title: Plasma PCSK9 measurement by liquid chromatography-Tandem mass spectrometry and comparison with conventional ELISA.
abstract: The combination of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and trypsin proteolysis is an effective tool for accurate quantitation of multiple proteins in a single run. However, expensive samples pre-treatment as immunoenrichment are often required to analyze low abundant proteins. Plasma proprotein convertase subtilisin/kexin type 9 (PCSK9), a circulating regulator of low-density lipoprotein metabolism, was studied as an example of a low abundant plasma protein. We investigated post-proteolysis solid-phase extraction (SPE) as an alternative strategy to improve its detection. After optimization of pretreatment, including denaturation, reduction, alkylation, tryptic digestion and selective SPE concentration, 91±7% of PCSK9 was recovered from human plasma samples and coefficients of variation were less than 13.2% with a lower limit of quantification of 37.5ng/ml. This LC-MS/MS method was compared with standard enzyme-linked immunosorbent assay in 30 human plasma samples with a broad range of PCSK9 concentrations. Both methods were significantly correlated (r=0.936, p<0.001) with less than 7% of the values out of the 95% confidence interval and similar concentrations were measured using either LC-MS/MS or ELISA methods (514.2±217.2 vs. 504.2±231.0ng/ml, respectively- p=NS). This method involving SPE is an effective measurement tool for low abundant plasma protein analysis that could be easily included in multiplexed assays.
authors.affiliation: INRA, UMR 1280, Physiologie des Adaptations Nutritionnelles, CHU Hôtel-Dieu, Nantes, France; CRNHO, West Human Nutrition Research Center, CHU Hôtel-Dieu, Nantes, France. Electronic address: mikael.croyal@univ-nantes.fr.
authors.forename: Mikaël
authors.initials: M
authors.lastname: Croyal
authors.affiliation: INRA, UMR 1280, Physiologie des Adaptations Nutritionnelles, CHU Hôtel-Dieu, Nantes, France. Electronic address: falfanta92@gmail.com.
authors.forename: Fanta
authors.initials: F
authors.lastname: Fall
authors.affiliation: INRA, UMR 1280, Physiologie des Adaptations Nutritionnelles, CHU Hôtel-Dieu, Nantes, France; CRNHO, West Human Nutrition Research Center, CHU Hôtel-Dieu, Nantes, France; Department of Endocrinology, Metabolic Diseases and Nutrition, G & R Laennec Hospital, Nantes, France. Electronic address: michel.krempf@univ-nantes.fr.
authors.forename: Michel
authors.initials: M
authors.lastname: Krempf
authors.affiliation: INRA, UMR 1280, Physiologie des Adaptations Nutritionnelles, CHU Hôtel-Dieu, Nantes, France; CRNHO, West Human Nutrition Research Center, CHU Hôtel-Dieu, Nantes, France. Electronic address: aurelie.thedrez@univ-nantes.fr.
authors.forename: Aurélie
authors.initials: A
authors.lastname: Thédrez
authors.affiliation: INRA, UMR 1280, Physiologie des Adaptations Nutritionnelles, CHU Hôtel-Dieu, Nantes, France; CRNHO, West Human Nutrition Research Center, CHU Hôtel-Dieu, Nantes, France. Electronic address: khadija.ouguerram@univ-nantes.fr.
authors.forename: Khadija
authors.initials: K
authors.lastname: Ouguerram
authors.affiliation: INRA, UMR 1280, Physiologie des Adaptations Nutritionnelles, CHU Hôtel-Dieu, Nantes, France; CRNHO, West Human Nutrition Research Center, CHU Hôtel-Dieu, Nantes, France. Electronic address: veronique.ferchaud-roucher@univ-nantes.fr.
authors.forename: Véronique
authors.initials: V
authors.lastname: Ferchaud-Roucher
authors.affiliation: INRA, UMR 1280, Physiologie des Adaptations Nutritionnelles, CHU Hôtel-Dieu, Nantes, France; CRNHO, West Human Nutrition Research Center, CHU Hôtel-Dieu, Nantes, France. Electronic address: audrey.aguesse@univ-nantes.fr.
authors.forename: Audrey
authors.initials: A
authors.lastname: Aguesse
authors.affiliation: INRA, UMR 1280, Physiologie des Adaptations Nutritionnelles, CHU Hôtel-Dieu, Nantes, France; CRNHO, West Human Nutrition Research Center, CHU Hôtel-Dieu, Nantes, France. Electronic address: stephanie.crossouard@univ-nantes.fr.
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Billon-Crossouard
authors.affiliation: Spanish Familial Hypercholesterolemia Foundation, Madrid, Spain. Electronic address: pemata@telefonica.net.
authors.forename: Pedro
authors.initials: P
authors.lastname: Mata
authors.affiliation: Spanish Familial Hypercholesterolemia Foundation, Madrid, Spain; Department of Nutrition, Clinica Las Condes, Santiago, Chile. Electronic address: rodrigoalonsok@gmail.com.
authors.forename: Rodrigo
authors.initials: R
authors.lastname: Alonso
authors.affiliation: INSERM, UMR 1188, Sainte-Clotilde, France; Université de la Réunion, Faculté de Médecine, Saint-Denis, France; CHU de la Réunion, Saint-Denis, France. Electronic address: gilles.lambert@univ-reunion.fr.
authors.forename: Gilles
authors.initials: G
authors.lastname: Lambert
authors.affiliation: INRA, UMR 1280, Physiologie des Adaptations Nutritionnelles, CHU Hôtel-Dieu, Nantes, France; CRNHO, West Human Nutrition Research Center, CHU Hôtel-Dieu, Nantes, France; Department of Endocrinology, Metabolic Diseases and Nutrition, G & R Laennec Hospital, Nantes, France. Electronic address: estelle.nobecourt-dupuy@univ-nantes.fr.
authors.forename: Estelle
authors.initials: E
authors.lastname: Nobécourt
mesh_terms: D000328:Adult; D002853:Chromatography, Liquid / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006801:Humans; D016014:Linear Models; D008297:Male; D008875:Middle Aged; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D000071449:Proprotein Convertase 9 / Q000097:blood*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D052616:Solid Phase Extraction / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
chemical_list: D010446:Peptide Fragments; C472125:PCSK9 protein, human; D000071449:Proprotein Convertase 9; D014357:Trypsin
keywords: LC–MS/MS; PCSK9; Proteotypic peptide analysis; Solid-phase extraction