#2749 quantification of urine proteins for discovery of biomarkers for kidney injuries by dia-proteomics

Abstract Background and Aims Recent advances in proteomics have enabled to quantify proteins comprehensively. We aimed to establish a platform of urine quantitative proteomics for selection of the biomarkers and assessment of health conditions. Since plasma proteins of varying amounts of were present in urine especially from proteinuric patients, we subtracted the plasma protein conteents from total urine proteomes after the quantitative proteomics for discovery of biomarkers for kidney injuries. On the other hand, urine proteins excreted from plasma may be indicators of pathophysiological conditions in the body, suggesting that the urine proteome profiles figure out health conditions and early discovery of any diseases in the future. Method More than 120,000 urine samples (∼10 ml each) have been collected from ∼15,000 patients with various diseases and healthy volunteers (HV) and stored in -20C freezers. In this study, urine samples were selected from HV, diabetic patients (DM) with or without microalbuminuria and other CKD patients. Proteins were precipitated by a methanol/chloroform precipitation method from 1 ml urine. The protein fractions were digested with trypsin and the tryptic peptides were purified by C18 column chromatography. The tryptic peptides of 200 ng each were analyzed by LC-MS (tims-TOFpro, Bruker) by quantitative Data-independent acquisition (DIA) proteomics and proteins were quantified by the DIA-nn tool. Major top 30 plasma proteins were removed from the quantitative proteomics data and kidney- and other urinary tract organ-derived proteins were normalized to compare their proportions in urine. Results Approximately 2,500 proteins were quantified by the DIA analysis. In urine from HV, proportions of plasma proteins were small, however, in urine from DM patients with microalbuminuria or CKD patients, the plasma protein contents were increased, resulting in relative decreases of urine proportions of other proteins derived from the kidney or other urinary tract organs. By subtraction of the plasma proteins from total urine proteins, protein proportions were comparable to select candidates of biomarkers for kidney injuries. Comparison of total urine protein profiles between HV and DM elucidated their differences, which might indicate changes of pathophysiological conditions. Conclusion By recent quantitative proteomics urine proteins were quantified comprehensively, facilitating discovery of biomarkers for kidney injuries and also evaluation of health conditions in the body.