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An in-gel digestion method of chymotrypsin to improve sequence coverage of membrane protein by mass spectrometry / 生物工程学报
Chinese Journal of Biotechnology ; (12): 2435-2442, 2020.
Article in Chinese | WPRIM | ID: wpr-878499
ABSTRACT
In recent years, mass spectrometry has been widely used to study membrane protein structure and function. However, the application of mass spectrometry to study integral membrane protein is limited because there are many hydrophobic amino acids in the trans-membrane domain of integral membrane protein to cause low sequence coverage detected by LC-MS/MS. Therefore, we used vitamin K epoxide reductase (VKORC1), a human integral membrane protein, as a model to optimize the digestion conditions of chymotrypsin, and developed an in-gel digestion method of chymotrypsin to improve sequence coverage of membrane protein by mass spectrometry. By exploring the effects of calcium concentration, pH value and buffer system on the percentage of sequence coverage, number of total detected and types of unique peptide, and the size of unique peptide, sequence coverage and peptide diversity could be considered under condition of Tris-HCl buffer with 5-10 mmol/L calcium ion concentration and pH value 8.0-8.5. This method could make the sequence coverage of membrane protein to reach more than 80%. It could be widely used in the study of membrane protein structure and function, identification of interaction site between membrane proteins, and identification of binding site between membrane protein and small molecular drug.
Subject(s)

Full text: Available Index: WPRIM (Western Pacific) Main subject: Chymotrypsin / Trypsin / Chromatography, Liquid / Amino Acid Sequence / Digestion / Tandem Mass Spectrometry / Vitamin K Epoxide Reductases / Membrane Proteins Limits: Humans Language: Chinese Journal: Chinese Journal of Biotechnology Year: 2020 Type: Article

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Full text: Available Index: WPRIM (Western Pacific) Main subject: Chymotrypsin / Trypsin / Chromatography, Liquid / Amino Acid Sequence / Digestion / Tandem Mass Spectrometry / Vitamin K Epoxide Reductases / Membrane Proteins Limits: Humans Language: Chinese Journal: Chinese Journal of Biotechnology Year: 2020 Type: Article