Aug 05, 2020
LC-MS/MS analysis
- 1domnic colvin
- UM-DAE Centre for Excellence in Basic Sciences
Document Citation: avinash.kale 2020. LC-MS/MS analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.bjdvki66
License: This is an open access document distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Created: August 05, 2020
Last Modified: August 05, 2020
Document Integer ID: 40085
LC-MS/MS analysis
Nano-LCMS using 6540 UHD Accurate Mass QTOF (CAMS, Venture Center, NCL Pune, India) was used to carry out all our mass spectrometric analysis. The peptides were analyzed using reversed-phase nanoscale liquid chromatography coupled to tandem mass spectrometry. The system consisted of a column; initially, the sample would be subjected to Agilent Binary LC1290 using column Polaris, high-performance chip, 360 nl enrichment chip (150mm X 75µm), and a separation column G4240-62030 with a thermostat temperature of 4°C. The mobile phase was Water (0.1% Formic Acid) and Acetonitrile: Water (90:10 v/v). The chromatography was carried out in a gradient manner (3% to 90% Acetonitrile: Water). The mass spec analysis was carried out on Agilent 6540 UHD QTOF MS (Gas temp: 250, Gas flow (l/minute): 8 minutes,) MS analysis was carried out in a data-dependent manner with survey scan resolution. Mass range: 250-1700(m/z), MS/MS: 50-1700(m/z), Data type: Centroid and software: Masshunter workstation software. Obtained data were searched against the UniProt protein databases of all the respective four Bacillus sp. using default parameters.