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Preparation of Tissue Samples for Large-scale Quantitative Mass Spectrometric Analysis

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 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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Acknowledgements

This work was supported by the Industrial Strategic Technology Development Program (#10079271 and #20000134), funded by the Ministry of Trade, Industry and Energy (MOTIE, Korea); the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (# HL19C0020); and the Collaborative Genome Program for Fostering New Post-Genome Industry (NRF-2017M3C9A5031597). This study was also supported by a grant from Seoul National University Hospital (2020).

The authors declare no conflict of interest.

Neither ethical approval nor informed consent was required for this study.

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Kim, Y., Yeo, I., Kim, H. et al. Preparation of Tissue Samples for Large-scale Quantitative Mass Spectrometric Analysis. Biotechnol Bioproc E 25, 551–561 (2020). https://doi.org/10.1007/s12257-019-0495-6

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Keywords

  • tissue
  • sample preparation method
  • homogenization
  • targeted proteomics
  • MRM-MS