A Peptide Mapping Method for Accurate Analysis of Non-Enzymatic Protein Modifications
Part # PS300
Abstract
Sergei Saveliev, Chris Hosfield, Mike Rosenblatt and Marjeta Urh
Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711
Non-enzymatic chemical modifications such as deamidation, disulfide bond scrambling and oxidation can affect the stability and efficacy of biotherapeutic proteins. Peptide mapping, the method of choice for monitoring these modifications, has a significant drawback, which is that peptide mapping sample preparation induces the same modifications. We developed a sample preparation method which prevented the above side effects. In our method, all sample preparation steps including reduction, alkylation and digestion are performed at low pH (mildly acidic conditions). Efficient reduction and alkylation at low pH utilized both specialized reagents and procedural modifications. The proteolytic step represented the major bottleneck since trypsin, the most commonly used protease in peptide mapping, is inhibited at acidic pH. We overcame this problem by supplementing trypsin with a low pH resistant Lys-C protease. We also selected a reagent with oxygen scavenging activity, which was successfully applied to suppress protein oxidation during sample preparation.
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