A hyphenated microLC‐Q‐TOF‐MS platform for exosomal lipidomics investigations: Application to RCC urinary exosomes

Abstract

Urinary exosomes are released from every renal epithelial cell type facing the urinary space and therefore, they may carry molecular markers of renal dysfunction and structural injury. Here, we present a hyphenated microLC‐Q‐TOF‐MS platform for lipidomics studies applied to investigate the urinary exosome lipid repertoire. Lipids were separated by reversed‐phase chromatography using a linear gradient of formic acid 0.2% and tetrahydrofuran, in 40 min of analysis. Features (m/z with associated own retention time) were extracted by MarkerLynxTM (Waters) and processed, demonstrating good analytical performance in terms of repeatability and mass accuracy of the microLC Q‐TOF MS platform. In particular, a stable retention time (RSD less than 4%) and relative intensity (RSD from 2.9% to 11%) were observed. Moreover, the method takes advantages by the use of a lock spray interface (Waters) that allows readjusting the m/z data after acquisition, obtaining inaccuracy below 6 ppm in measuring the m/z value of the reference compound during chromatographic run. The method was employed in a preliminary application to perform comparative analysis from healthy control subjects and renal cell carcinoma (RCC) patients, in order to possibly highlight differences in lipid composition to be exploited as potential tumor biomarker. Differential lipid composition in RCC urinary exosomes was achieved and tentatively identified by accurate mass, providing a preliminary indication of a relationship between lipid composition of urinary exosomes and RCC disease. Among the total features significantly different in RCC exosomes, the ion at m/z 502.3 was taken as an example for molecular confirmation by MS/MS fragmentation analysis.

Urinary Signatures of Renal Cell Carcinoma Investigated by Peptidomic Approaches

“…Biomarker discovery studies are widely performed on urine samples, with the aim of developing a non-invasive diagnostic tool for prostate cancer [21], diabetic nephropathy [22], chronic kidney disease [23] as well as for RCC [3], [10], [24].…”

Lipidomics

“…Micro-LC-QTOF/MS was used to investigate urine lipids in RCC versus healthy subjects. Thirty-five lipid species were tentatively identified including lipidomic changes in urinary exosomes [109]. Tissue GP and their enzymatic by-products appear related to malignant transformation [110,111] and significantly increased PI has been observed in transformed cells [112].…”

Differential protein profiling of renal cell carcinoma urinary exosomes

“…10). It has to be underlined that also the RCC UE lipid composition was recently demonstrated to be differential, 54 providing further evidence for a relationship between UE composition and RCC disease. Western blot results were then used to generate ROC curves, to predict the potential impact for use of the biomarker candidates in discriminating between the RCC group and the controls; the AUC values were determined for each protein and range between 0.73-1.…”