Faster Mass Spec: Same-Day Sample Prep Now a Reality
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Mike is the leader of the Mass Spec Reagents group at Promega Corporation. He received his B.S. degree from Towson University and PhD from the University of Illinois at Urbana-Champaign. After completing an NIH post-doctoral fellowship at the University of Pennsylvania, he founded the Proteomics Core facility at the Children’s Hospital of Philadelphia. Prior to joining Promega he worked with Thermo-Fisher Scientific where he was a Senior Scientist focusing on the development of reagents for the preparation of samples for biological mass spectrometry.
Micheal M. Rosenblatt, PhD
R&D Group Leader, Mass Spec Reagents
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- Original Webinar Date: Tuesday, October 25, 2016
Traditional protocols require digestion times of up to 18 hours, as well as reduction and alkylation prior to digestion. These denaturants often inhibit enzymes and modify proteins, and require removal prior to analysis. What if you could shorten digestion times to ~30 minutes, without denaturants or reductants or alkylating agents? Learn more below!
Preparation of protein samples for mass spec analysis is often the first of several bottlenecks in bottom-up proteomics workflow. Recent LC-MS/MS methods are as short as 10-minutes per run, and data analysis is more streamlined. However, the proteolysis step is often carried out overnight, extending mass spec analysis to 2 days. This bottleneck is challenging when preparing samples for drug metabolism (Pharmacokinetics) studies as well as complex protein samples (i.e., analysis of biomarkers). We have developed a modified protease mix of trypsin/lys-C and an optimized digestion buffer to shorten proteolysis to <1 hour. No reduction or alkylation is required, nor is the critical filtration step required when using immobilized resins that are purported to speed up proteolysis. While 1 hour is the average time suggested for rapid digestion, we have found that many proteins (e.g., IgG1) undergo proteolysis in as little as 15 minutes even in the absence of reduction or alkylation. We have observed efficiencies of various protein substrates >90% (at both lysine and arginine). The sequence coverage and total spectra produced are highly comparable to overnight digestions performed at 37°C. The new rapid digestion protease mixture is compatible with reduction, alkylation and denaturation. The protease mixture is highly scalable with respect to sample volumes. We will present examples of proteolysis of purified proteins, defined protein mixture and complex protein samples.