title: Plasma biomarker discovery in preeclampsia using a novel differential isolation technology for circulating extracellular vesicles.
abstract: OBJECTIVE
  To circumvent the complex protein milieu of plasma and discover robust predictive biomarkers for preeclampsia (PE), we investigate if phospholipid-binding ligands can reduce the milieu complexity by extracting plasma extracellular vesicles for biomarker discovery.
  
  
  METHODS
  Cholera toxin B chain (CTB) and annexin V (AV) which respectively binds GM1 ganglioside and phosphatidylserine were used to isolate extracellular vesicles from plasma of PE patients and healthy pregnant women. The proteins in the vesicles were identified using enzyme-linked immunosorbent assay, antibody array, and mass spectrometry.
  
  
  RESULTS
  CTB and AV were found to bind 2 distinct groups of extracellular vesicles. Antibody array and enzyme-linked immunosorbent assay revealed that PE patients had elevated levels of CD105, interleukin-6, placental growth factor, tissue inhibitor of metallopeptidase 1, and atrial natriuretic peptide in cholera toxin B- but not AV-vesicles, and elevated levels of plasminogen activator inhibitor-1, pro-calcitonin, S100b, tumor growth factor β, vascular endothelial growth factor receptor 1, brain natriuretic peptide, and placental growth factor in both cholera toxin B- and AV-vesicles. CD9 level was elevated in cholera toxin B-vesicles but reduced in AV vesicles of PE patients. Proteome analysis revealed that in cholera toxin B-vesicles, 87 and 222 proteins were present only in PE patients and healthy pregnant women respectively while in AV-vesicles, 104 and 157 proteins were present only in PE and healthy pregnant women, respectively.
  
  
  CONCLUSIONS
  This study demonstrated for the first time that CTB and AV bind unique extracellular vesicles, and their protein cargo reflects the disease state of the patient. The successful use of these 2 ligands to isolate circulating plasma extracellular vesicles for biomarker discovery in PE represents a novel technology for biomarker discovery that can be applied to other specialties.
authors.affiliation: Department of Maternal-Fetal Medicine, KK Women's and Children's Hospital, Republic of Singapore; Duke-NUS Graduate Medical School Singapore, Republic of Singapore. Electronic address: tan.kok.hian@kkh.com.sg.
authors.forename: Kok Hian
authors.initials: KH
authors.lastname: Tan
authors.affiliation: A∗STAR Institute of Medical Biology, Republic of Singapore.
authors.forename: Soon Sim
authors.initials: SS
authors.lastname: Tan
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Republic of Singapore.
authors.forename: Siu Kwan
authors.initials: SK
authors.lastname: Sze
authors.affiliation: Department of Maternal-Fetal Medicine, KK Women's and Children's Hospital, Republic of Singapore.
authors.forename: Wai Kheong Ryan
authors.initials: WK
authors.lastname: Lee
authors.affiliation: Department of Maternal-Fetal Medicine, KK Women's and Children's Hospital, Republic of Singapore.
authors.forename: Mor Jack
authors.initials: MJ
authors.lastname: Ng
authors.affiliation: A∗STAR Institute of Medical Biology, Republic of Singapore; Department of Surgery, YLL School of Medicine, NUS, Republic of Singapore.
authors.forename: Sai Kiang
authors.initials: SK
authors.lastname: Lim
mesh_terms: D000328:Adult; D017304:Annexin A5; D015415:Biomarkers / Q000097:blood*; D016022:Case-Control Studies; D002772:Cholera Toxin; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D055354:Exosomes / Q000378:metabolism*; D005260:Female; D006801:Humans; D013058:Mass Spectrometry; D011225:Pre-Eclampsia / Q000097:blood / Q000175:diagnosis*; D011247:Pregnancy; D011263:Pregnancy Trimester, Third; D020543:Proteome
chemical_list: D017304:Annexin A5; D015415:Biomarkers; D020543:Proteome; D002772:Cholera Toxin
keywords: annexin V; biomarker discovery; cholera toxin B chain; extracellular vesicles; preeclampsia