期刊
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
卷 409, 期 8, 页码 2179-2187出版社
SPRINGER HEIDELBERG
DOI: 10.1007/s00216-016-0163-z
关键词
Covalent organic framework; Immobilized trypsin; Magnetic graphene
资金
- National Natural Science Foundation of China [21675125, 21606181, 21275159, 21235001]
- National Key Program for Basic Research of China [2013CB911204, 2016YFA0501403]
- National Key Program for Scientific Instrument and Equipment Development [2012YQ12004407, 2011YQ06008408, 2013YQ14040506]
- Amygdalus pedunculata Engineering Technology Research Center of State Forestry Administration
- Key Laboratory of Yulin Desert Plants Resources
Deep and efficient proteolysis is the critical premise in mass spectrometry-based bottom-up proteomics. It is difficult for traditional in-solution digestion to meet the requirement unless prolonged digestion time and enhanced enzyme dosage are employed, which makes the whole workflow time-consuming and costly. The abovementioned problems could be effectively ameliorated by anchoring many proteases on solid supports. In this work, covalent organic framework-coated magnetic graphene (MG@TpPa-1) was designed and prepared as a novel enzyme carrier for the covalent immobilization of trypsin with a high degree of loading (up to 268 mu g mg(-1)). Profiting from the advantages of magnetic graphene and covalent organic frameworks, the novel trypsin bioreactor was successfully applied for the enzymatic digestion of a model protein with dramatically improved digestion efficiency, stability, and reusability. Complete digestion could be achieved in a time period as short as 2 min. For the digestion of proteins extracted from Amygdalus pedunculata, a total of 2833 protein groups were identified, which was slightly more than those obtained by 12 h of in-solution digestion (2739 protein groups). All of the results demonstrate that MG@TpPa-1-trypsin is an excellent candidate for sample preparation in a high-throughput proteomics analysis.
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