Preparation of Tissue Samples for Large-scale Quantitative Mass Spectrometric Analysis
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pISSN 1226-8372 eISSN 1976-3816
RESEARCH PAPER
Preparation of Tissue Samples for Large-scale Quantitative Mass Spectrometric Analysis Yoseop Kim, Injoon Yeo, Hyunsoo Kim, Minsoo Son, and Youngsoo Kim
Received: 21 December 2019 / Revised: 27 February 2020 / Accepted: 27 February 2020 © The Korean Society for Biotechnology and Bioengineering and Springer 2020
Abstract 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 monitoringmass 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.
Yoseop Kim†, Injoon Yeo†, Minsoo Son, Youngsoo Kim* Department of Biomedical Engineering, Seoul National University College of Medicine, Seoul 03080, Korea Tel: 82-2-740-8073; Fax: 82-2-741-0253 E-mail: [email protected] Hyunsoo Kim, Youngsoo Kim Institute of Medical and Biological Engineering, Medical Research Center, Seoul National University College of Medicine, Seoul 03080, Korea †
These authors contributed equally to this work.
Keywords: tissue, sample preparation method, homogenization, targeted proteomics, MRM-MS
1. Introduction Mass spectrometry based targeted proteomics (e.g., multiple reaction monitoring-mass spectrometry (MRM-MS)) has been proved as a powerful tool for quantitative protein analysis since it allows measurement of hundreds of target peptides with possibly high sensitivity and selectivity and multiplexing capabilities at impressive speed [1-4]. Particularly, MRM-MS facilitates the highly specific and precise quantification of multiple proteins, simultaneously [5]. The use of synthetic, stable isotope-labeled standard peptides (SIS) as internal standards allows accurate quantification when using MRM-MS [6]. Compared with other conventional protein measurement techniques (e.g., immunohistochemistry, IHC), MRM-MS has the advantage of not requiring expensive antibodies while being capable of quantifying more proteins [7-10]. Given
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