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KMID : 1094720200250040551
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Biotechnology and Bioprocess Engineering
2020 Volume.25 No. 4 p.551 ~ p.561
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Preparation of Tissue Samples for Large-scale Quantitative Mass Spectrometric Analysis
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Kim Yo-Seop
Yeo In-Joon
Kim Hyun-Soo
Son Min-Soo
Kim Young-Soo
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Abstract
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Tissues contain more tumor-type specific information than biofluids, such as blood, rendering them valuable resources for biomarker studies. However, considering the characteristics of tissue homogenization, it is difficult to obtain reproducible samples and analyze many samples simultaneously. To address these issues, we developed a robust and reproducible method for preparing tissues for targeted proteomics?multiple reaction monitoring-mass spectrometry (MRM-MS)?using a Bioruptor Pico sonicator. This approach uses sodium deoxycholate (SDC) as a detergent and can extract proteins from up to 20 mg of tissue using a lysis buffer volume of 300 μL and a sonication time of 30 s, with 30 on/off cycles. The tryptic digestion was optimized as follows: digestion base buffer, ammonium bicarbonate (ABC); reduction and alkylation reagent, dithiothreitol (DTT) and iodoacetamide (IAA), respectively; and trypsin amount and incubation time, 1:50 (enzyme: substrate) and 10 h, respectively. With regard to reproducibility, the intra-assay and inter-assay CVs for the target peptides were less than 20% (intra-CV, 0.87% to 19.13%; inter-CV, 2.3% to 13.62%). Our method was robust and reproducible in the quantitative analysis of tissue by MRM-MS, rendering it applicable to the large-scale study of tissue-based biomarkers.
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KEYWORD
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tissue, sample preparation method, homogenization, targeted proteomics, MRM-MS
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