title: Development of new quantitative mass spectrometry and semi-automatic isofocusing methods for the determination of Apolipoprotein E typing.
abstract: BACKGROUND
  Apolipoprotein E (Apo E) is a 36 Kda glycoprotein involved in lipid transport. It exists in 3 major isoforms: E2, E3 and E4. ApoE status is known to be a major risk factor for late-onset Alzheimer's and cardiovascular diseases. Genotyping is commonly used to obtain ApoE status but can show technical issues with ambiguous determinations. Phenotyping can be an alternative, not requiring genetic material. We evaluated the ability to accurately type ApoE isoforms by 2 phenotyping tests in comparison with genotyping.
  
  
  METHODS
  Two phenotyping techniques were used: (1) LC-MS/MS detection of 4 ApoE specific peptides (6490 Agilent triple quadripole): After its denaturation, serum was either reduced and alkylated, or only diluted, and then trypsin digested. Before analysis, desalting, evaporation and resuspension were performed. (2) Isoelectric focusing and immunoprecipitation: serum samples were neuraminidase digested, delipidated and electrophoresed on Hydragel ApoE (Sebia agarose gel) using Hydrasys 2 Scan instrument (Sebia, Lisses, France). ApoE isoforms bands were directly immunofixed in the gel using a polyclonal anti human ApoE antibody. Then, incubation of the gel with HRP secondary antibody followed by TTF1/TTF2 substrate allowed the visualization of ApoE bands. The results of the two techniques were compared to genotyping.
  
  
  RESULTS
  Sera from 35 patients previously genotyped were analyzed with the 2 phenotyping techniques. 100% concordance between both phenotyping assays was obtained for the tested phenotypes (E2/E2, E2/E3, E2/E4, E3/E3, E3/E4, E4/E4). When compared to genotyping, 3 samples were discordant. After reanalyzing them by both phenotyping tests and DNA sequencing, 2/3 discrepancies were confirmed. Those can be explained by variants or rare ApoE alleles or by unidentified technical issues. 102 additional samples were then tested on LC-MS/MS only and compared to genotyping. The data showed 100% concordance.
  
  
  CONCLUSIONS
  Our 2 phenotyping methods represent a valuable alternative to genotyping. LC-MS/MS has the advantage of being fully specific, with identification of the different isoforms and can be considered as a reference method. Sebia isofocusing technique was concordant with LC-MS/MS. Plus, it is a rapid, semi-automated assay that can be easily implemented in clinical laboratories.
authors.affiliation: CHRU de Montpellier, Hôpital St Eloi Université de Montpellier, INSERM U1183, IRMB, Laboratoire de Biochimie Protéomique Clinique, Montpellier, France.
authors.forename: Christophe
authors.initials: C
authors.lastname: Hirtz
authors.affiliation: CHRU de Montpellier, Hôpital St Eloi Université de Montpellier, INSERM U1183, IRMB, Laboratoire de Biochimie Protéomique Clinique, Montpellier, France.
authors.forename: Jerome
authors.initials: J
authors.lastname: Vialaret
authors.affiliation: SEBIA, 91008 Evry, France.
authors.forename: Georges
authors.initials: G
authors.lastname: Nouadje
authors.affiliation: Inserm U837, Lille, FRANCE Université Paris 7-Denis Diderot, France.
authors.forename: Susanna
authors.initials: S
authors.lastname: Schraen
authors.affiliation: Université Lille 2 and UMR CNRS 8199 Integrative Genomics and Metabolic Disease Modeling, LILLE France; CHRU de Lille, Unité de Médecine Moléculaire des Maladies Métaboliques (U4M), Lille, FRANCE.
authors.forename: Pascale
authors.initials: P
authors.lastname: Benlian
authors.affiliation: Université Lille 2 and UMR CNRS 8199 Integrative Genomics and Metabolic Disease Modeling, LILLE France.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Mary
authors.affiliation: CHRU de Montpellier, Service de Biochimie et d'Hormonologie, Montpellier, France.
authors.forename: Pascal
authors.initials: P
authors.lastname: Philibert
authors.affiliation: CHRU de Montpellier, Hôpital St Eloi Université de Montpellier, INSERM U1183, IRMB, Laboratoire de Biochimie Protéomique Clinique, Montpellier, France.
authors.forename: Laurent
authors.initials: L
authors.lastname: Tiers
authors.affiliation: CHRU de Montpellier, Hôpital St Eloi Université de Montpellier, INSERM U1183, IRMB, Laboratoire de Biochimie Protéomique Clinique, Montpellier, France.
authors.forename: Pauline
authors.initials: P
authors.lastname: Bros
authors.affiliation: CHRU de Montpellier, Hôpital St Eloi Université de Montpellier, INSERM U1183, IRMB, Laboratoire de Biochimie Protéomique Clinique, Montpellier, France.
authors.forename: Constance
authors.initials: C
authors.lastname: Delaby
authors.affiliation: CHRU de Montpellier, Hôpital St Eloi Université de Montpellier, INSERM U1183, IRMB, Laboratoire de Biochimie Protéomique Clinique, Montpellier, France; Centre Mémoire de Ressources et de Recherche Languedoc-Roussillon, Département de Neurologie, CHRU de Montpellier, Université de Montpellier, Montpellier, France.
authors.forename: Audrey
authors.initials: A
authors.lastname: Gabelle
authors.affiliation: CHRU de Montpellier, Hôpital St Eloi Université de Montpellier, INSERM U1183, IRMB, Laboratoire de Biochimie Protéomique Clinique, Montpellier, France. Electronic address: s-lehmann@chu-montpellier.fr.
authors.forename: Sylvain
authors.initials: S
authors.lastname: Lehmann
mesh_terms: D053329:Apolipoprotein E2 / Q000097:blood / Q000235:genetics*; D053318:Apolipoprotein E3 / Q000097:blood / Q000235:genetics*; D053327:Apolipoprotein E4 / Q000097:blood / Q000235:genetics*; D001331:Automation; D005838:Genotype; D006801:Humans; D007525:Isoelectric Focusing / Q000295:instrumentation / Q000379:methods*; D010641:Phenotype; D012984:Software; D053719:Tandem Mass Spectrometry / Q000295:instrumentation / Q000379:methods*
chemical_list: D053329:Apolipoprotein E2; D053318:Apolipoprotein E3; D053327:Apolipoprotein E4
keywords: Alzheimer's disease; Apolipoprotein E; Genotype; Immunofixation; Immunoprecipitation; Isoelectric focusing; Mass spectrometry; Phenotype