A multiplex quantitative proteomics strategy for protein biomarker studies in urinary exosomes

Abstract

Urinary exosomes have received considerable attention as a potential biomarker source for the diagnosis of renal diseases. Notwithstanding, their use in protein biomarker research is hampered by the lack of efficient methods for vesicle isolation, lysis, and protein quantification. Here we report an improved ultracentrifugation-based method that facilitates the solubilization and removal of major impurities associated with urinary exosomes. A double-cushion sucrose/D(2)O centrifugation step was used after a two-step differential centrifugation to separate exosomes from the heavier vesicles. After the removal of uromodulin, 378 and 79 unique proteins were identified, respectively, in low- and high-density fractions. Comparison of our data with two previously published data sets helped to define proteins commonly found in urinary exosomes. Lysis, protein extraction, and in-solution digestion of exosomes were then optimized for MudPIT application. More than a hundred exosomal proteins were quantified by four-plex iTRAQ analysis of single and pooled samples from two different age groups. For healthy men, six proteins (TSN1, PODXL, IDHC, PPAP, ACBP, and ANXA5) showed significant expression differences between exosome pools of those aged 25-50 and 50-70 years old. Thus, exosomes isolated by our method provide the basis for the development of robust quantitative methods for protein biomarker research.

Urinary Exosomes for Protein Biomarker Research

“…Recently, we have developed protocols for lysis, protein extraction and in-solution digestion of UEs for MudPIT application to quantitative proteomics (Raj et al, 2011a). For the solubilization of exosomal membrane proteins the use of an acid cleavable detergent was found to be particularly useful.…”

Cellular Phenotype and Extracellular Vesicles: Basic and Clinical Considerations

“…Other factors such as gender, age, circadian rhythms, fasting state, medication exposure, and physical activity also may influence the number and nature of vesicles under different conditions [6][7][8][9][10][11]. Vesicles have been studied most extensively in plasma and serum [12], but have also been isolated from pleural effusions [13], ocular effluent and aqueous humor [14], breast milk [15], ascites [16], amniotic fluid [17], semen [18], saliva [19], cerebrospinal fluid [20], and urine [21]. In addition, vesicles derive from virtually all tissues in culture.…”

High-purified Isolation and Proteomic Analysis of Urinary Exosomes from Healthy Persons

“…In the last decade, urinary exosomes have received a considerable attention for their potential as non-invasive renal disease biomarkers due to their enrichment protein [2], mRNA [3] and microRNA complex [4]. However, researches on urinary exosomes have been hampered by lacking a facile and lowcost isolation technique for highly-purified exosomes [5]. Currently, the existing methods are mainly based on ultracentrifugation [1], ultrafiltration [6], size exclusion [7], polyethylene glycol (PEG) [8] or commercial precipitation reagent [9].…”