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Related Concept Videos

Protein Digestion01:02

Protein Digestion

Protein digestion begins in the stomach, where the highly acidic environment can easily disrupt protein structure by exposing the peptide bonds of polypeptide chains. After polypeptide chains are broken into individual amino acids by a series of digestive enzymes, the amino acids are transported to the liver via the bloodstream to produce energy.

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Simultaneous Affinity Enrichment of Two Post-Translational Modifications for Quantification and Site Localization
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An improved trypsin digestion method minimizes digestion-induced modifications on proteins.

Da Ren1, Gary D Pipes, Dingjiang Liu

  • 1Department of Formulation and Analytical Resources, Amgen, Thousand Oaks, CA 91320, USA. dren@amgen.com

Analytical Biochemistry
|May 22, 2009
PubMed
Summary

Minimizing trypsin digestion time by removing inhibitors significantly reduces artificial protein modifications like deamidation. This optimized 30-minute method yields cleaner protein maps and improves throughput.

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Area of Science:

  • Proteomics
  • Analytical Chemistry
  • Biochemistry

Background:

  • Trypsin digestion can cause artificial protein modifications, including asparagine deamidation and N-terminal glutamine cyclization.
  • These modifications arise from prolonged exposure to alkaline pH and high temperatures during digestion.
  • Incubation time in reduction/alkylation and digestion buffers directly correlates with the extent of artificial modifications.

Purpose of the Study:

  • To minimize protocol-induced artificial modifications during protein digestion.
  • To enhance trypsin activity by removing inhibitors, thereby reducing digestion time.
  • To improve the quality of tryptic maps and overall throughput in proteomic sample preparation.

Main Methods:

  • Optimized trypsin digestion by removing guanidine, a known inhibitor, from the digestion buffer.
  • Achieved near-complete digestion of reduced and alkylated immunoglobulin gamma molecules within 30 minutes.
  • Utilized reversed-phase liquid chromatography/tandem mass spectrometry (LC-MS/MS) with an LTQ Orbitrap for analysis.
  • Minimized reduction and alkylation reaction times by monitoring completion with high-resolution time-of-flight mass spectrometry.

Main Results:

  • Observed minimal protocol-induced deamidation and N-terminal glutamine cyclization with the 30-minute digestion method.
  • Obtained cleaner tryptic maps due to reduced trypsin self-digestion and fewer nonspecific cleavages.
  • Significantly improved the throughput of trypsin digestion compared to conventional methods.

Conclusions:

  • A rapid, 30-minute in-solution trypsin digestion protocol effectively minimizes artificial protein modifications.
  • Removing trypsin inhibitors enhances enzyme activity, leading to faster and more complete digestion.
  • This optimized method provides cleaner peptide profiles and increases sample preparation efficiency for mass spectrometry-based proteomics.