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X-ray footprinting with Mass Spectrometry (XFMS) is a method consisting of X-ray irradiation at the beamline, and subsequent collection and analysis of the Liquid Chromatography Mass Spectrometry (LCMS) data.
Generally you will work with the beamline scientist to irradiate your samples, digest the samples, and then run LCMS.
Here we cover the LCMS analysis portion of the method:
- Accessing the software server for data analysis
- Your beamline scientist will give you an account on the PMI software server, which is located at the Molecular Foundry at Berkeley Lab, and is remote accessible
- Once you have an account, view this video to learn how to reserve time on the server, how to log in, and how to create your first project
- With project creation, the PMI software auto-analyzes your LCMS data and makes assignments of the peaks in the LC
- This PDF document gives you additional information about modifications you can tell the software look for during project creation
- Getting started with LCMS data analysis
- This video shows you how to start analyzing your data, and includes an overview of the dose response plot, as well as an overview of the various panels in the PMI software.
- Further analysis of the LCMS data
- After you have created your project and are comfortable with the layout of the PMI software, you will start validating your data. The first step uses the fragment table, and this video shows you how to read the fragment table
- After you are confident with the assignment of the peaks in the LC, you can set the integration limits in the XICs for each peptide
- For XFMS data consisting of two states
- For XFMS data consisting of two states (eg, protein with ligand and protein without ligand) you can use our dose response script to calculate the fold change between the two states automatically. This script assumes you have already validated your data and set the integration limits on your LC peaks. This video explains how to use the automated dose response, and this PDF document gives more information about how the script works
- More information about XFMS in general and data processing can be found here:
- A detailed data processing overview from Line Kristensen can be found here
- The 2020 workshop at the ACA highlighted different ways to do footprinting and gave advice on data analysis. The recording of the workshop can be found here