Isolation and mass spectrometry analysis of urinary extraexosomal proteins
نویسندگان
چکیده
The aim of the present study was to develop a LC-MS/MS-based proteomic analysis method of urinary exosomal proteins that has the potential to discover disease biomarkers. In short, urinary exosomes from healthy subjects were isolated by immunocapture on magnetic beads, detected by immunofluorescence and TEM, trypsin digested directly on the beads for an accelerated time with no addition of detergents before performing an LC-MS analysis of the trypsinate. To our knowledge, this is the first proteomic analysis of proteins displayed on the outer surface of exosomes. The outer exosome proteome may contain proteins that are of higher biomarker value compared to soluble cargo protein as the proteins projecting into the extracellular milieu might be more directly involved in physiological functions of exosomes. The proteomic analysis identified 49 proteins that were considered significant; the majority is involved in carbohydrate and lipid metabolism or in immune responses. Thirty of the proteins are linked to diseases. The developed proteomic method exploiting urinary exosomes might be of great value in search for diagnostic or prognostic biomarkers of especially metabolic and immune-related diseases.
منابع مشابه
Proteome analysis of Cryptosporidium parvum and C. hominis using two-dimentional electrophoresis, image analysis and tandem mass spectrometry
Until recently, Cryptosporidium was thought to be a single species genus. Molecular studies now showthat there are at least 10 valid species of this parasite. Among them, two morphologically identical species, C.hominis and C. parvum are the most pathogenic identified to date and share 97% of identical genomes.Post-genomic analyses is therefore necessary to explore further the...
متن کاملProteomic Analysis of Gene Expression in Basal Cell Carcinoma
Background: Basal Cell Carcinoma (BCC) is a type of non-melanoma skin cancer. Alteration in gene expression is the important event that happens in cancer cell. Detection of this event is possible by proteomics techniques. Methods: Normal and tumor tissues were taken from BCC patient. Total proteins were purified by standard methods, and proteins were separated by two-dimensional electrophoresis...
متن کاملProtein profiling and analysis of drug sensitive and multidrug resistant isolates of Mycobacterium tuberculosis by native polyacrylamide gel electrophoresis and mass spectrometry
Introduction: Tuberculosis (TB) remains a deadly infectious disease despite all the efforts to reduce its incidence. Spread of multidrug resistant TB has seriously undermined the efforts to control the disease globally. In this study protein expression profile of MDR and sensitive isolates of MTB were analyzed and compared in order to identify proteins, which could be used in prevention, diagno...
متن کاملFeasibility of urinary extracellular vesicle proteome profiling using a robust and simple, clinically applicable isolation method
Extracellular vesicles (EVs) secreted by prostate cancer (PCa) cells contain specific biomarkers and can be isolated from urine. Collection of urine is not invasive, and therefore urinary EVs represent a liquid biopsy for diagnostic and prognostic testing for PCa. In this study, we optimised urinary EV isolation using a method based on heat shock proteins and compared it to gold-standard ultrac...
متن کاملAn improved method for the analysis of membrane proteins by mass spectrometry
Membrane-bound and membrane-associated proteins are difficult to analyze by mass spectrometry since the association with lipids impedes the isolation and solubilization of the proteins in buffers suitable for mass spectrometry and the efficient generation of positively charged peptide ions by electrospray ionization. Current methods mostly utilize detergents for the isolation of proteins from m...
متن کامل