Hi Members,
I am doing analysis of Mab (Herceptin) I was curious to know the protocol for sample preparation of Mab for
1. Intact Mass analysis on Q Tof
2. Peptide mapping
3. Sub unit analysis.
Can someone guide me in this regards
Hi,
Here are some starting points:
1. You could run reversed phase LC-MS of an aliquot of Herceptin without any sample preparation (other than dilution perhaps). This will desalt your sample and concentrate the protein in a plug which allows you to record a nice intact protein spectrum on your Q-ToF. If you don't have an LC, I would advise you to desalt (and buffer exchange to 50 mM ammonium bicarbonate) the sample prior to direct infusion to get rid of excipients that can suppress ionization and contaminate your source;
2. For peptide mapping, you should reduce and alkylate the antibody, for example using dithiothreitol and iodoacetamide, respectively, followed by proteolytic digestion using trypsin. The resulting peptide mixture can then be separated and analyzed using reversed phase LC-MS/MS;. If you search for 'tryptic peptide mapping protocol' you will have ample examples to choose from;
3. For subunit analysis, you can reduce the antibody using dithiothreitol (DTT) or TCEP (at ~10 mM final concentrations, incubate for 30' at 37oC) and analyse the resulting light and heavy chains using reversed phase LC coupled to your Q-ToF. If you want slightly more detailed information, you could also consider using an enzyme like FabRICATOR; visit the website of the manufacturer (Genovis) for more information.
Good luck!
PS. What aspect(s) of Hercepting are you looking to investigate?
Hi Thanks for your prompt response. Do you have any printed protocol for sample prep to refer. I would love to have that to corroborate..!
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