Journal
CHEMPLUSCHEM
Volume 78, Issue 5, Pages 407-412Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/cplu.201200323
Keywords
digestion; enzymes; mass spectrometry; proteins; proteomics
Categories
Funding
- 863 Program of China [2012AA06A303, 2013NC190D01089, SS2013AA100204]
- Ministry of Science and Technology of China [2012YQ090194]
- Natural Science Foundation of China [20976125, 31071509, 51173128, 21206113]
- Program of Introducing Talents of Discipline to Universities of China [B06006]
- [10JCYBJC05100]
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In this study, cross-linked enzyme aggregates of tissue-culture-grade trypsin (CLEAs-T) were prepared and introduced to the proteomics analysis instead of the expensive proteomics-grade trypsin, which benefits from the protein purification during the preparation of the CLEAs. Bovine serum albumin, lysozyme, bovine hemoglobin, and -casein were used as model proteins, and three intensive strategies (high-enzyme-concentration, high-temperature, and ultrasound-assisted digestion) were applied to the rapid and efficient digestion of model proteins by CLEAs-T. The peptide fragments were then identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, which revealed the higher sequence coverage and sharply reduced digestion time of these strategies compared with the conventional in-solution 12h digestion method. Morphology studies demonstrated that the excellent performance of CLEAs-T in proteomics analysis came from the scaffoldlike supermolecular structure, which provided a high resistance to the autolysis and denaturation of trypsin under high-enzyme-concentration, high-temperature, and ultrasound-assisted digestion.
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