title: Intraluminal proteome and peptidome of human urinary extracellular vesicles.
abstract: OBJECTIVE
  Urinary extracellular vesicles (UEVs) are a novel source for disease biomarker discovery. However, Tamm-Horsfall protein (THP) is still a challenge for proteomic analysis since it can inhibit detection of low-abundance proteins. Here, we introduce a new approach that does not involve an ultracentrifugation step to enrich vesicles and that reduces the amount of THP to manageable levels.
  
  
  METHODS
  UEVs were dialyzed and ultrafiltered after reduction and alkylation. The retained fraction was digested with trypsin to reduce the remaining THP and incubated with deoxycholate (DOC). The internal peptidome and internal proteome were analyzed by LC-ESI-MS.
  
  
  RESULTS
  A total of 942 different proteins and 3115 unique endogenous peptide fragments deriving from 973 different protein isoforms were identified. Around 82% of the key endosomal sorting complex required for transport components of UEVs generation could be detected from the intraluminal content.
  
  
  CONCLUSIONS
  Our UEVs preparation protocol provides a simplified way to investigate the intraluminal proteome and peptidome, in particular the subpopulation of UEVs of the trypsin-resistant class of exosomes (positive for tumor susceptibility gene101) and eliminates the majority of interfering proteins such as THP. This method allows the possibility to study endoproteome and endopeptidome of UEVs, thus greatly facilitating biomarker discovery.
authors.affiliation: Centre for BioAnalytical Sciences, Dublin City University, Dublin, Ireland.|Institute of Nephrology and Urology, The Third Affiliated Hospital of Southern Medical University, Guangzhou, China.
authors.forename: Xinyu
authors.initials: X
authors.lastname: Liu
authors.affiliation: Department of Health Science, University of Milano-Bicocca, Monza, Italy.
authors.forename: Clizia
authors.initials: C
authors.lastname: Chinello
authors.affiliation: Centre for BioAnalytical Sciences, Dublin City University, Dublin, Ireland.
authors.forename: Luca
authors.initials: L
authors.lastname: Musante
authors.affiliation: Department of Health Science, University of Milano-Bicocca, Monza, Italy.
authors.forename: Marta
authors.initials: M
authors.lastname: Cazzaniga
authors.affiliation: Centre for BioAnalytical Sciences, Dublin City University, Dublin, Ireland.
authors.forename: Dorota
authors.initials: D
authors.lastname: Tataruch
authors.affiliation: Centre for BioAnalytical Sciences, Dublin City University, Dublin, Ireland.
authors.forename: Giulio
authors.initials: G
authors.lastname: Calzaferri
authors.affiliation: Department of Health Science, University of Milano-Bicocca, Monza, Italy.
authors.forename: Andrew
authors.initials: A
authors.lastname: James Smith
authors.affiliation: Department of Health Science, University of Milano-Bicocca, Monza, Italy.
authors.forename: Gabriele
authors.initials: G
authors.lastname: De Sio
authors.affiliation: Department of Health Science, University of Milano-Bicocca, Monza, Italy.
authors.forename: Fulvio
authors.initials: F
authors.lastname: Magni
authors.affiliation: Institute of Nephrology and Urology, The Third Affiliated Hospital of Southern Medical University, Guangzhou, China.
authors.forename: Hequn
authors.initials: H
authors.lastname: Zou
authors.affiliation: Centre for BioAnalytical Sciences, Dublin City University, Dublin, Ireland.
authors.forename: Harry
authors.initials: H
authors.lastname: Holthofer
mesh_terms: D000067128:Extracellular Vesicles / Q000378:metabolism*; D006801:Humans; D010446:Peptide Fragments / Q000302:isolation & purification / Q000652:urine*; D020543:Proteome / Q000302:isolation & purification / Q000378:metabolism*; D014462:Ultrafiltration; D016482:Urinalysis / Q000379:methods*
chemical_list: D010446:Peptide Fragments; D020543:Proteome
keywords: Exosome; Peptidome; Proteome; Tamm-Horsfall protein; Urinary extracellular vesicles