May 03, 2024

Public workspaceLC-MS of Native Nanodiscs V.2

DOI
dx.doi.org/10.17504/protocols.io.e6nvw1k97lmk/v2
  • 1Nanobiology Institute, Yale University, West Haven, CT, USA;
  • 2Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA;
  • 3Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815
Caroline Brown
Open access
DOI: dx.doi.org/10.17504/protocols.io.e6nvw1k97lmk/v2
Protocol Citation: Caroline Brown, Snehasish Ghosh, Kallol Gupta 2024. LC-MS of Native Nanodiscs. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw1k97lmk/v2Version created by Caroline Brown
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: May 02, 2024
Last Modified: May 03, 2024
Protocol Integer ID: 99224
Abstract
This protocol provides a step-by-step guide for conducting LC-MS analysis of native nanodiscs extracted from biological membranes.
Liquid Chromatography
Liquid Chromatography
Resuspend peptides in water + 0.1 formic acid varying amount of water so that 500ng of peptide can be loaded in a reasonable volume.
Chromatography is subsequently conducted using home-packed C18 columns (15cm x 75uM ID) for separation of peptides by hydrophobicity.
Use a 75 minute separation gradient with a buffer system of water/acetonitrile with 0.1% formic acid beginning at 6% acetonitrile and ending at 100% acetonitrile.
Mass Spectrometry
Mass Spectrometry
Data Dependent Acquisition (DDA) mass spectrometry is conducted using an Orbitrap Eclipse instrument.
MS1 scans a acquired using the Orbitrap detector at a resolution of 120000 and stored in profile mode using a mass range of 350-1400 m/z and and intensity threshold of 1.0e3.
Use a cycle time of 1 second for ion isolation and MS2 fragmentation isolating ions with a 1.4 m/z window and fragmented with CID energy at 30% activation.
Product ions are detected at the ion trap set to rapid detection mode with precursors ions being dynamically excluded for 20 seconds after one instance of detection.