Dr Ning Tang, Biopharma Discovery Segment Marketing Manager, and Dr Randy Bolger, Workflow Solutions Sales Manager, both of Agilent Technologies
In this webinar, Dr Ning Tang, Biopharma Discovery Segment Marketing Manager, and Dr Randy Bolger, Workflow Solutions Sales Manager, Agilent Technologies, discussed automation and streamlining of peptide mapping using the Agilent AssayMAP Bravo’s liquid handling robotics and gave tips on how you can save time and boost analytical productivity.
Read on for highlights from the Q&A session of the webinar, and if you missed it, watch the webinar on-demand.
Q: How many times does the user have to interact with the AssayMAP Bravo starting from a protein sample and performing all the steps needed for peptide mapping sample prep?
A: (Dr Randy Bolger) It depends on the approach you take. If you use a conventional workflow, assuming that you’ve already purified your protein and now need to do a digestion and clean up before analysis, typically you let the AssayMAP Bravo do all of the liquid additions including adding the protease. Then you perform an overnight digestion at 37 degrees, so you take it off, seal it, put in the incubator and leave it. You come back the next morning and quench, clean up the peptide and then inject into the LCMS. We also have another process that I didn’t talk about during the presentation that is also commonly used in biopharma and that involves cleaning the protein on a C4-type column, then you can perform the digest in about 30 minutes. This approach still requires two interactions with the AssayMAP Bravo but the whole process takes less than 4 hours.
Q: What is the sensitivity of the peptide mapping workflow and how low can you see the modifications?
A: (Dr Ning Tang) The typical amount of protein digest that we load on the columns is about 500 ng. Within this range we can typically observe bound ppms as low as 0.1% of the regular peptide, identified by MS-MS and relative quantitation, obtained using the precursor maps.
Q: What should be the acceptable mass error for each peptide and also ppm? Also what should be the percentage match first generation primary ions range?
A: (Dr Ning Tang) The parameters that we used in the example presented was 5 ppm for the mass error for the precursor mass and 20 ppm for the MS-MS fragment ions to match the free radical. For sequence coverage, this depends on customers’ preference. We typically set it up to be greater than 90% but you can set it up to the specific criteria that you require.
Q: How can I specify the kit for a specific protein that is not a monoclonal antibody?
A: (Dr Randy Bolger) It depends on what part of the workflow you are talking about. At the beginning of the workflow when you are doing an enrichment, for example when you are purifying your monoclonal out of serum, you’d probably be using a standard of purification, like Protein A or Protein G or perhaps streptavidin with a biotin labelled ligand. The second half of the process, doing the digestion and the peptide clean-up, is very generic for a wide range of glycoprotein drugs.
For Research Use Only. Not for use in diagnostic procedures.