Agilent masshunter qualitative analysis b 07

The protein was loaded onto an Agilent Zorbax Eclipse XDB-C18 chip column mounted on the Agilent 1290 Infinity LC system coupled to the Agilent 6520 Accurate-Mass Q-TOF mass spectrometer with a dual ESI source (Agilent Technologies, Santa Clara, CA, USA). The chip column was run at 0.5 mL/min using 0.1% formic acid in water (solution A) and 0.1% formic acid in acetonitrile (solution B). The chip was equilibrated with 5% solution B, and the gradient used during the run was 5–100% solution B for 5 to 20 min and maintained with 100% buffer B for another 5 min. The polarity of the Q-TOF was set at positive, the capillary voltage at 4000 V, the fragmentor voltage at 125 V, the drying gas flow at 10 L/min, and a gas temperature of 300 °C. The intact protein spectrum was analyzed in MS-only mode with a range of 100–3200 m/z. The spectrum was deconvoluted using Agilent MassHunter Qualitative Analysis B.07.00 (Agilent Technologies, Santa Clara, CA, USA).

The in-gel digested sample was analyzed using the Agilent 1200 HPLC-Chip/MS Interface, coupled with Agilent 6550 iFunnel Q-TOF LC/MS. The digested peptides were loaded onto an Agilent C18 300 Å Large Capacity Chip (Agilent Technologies, Santa Clara, CA, USA). The column was equilibrated with 0.1% formic acid in water (solution A). Peptides were eluted with an increasing gradient of 90% acetonitrile (ACN) in 0.1% formic acid (solution B) by the following gradient: 5–75% solution B from 0 to 30 min and 75% solution B from 39 to 47 min. The polarity of the Q-TOF was set at positive, the capillary voltage at 1800 V, the fragmentor voltage at 360 V, the drying gas flow at 11 L/min, and a gas temperature of 280 °C. The spectrum was obtained from Agilent MassHunter Qualitative Analysis B.07.00 (Agilent Technologies, Santa Clara, CA, USA).

Data acquisition was done using Agilent MassHunter LCMS Acquisition software 10.0. For the optimization of the QQQ-MS parameters, MassHunter Optimizer 10.0 and MassHunter Source Optimizer 10.0 were used. The software Agilent MassHunter Qualitative Analysis B.07.00 and Agilent MassHunter Quantitative Analysis 10.1 were employed for data evaluation.

For data, recordings of Agilent MassHunter Workstation LC/MS Data Acquisition for 6400 Series Triple Quadrupole (Version 10.0 SR1) and Agilent MassHunter Optimizer (Version 10.0 SR1) were used. Quantitative analysis was done using Agilent MassHunter Quantitative Analysis for QQQ (Version 10.1).
For qualitative data evaluation, Agilent MassHunter Qualitative Analysis B.07.00 was used.

The peak-picking procedure was initially performed using “Find by Molecular Feature” algorithm in Agilent MassHunter Qualitative Analysis B.07.00 (Agilent Technologies). Centroid spectra peaks higher than 400 counts were subjected against ion restriction: negative ions [M-H]⁻, [M + Cl]⁻, and [M + HCOO]⁻, and positive ions [M + H]⁺ and [M + Na]⁺. The data (.cef) files were then aligned and combined into a reference (.cef) file against which the original raw data was reanalysed in Mass Profiler Professional (MPP) software version 2.2 (Agilent Technologies). For this recursive analysis, we restricted the compounds with a maximum mass of 1000 Da and minimum absolute abundance of 5000 counts in negative modes or 8000 counts in positive ones. The tolerance for compound mass was 15 ppm ± 2 mDa, retention time ± 0.15 min, and symmetric expansion value ± 10 ppm for chromatograms. The output was re-exported to MPP for peak alignment and data cleanup. Subsequent filtering included only features that were present in at least 50% of samples in at least one study group. As the results, we got 2372 and 504 features from HILIC, and 3274 and 1334 features from RP, in positive and negative mode, respectively. The combined data matrix, which consisted of 7484 features from 248 samples with respective raw peak areas henceforth underwent data pre-processing.