Analysis of IP-MassSpec data using ProteinGURU’s Online MassSpec

data Analysis Tool (OLMAT)

ProteinGURU.com

Step 1: Do the IP and run it on the gel

• In this experiment, U2OS cells stably expressing GFP alone or GFP-protein fusion were treated with either DMSO or an inhibitor (drug) of interest.

• The proteins were immunoprecipitated from the lysates using anti-GFP antibody and samples were run on a 4-12% gradient gel.

Step 2: Cut your gel into slices and submit for Orbitrap analysis

• It is important to cut the slices in the negative control lanes (GFP alone) corresponding to the MW of the slices cut from the test samples.

Step 3: Archive the Orbitrap analysis files

• Select all the HTML files that came from the Orbitrap analysis and compress them into a ZIP file (don’t select the folders or any other files).

• For Windows:– Select all the files– Right-click– Send to– Compressed (zipped) folder

• For Mac:– Select all the files– Ctrl-click– Compress (an Archive.zip file

will be created)

Step 4: Upload the ZIP file to OLMAT and Process the Experiment

1. Go to:http://www.proteinguru.com/MassSpec/OLMAT/

2. Follow the OLMAT Demo page (3 videos).

3. Download your analysis package.