title: Multiple reaction monitoring assay for pre-eclampsia related calcyclin peptides in formalin fixed paraffin embedded placenta.
abstract: Although the cause of pre-eclampsia during pregnancy has not been elucidated yet, it is evident that placental and maternal endothelial dysfunction is involved. We previously demonstrated that in early onset pre-eclampsia placental calcyclin (S100A6) expression is significantly higher compared to controls ( De Groot , C. J. ; Clin. Proteomics 2007 , 1 , 325 ). In the current study, the results were confirmed and relatively quantified by using multiple reaction monitoring (MRM) on two peptide fragments of calcyclin. Cells were obtained from control (n = 5) and pre-eclamptic placental (n = 5) tissue collected by laser capture microdissection from formalin-fixed paraffin-embedded (FFPE) material treated with a solution to reverse formalin fixation. Two calcyclin peptides with an extra glycine inserted in the middle of the amino acid sequence were synthesized and used as an internal reference. Data presented show that MRM on laser microdissected material from FFPE tissue material is possible. The developed MRM assay to study quantitative levels of proteins in FFPE laser microdissected cells using nonisotopic-labeled chemical analogs of mass tagged internal references showed that in pre-eclamptic patients elevated levels of calcyclin is observed in placental trophoblast cells compared to normal trophoblast cells. By immunohistochemistry, we were able to confirm this observation in a qualitative manner.
authors.affiliation: Department of Neurology, Erasmus MC, Rotterdam, The Netherlands.
authors.forename: Coşkun
authors.initials: C
authors.lastname: Güzel
authors.forename: Nicolette T C
authors.initials: NT
authors.lastname: Ursem
authors.forename: Lennard J
authors.initials: LJ
authors.lastname: Dekker
authors.forename: Pieter
authors.initials: P
authors.lastname: Derkx
authors.forename: Jos
authors.initials: J
authors.lastname: Joore
authors.forename: Evert
authors.initials: E
authors.lastname: van Dijk
authors.forename: Gerard
authors.initials: G
authors.lastname: Ligtvoet
authors.forename: Eric A P
authors.initials: EA
authors.lastname: Steegers
authors.forename: Theo M
authors.initials: TM
authors.lastname: Luider
mesh_terms: D000328:Adult; D001681:Biological Assay; D018797:Cell Cycle Proteins / Q000096:biosynthesis / Q000235:genetics; D002853:Chromatography, Liquid; D005260:Female; D005404:Fixatives / Q000737:chemistry; D005557:Formaldehyde / Q000737:chemistry; D015870:Gene Expression; D006801:Humans; D007150:Immunohistochemistry; D007834:Lasers; D013058:Mass Spectrometry; D042282:Microdissection; D016612:Paraffin Embedding; D010446:Peptide Fragments / Q000032:analysis* / Q000737:chemistry; D010920:Placenta / Q000378:metabolism* / Q000473:pathology; D011225:Pre-Eclampsia / Q000175:diagnosis / Q000235:genetics / Q000378:metabolism*; D011247:Pregnancy; D040901:Proteomics / Q000379:methods*; D012015:Reference Standards; D000075383:S100 Calcium Binding Protein A6; D009418:S100 Proteins / Q000096:biosynthesis / Q000235:genetics; D014327:Trophoblasts / Q000378:metabolism* / Q000473:pathology; D014357:Trypsin / Q000378:metabolism
chemical_list: D018797:Cell Cycle Proteins; D005404:Fixatives; D010446:Peptide Fragments; D000075383:S100 Calcium Binding Protein A6; D009418:S100 Proteins; C055351:S100A6 protein, human; D005557:Formaldehyde; D014357:Trypsin