Jul 10, 2024
LC-MS/MS Analysis
- 1Duke University
Protocol Citation: Shiyi Wang 2024. LC-MS/MS Analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g71388gwz/v1
License: This is an open access protocol 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
Protocol status: Working
We use this protocol and it's working
Created: July 10, 2024
Last Modified: July 10, 2024
Protocol Integer ID: 103179
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-020607
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Abstract
LC-MS/MS Analysis
**Sample Injection** - Inject 3 μL of each sample into the MClass UPLC system (Waters Corp).
**Sample Trapping** - Trap the sample on a Symmetry C18 20 mm × 180 μm trapping column at a flow rate of 5 μL/min with a solvent composition of 99.9/0.1 v/v water/acetonitrile.
**Analytical Separation** - Perform analytical separation using a 1.8 μm Acquity HSS T3 C18 75 μm × 250 mm column (Waters Corp.). - Use a 90-minute linear gradient of 5 to 30% acetonitrile with 0.1% formic acid at a flow rate of 400 nanoliters/minute (nL/min). - Maintain the column temperature at 55°C.
**Mass Spectrometry Data Collection** - Perform data collection on the Thermo Orbitrap Fusion Lumos mass spectrometer equipped with a FAIMSPro device via a nanoelectrospray ionization source. - Use three different compensation voltages (-40V, -60V, -80V).
**Full MS Scan** - Use data-dependent acquisition (DDA) mode for each compensation voltage (CV). - Perform full MS scan from m/z 375 – 1500 with a resolution of 120,000 (@ m/z 200) and a target AGC value of 4e5 ions. - Set the max fill time to 35 ms.
**MS/MS Scan** - Acquire MS/MS scans with HCD settings of 30% in the linear ion trap in “rapid” mode. - Use a target AGC value of 1e4. - Set the max fill time to 35 ms. - Employ a 20-second dynamic exclusion to increase the depth of coverage.
**Cycle Time** - Set the total cycle time for each CV to 0.66 seconds. - Set total cycle times to 2 seconds between like full MS scans.
**Total Analysis Time** - The total analysis cycle time for each sample injection is approximately 2 hours.