engleski

An ion mobility enabled data independent multi-omics approach to quantitatively characterize urine from children diagnosed with idiopathic nephrotic syndrome

Idiopathic nephrotic syndrome (INS) is the most prevalent glomerular disease in children. In spite of some progress, its pathogenesis is still unknown and the therapy options are confined to gross immune modulation. A variety of methods for diagnostic and treatment purposes are available for the patients ; however, the lack of understanding regarding the pathogenic mechanisms underlying INS can lead to poor therapeutic response and adverse side-effects. Here, we describe quantitative proteomic approach to reveal new molecular factors involved in pathogenesis of INS with potential diagnostic and therapeutic significance. Urine samples were collected from 10 children diagnosed with INS receiving no therapy and 10 healthy children. All samples were purified using spin filters followed by affinity depletion of albumin. The purified proteins were recovered and digested with trypsin overnight. Label-free protein expression data were acquired with Synapt G2 using an ion mobility data independent approach, whereby the collision energy was switched between low and elevated energy state during alternate scans and associate precursor and product ions by means of retention and drift time alignment. The acquired data was processed using ProteinLynx Global SERVER and searched against a human database, which was amended to account for N-terminal processed peptides. Normalized label-free quantitation results were generated using Progenesis software. In a similar fashion the diluted neat urine samples were analysed using a small molecule profiling approach. The resulting data was analysed using MarkerLynx XS. The results of this study showed a significant number of proteins to be over-expressed in the urine from INS patients. More interestingly, the majority of proteins identified as being regulated were secreted glycosylated proteins (e.g. zinc-alpha-2-glycoprotein and cerruloplasmin) in relatively low abundance. Metabolomics analysis showed some statistically significant changes in small molecule profile of the INS patients, with homocysteine, and uridine higher and glutamate, indoline, phenylalanine, and glucose lower.

idiopatic nephrotic syndrome; urine; proteomics

nije evidentirano