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Related Concept Videos

Protein Digestion01:02

Protein Digestion

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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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Physiology of the Gastrointestinal System II: Digestion and Absorption01:22

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Mechanical and Chemical Digestion in the Small Intestine01:30

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The small intestine plays a crucial role in our digestive system, performing both mechanical and chemical digestion.
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Accessory organs are those that participate in the digestion of food but do not come into direct contact with it like the mouth, stomach, or intestine do. Accessory organs secrete enzymes into the digestive tract to facilitate the breakdown of food.
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Evaluation of Protein–Protein Interactions using an On-Membrane Digestion Technique
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Evaluation of Protein–Protein Interactions using an On-Membrane Digestion Technique

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Protein Digestion for DIGE Analysis.

Sandra Murphy1, Kay Ohlendieck2

  • 1Department of Biology, Maynooth University, Maynooth, Co. Kildare, Ireland.

Methods in Molecular Biology (Clifton, N.J.)
|October 12, 2017
PubMed
Summary
This summary is machine-generated.

This study explores in-gel digestion techniques for mass spectrometry-based proteomics. It reviews alternative enzymes to trypsin for improved protein identification in comparative studies.

Keywords:
Alternative proteolytic enzymesMass spectrometryProtein digestionTrypsinTwo-dimensional gel electrophoresis

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Area of Science:

  • Proteomics
  • Mass Spectrometry
  • Biochemistry

Background:

  • In-gel digestion of 2D gel protein spots is crucial for mass spectrometry-based proteomics.
  • This technique aids in comparative proteome mapping and identifying differentially expressed proteins in health and disease.
  • Standard protocols involve spot excision, de-staining, reduction/alkylation, dehydration, and enzymatic digestion.

Purpose of the Study:

  • To discuss alternative proteolytic enzymes for in-gel digestion.
  • To highlight the limitations of trypsin in peptide digestion.
  • To describe the in-gel digestion process using trypsin.

Main Methods:

  • Protein spot excision from 2D gels.
  • Standard sample preparation steps (de-staining, reduction, alkylation, dehydration).
  • Enzymatic digestion of proteins using proteolytic enzymes, focusing on trypsin.

Main Results:

  • Trypsin, while widely used, can lead to incomplete peptide digestion.
  • Alternative enzymes are gaining popularity, used alone or in combination with trypsin.
  • The chapter details the established in-gel digestion protocol using trypsin.

Conclusions:

  • Optimizing in-gel digestion is key for accurate protein identification in proteomics.
  • Exploring alternative enzymes can overcome trypsin's limitations for more comprehensive proteome analysis.
  • Understanding standard trypsin digestion protocols remains fundamental for proteomic research.