. Charged peptides ranging from + 2 to + 5 were screened for the MS/MS analysis. The collision energy (CE) for the collision-induced dissociation (CID) was automatically controlled using an Information-Dependent Acquisition (IDA) CE parameter script to achieve optimum fragmentation efficiency.
To identify the proteins within samples, theoretical peptide spectra of proteins deposited in a protein database were matched to the acquired tandem mass spectra using the search engine MASCOT. Then, the approach was termed spectral counting, implying counting and comparison of the number of fragment-ion spectra (MS/MS) acquired for peptides of a special protein.

A NanoLC system (NanoLC-2D Ultra, Eksigent, Dublin, CA) equipped with a Triple TOF 5600 mass spectrometer (AB SCIEX, USA) was used for analysis. Peptides were trapped on a NanoLC pre-column (Chromxp C18-LC-3 μm, size 0.35 × 0.5 mm, Eksigent, Dublin, CA) and then eluted onto an analytical column (C18-CL-120, size 0.075 × 150 mm, Eksigent, Dublin, CA). The NanoLC gradient was 5–35% Buffer B (98% ACN, 2% H2O, 0.1% FA) over 120 min at a flow rate of 300 nL/min. Full-scan MS was performed in positive ion mode with a nano-ion spray voltage of 2.2 kV. Survey scans were acquired from 350 to 1500 (m/z) with up to 40 precursors selected for MS/MS (m/z 100–1500). The collision energy (CE) for collision-induced dissociation was automatically controlled using an Information-Dependent Acquisition CE parameter script to achieve optimum fragmentation efficiency. The mass spectrometer was calibrated using beta galactosidase tryptic peptides.