title: Assessment of peptide chemical modifications on the development of an accurate and precise multiplex selected reaction monitoring assay for apolipoprotein e isoforms.
abstract: Apolipoprotein E (ApoE) is a polymorphic protein that plays a major role in lipid metabolism in the central nervous system and periphery. It has three common allelic isoforms, ApoE2, ApoE3, and ApoE4, that differ in only one or two amino acids. ApoE isoforms have been associated with the occurrence and progression of several pathological conditions, such as coronary atherosclerosis and Alzheimer's disease. The aim of this study was to develop a mass spectrometry (MS)-based assay for absolute quantification of ApoE isoforms in cerebrospinal fluid and plasma samples using isotope-labeled peptides. The assay included five tryptic peptides: CLAVYQAGAR (ApoE2), LGADMEDVCGR (ApoE2 and 3), LAVYQAGAR (ApoE3 and 4), LGADMEDVR (ApoE4), and LGPLVEQGR (total ApoE). Both cerebrospinal fluid and plasma samples were assayed to validate the method. The digestion yield and the extension of chemical modifications in selected amino acid residues (methionine oxidation, glutamine deamidation, and cyclization of N-terminus carbamidomethylcysteine) were also studied. The ApoE phenotype was successfully assigned to all samples analyzed in a blinded manner. The method showed good linearity (R(2) > 0.99) and reproducibility (within laboratory imprecision <13%). The comparison of the MS-based assay with an ELISA for total ApoE concentration showed a moderate correlation (R(2) = 0.59). This MS-based assay can serve as an important tool in clinical studies aiming to elucidate the association between ApoE genotype, total ApoE, and ApoE isoform concentrations in various disorders related to ApoE polymorphisms.
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Joseph and Wolf Lebovic Health Complex, Mount Sinai Hospital , 60 Murray Street, Toronto, Ontario, M5T 3L9 Canada.
authors.forename: Eduardo
authors.initials: E
authors.lastname: Martínez-Morillo
authors.forename: Henrietta M
authors.initials: HM
authors.lastname: Nielsen
authors.forename: Ihor
authors.initials: I
authors.lastname: Batruch
authors.forename: Andrei P
authors.initials: AP
authors.lastname: Drabovich
authors.forename: Ilijana
authors.initials: I
authors.lastname: Begcevic
authors.forename: Mary F
authors.initials: MF
authors.lastname: Lopez
authors.forename: Lennart
authors.initials: L
authors.lastname: Minthon
authors.forename: Guojun
authors.initials: G
authors.lastname: Bu
authors.forename: Niklas
authors.initials: N
authors.lastname: Mattsson
authors.forename: Erik
authors.initials: E
authors.lastname: Portelius
authors.forename: Oskar
authors.initials: O
authors.lastname: Hansson
authors.forename: Eleftherios P
authors.initials: EP
authors.lastname: Diamandis
mesh_terms: D000595:Amino Acid Sequence; D001057:Apolipoproteins E / Q000032:analysis / Q000737:chemistry*; D002853:Chromatography, Liquid; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry*; D020033:Protein Isoforms / Q000032:analysis / Q000737:chemistry*; D014357:Trypsin / Q000737:chemistry
chemical_list: D001057:Apolipoproteins E; D010455:Peptides; D020033:Protein Isoforms; D014357:Trypsin