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Abstract: SA-PO999

Establishment of Quantitative Proteomics Platform for Urine Biomarker Discovery and Validation

Session Information

  • CKD: Pathobiology - II
    November 05, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
    Abstract Time: 10:00 AM - 12:00 PM

Category: CKD (Non-Dialysis)

  • 2203 CKD (Non-Dialysis): Mechanisms

Authors

  • Yamamoto, Keiko, Niigata Daigaku Igakubu Igakuka Daigakuin Ishigaku Sogo Kenkyuka, Niigata, Niigata, Japan
  • Yamamoto, Tadashi, Niigata Daigaku Igakubu Igakuka Daigakuin Ishigaku Sogo Kenkyuka, Niigata, Japan
Background

Proteomics has been introduced for discovery of biomarkers in biological fluids such as plasma, urine and others. Recent advance of the data-independent acquisition (DIA) method of proteomics has improved the quantitative evaluation. We employed the method for urine biomarker discovery and adapted it for validation of the biomarkers.

Methods

Urine samples were collected in Biofluid Biomarker Center (BBC), Niigata University. Japan from patients with various diseases and healthy persons received medical examinations at several hospitals according to a guide proposed by Human Kidney & Urine Proteome Project of Human Proteome Organization and stored in -20C freezers.
Urine proteins were separated from 1ml of the frozen urine by a methanol/chloroform precipitation method and digested with trypsin to prepare peptides, purified through C-14 spin column.
The urine peptide samples of 200 ng each were analyzed by liquid chromatography-mass spectrometry (LC-MS, tims-TOFpro, Bruker Daltonics) sequentially in data-dependent acquisition (DDA) and data-independent acquisition (DIA) manners.
The platform was applied to urine samples from diabetic patients (n=200) and healthy volunteers (n=200).

Results

More than 100,000 urine samples have been collected from about 12,000 patients and healthy persons, indicating serial urine collections from the same patients. Approx. 2,000 proteins were identified by the DDA LC-MS and quantified by the DIA LC-MS using 200 ng peptides each. Qualities of the peptide samples and LC-MS analysis were certified by consistent identification and quantification of quality control proteins of high to low abundance, selected in BBC.
The urine biomarker candidates were selected by comparing quantities of proteins, evaluated in about 50-100 sample analyses by the DIA-LC-MS between men and foemen and between DM patients and healthy people. The selected biomarker candidates were examined in another set of about 50-100 sample analysis data for validation of the urine biomarkers.
Several urine biomarkers for gender identification and for DM-induced kidney injuries are demonstrated in the presentation.

Conclusion

The quantitative proteomics platform is powerful to find and validate new urine biomarkers since it is efficient to quantitate thousands of proteins in a single analysis and also consistent for quantitation by using several quality controls.

Funding

  • Commercial Support