1. Analysis of IP-MassSpec data using ProteinGURU’s Online MassSpec data Analysis Tool (OLMAT) ProteinGURU.com

2. 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.

3. 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.

4. 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)

5. 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.