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Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor
Published on: April 6, 2016
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Achieving efficient digestion faster with Flash Digest: potential alternative to multi-step detergent assisted
Vinit Shah1, Michael E Lassman2, Ying Chen1
1Cardiometabolic Disease, Merck Research Laboratories, Kenilworth, NJ, USA.
Rapid Communications in Mass Spectrometry : RCM
|October 30, 2016
Summary
Immobilized enzyme reactors (IMERs) significantly reduce protein digestion time for quantitative proteomics, offering a faster alternative to traditional in-solution methods for analyzing biomarkers like apolipoproteins.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Proteomics
Background:
- Quantitative analysis of protein biomarkers and therapeutic proteins commonly uses liquid chromatography/mass spectrometry (LC/MS).
- Protein digestion into peptides is crucial for LC/MS, but proteolytically resistant proteins require lengthy digestion times with in-solution methods.
- Immobilized enzyme reactors (IMERs) offer a potential solution to reduce digestion times and increase throughput.
Purpose of the Study:
- To evaluate the performance of an immobilized enzyme reactor (IMER) for protein digestion in quantitative proteomics.
- To compare the digestion time, efficiency, and method development time of an IMER (Flash Digest) versus traditional in-solution digestion.
- To assess the impact of IMER use on the analysis of apolipoprotein biomarkers in human plasma.
Main Methods:
- Human plasma samples were digested using a commercial IMER (Flash Digest) and compared to in-solution digestion.
- Three apolipoprotein biomarkers (APOE, APOC2, APOC3) were analyzed using LC/MS with electrospray ionization (ESI) and multiple reaction monitoring (MRM).
- Method performance was evaluated based on digestion time, digestion efficiency, linearity, and precision (% CV).
Main Results:
- Flash Digest reduced digestion time for the resistant APOC3 protein to 30 minutes, compared to overnight for in-solution digestion.
- The IMER achieved comparable digestion efficiency with minimal method development and reduced sample preparation time.
- Both methods demonstrated linearity over a relevant concentration range, with Flash Digest achieving % CV < 8% for intra-day reproducibility.
Conclusions:
- Immobilized enzyme reactors (IMERs) like Flash Digest can accelerate the digestion of proteolytically resistant proteins in quantitative proteomics.
- IMERs offer a potential alternative to in-solution digestion, reducing method development time and increasing sample throughput.
- This technology can improve the efficiency of quantitative proteomics experiments, particularly for challenging protein targets.
