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How to Avoid Artificial Non-Enzymatic PTMs During Peptide Sample Preparation

Topics covered include:

  • Sources of artificial protein modifications and procedural optimizations to suppress PTMs
  • Selection of appropriate denaturing agents and 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

Summary

Non-enzymatic chemical modifications such as deamidation, disulfide bond scrambling and oxidation have negatively affect efficacy and stability of biotherapeutic proteins. Peptide mapping is the primary analytical tool used to monitor these modifications, and the process involves proteolysis of the biotherapeutic protein into peptides followed by downstream reverse phase HPLC and mass spectrometry analyses. 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 trypsin and other proteases used in peptide mapping.  Excipients and impurities possessing protein oxidation activity cause the third major non-enzymatic PTM, oxidation. These issues complicate analysis of the PTMs. 

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 represented the major bottleneck since trypsin, the most commonly used protease in peptide mapping, 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. The pre-digestion step assured digestion of tightly folded proteins and protein domains. To suppress artificial protein oxidation during sample preparation, we selected a compound with high oxygen scavenging activity. In a model study, we successfully applied the compound to suppress protein oxidation caused by hydrogen peroxide. 


Speaker

sergei-saveliev-125x125

Sergei Saveliev, PhD
Senior Research Scientist

Sergei Saveliev is a Senior Research Scientist at Promega. Prior to joining the 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 overcome address the major challenges in  needs of protein mass spec sample analysis including efficient proteolysis, standardization, suppressing of side effects of protein mass spec sample preparation and others.

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