Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Proteomic analysis of drug metabolising enzymes in extracellular vesicles   (#894)

Madelé Dr van Dyk 1 , Andrew A Prof Rowland 1
  1. Flinders University, Bedford Park, SOUTH AUSTRALIA, Australia


Variability in drug exposure as a result of variability in drug absorption, distribution, metabolism and excretion can be accounted for by understanding the enzyme activity and expression. Small extracellular vesicles (sEVs) are released into the bloodstream by organs, containing functional proteins and nucleic acids, and reflect the functional state of that organ. This study aims to quantify activity and expression of Cytochrome P450s (CYPs) and UDP-glucuronosyltransferases (UGTs) in sEVs derived from blood as a source for potential biomarkers.



For peptide screening, in-gel trypsin digestion was performed. Peptides were separated by liquid chromatography (LC) with a 45 min acetonitrile gradient (BSciexEkspert400nanoHPLC). Column elutant was monitored by an AB Sciex 5600+ triple time of flight mass spectrometer (MS). De novo sequencing was performed on raw MS data (Peaks Studio v7.0 software).


Endogenous and labelled peptides were separated by LC (Agilent 1290 Infinity II HPLC) with a 17 min 0.1% formic acid in acetonitrile gradient. Column eluant was monitored by an Agilent 6495B Triple Quadrupole MS (ESI+ mode). Multiple reaction monitoring was performed with a single quantifier and two qualifier ion transitions. Endogenous peptide identities were confirmed by comparison of retention time, and quantifier/qualifier transition ratios of the respective labelled peptide standards. 



188 unique peptides originating from CYP 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2J2, 3A4 and 3A5, and UGT 1A1, 1A3, 1A4, 1A6, 1A9, 2B4, 2B7, 2B10 and 2B15 were detected. The number of unique peptides detected for each protein ranged between 2 and 19, with a mean of 9.65. By way of example, mean (range) CYP2D6 and CYP3A4 protein abundances in sEV were 192 (79 to 347) fmol/mL and 1094 (713 to 1523) fmol/mL, respectively. 



This study demonstrated the quantification of CYPs and UGTs in sEVS derived from blood which may be used as a potential source of clinical biomarkers. Additionally, it may complement existing drug probe-based approaches, while possibly circumventing the need for tissue biopsy.