Tof 5600 mass spectrometer

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.

The sample was separated by an Agilent 1290 Infinity LC ultra-high performance liquid chromatography system hilic column, and the separation conditions are shown in Table 1.
Mass spectrometry was detected in two modes of ESI+ and ESI-. The samples were separated by UHPLC and analyzed by TOF 5600 mass spectrometer(AB SCIEX). The secondary mass spectrometry was obtained by IDA, which needs to exclude isotopes within 4Da. ESI source conditions, MS/MS conditions and IDA conditions are shown in Table 2. The QC samples were mixed in equal amounts from the samples to be tested, and were tested before, during and after the LC-MS/MS injection. 2 uL of QC was injected, and the QC spacing in the samples was 8 spacings.