How to Avoid Artificial Non-Enzymatic PTMs During the Peptide Sample Preparation Process
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Prior to joining industry Dr. Saveliev worked on various academic projects including epigenomics, DNA repair and recombination, enzymology, mechanisms of radioresistance and circadian rhythm. Dr. Saveliev’s research efforts were supported by US government funding, and the results were published in the leading academic journals such as the EMBO Journal, PNAS, PLOS, and Genes & Development. After joining Promega Corporation in 2004, Dr. Saveliev utilized his expertise to develop tools for life sciences. Lately, his product development efforts have been focused on tools for protein mass spectrometry sample preparation. Sergei has played the key role in building mass spectrometry protein analytical portfolio at Promega. His efforts are directed toward the development of solutions that help address the major needs of protein mass spec sample analysis including efficient proteolysis, standardization, suppressing of side effects of protein mass spec sample preparation and others.
Sergei Saveliev, PhD
Sr. Research Scientist
- See speaker bio
- Original Webinar Date: Tuesday, June 13, 2017
Non-enzymatic post-translational modifications (PTMs) spontaneously occur in biotherapeutic proteins during manufacturing and storage. These modifications negatively affect efficacy and stability of biotherapeutic proteins. Major non-enzymatic PTMs are deamidation, disulfide bond scrambling and oxidation. These non-enzymatic PTMs are also introduced during protein preparation for peptide mapping and compromise the analysis. In the webinar recording Dr. Saveliev discusses sources of these artificial protein modifications as well as procedural optimizations to suppress these PTMs.
Non-enzymatic chemical modifications such as deamidation, disulfide bond scrambling and oxidation negatively affect efficacy and stability of biotherapeutic proteins. Peptide mapping is the primary analytical tool used to monitor these modifications. Unfortunately, steps involved in peptide mapping sample preparation are also a source of PTMs. In fact, deamidation and disulfide bond scrambling are induced at alkaline pH, which is favored by proteases used in peptide mapping. Excipients and impurities possessing protein oxidation activity cause the third major non-enzymatic PTM, oxidation.
To address these problems, we developed a sample preparation procedure according to which all sample preparation steps are performed at acidic conditions. To achieve efficient reduction and alkylation at these conditions, we selected suitable modifying chemicals and introduced special procedural modifications. The proteolytic step has been a major bottleneck since trypsin is inhibited at acidic pH. We solved this problem by supplementing trypsin with a specialized, low pH resistant Lys-C protease. Using this approach we achieved robust digestion at acidic conditions while suppressing deamidation and disulfide bond scrambling. We were able to further optimize digestion by introducing a pre-digestion step under strong denaturing conditions. To suppress artificial protein oxidation during sample preparation, we selected a compound with high oxygen scavenging activity.
This webinar includes:
- Selection of appropriate denaturing agents and the approaches to minimize their concentration prior to digestion
- Critical parameters of the reaction buffer for sample preparation under acidic conditions
- Optimizations required to minimize baseline noise and shorten digestion period
- Test studies showing advantages of the described procedural optimizations