LC-MS of Native Nanodiscs
Caroline Brown, Snehasish Ghosh, Kallol Gupta
Abstract
This protocol provides a step-by-step guide for conducting LC-MS analysis of native nanodiscs extracted from biological membranes.
Steps
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
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.
