Total ApoE and ApoE4 Isoform Assays in an Alzheimer's Disease Case-control Study by Targeted Mass Spectrometry (n = 669): A Pilot Assay for Methionine-containing Proteotypic Peptides

Abstract

Allelic polymorphism of the apolipoprotein E (ApoE) gene (ApoE 2, ApoE 3 and ApoE 4 alleles) gives rise to three protein isoforms (ApoE2, ApoE3 and ApoE4) that differ by 1 or 2 amino acids. Inheritance of the ApoE 4 allele is a risk factor for developing Alzheimer's disease (AD). The potential diagnostic value of ApoE protein levels in biological fluids (i.e. cerebrospinal fluid, plasma and serum) for distinguishing between AD patients and healthy elderly subjects is subject to great controversy. Although a recent study reported subnormal total ApoE and ApoE4 levels in the plasma of AD patients, other studies have found normal or even elevated protein levels (versus controls). Because all previously reported assays were based on immunoenzymatic techniques, we decided to develop an orthogonal assay based on targeted mass spectrometry by tracking (i) a proteotypic peptide common to all ApoE isoforms and (ii) a peptide that is specific for the 4 allele. After trypsin digestion, the ApoE4-specific peptide contains an oxidation-prone methionine residue. The endogenous methionine oxidation level was evaluated in a small cohort (n ‫؍‬ 68) of heterozygous 34 carriers containing both healthy controls and AD patients. As expected, the proportion of oxidized residues varied from 0 to 10%, with an average of 5%. We therefore developed a standardized strategy for the unbiased, absolute quantification of ApoE4, based on performic acid oxidization of methionine. Once the sample workflow had been thoroughly validated, it was applied to the concomitant quantification of total ApoE and ApoE4 isoform in a large casecontrol study (n ‫؍‬ 669). The final measurements were consistent with most previously reported ApoE concentration values and confirm the influence of the different alleles on the protein expression level. Our results illustrate (i) the reliability of selected reaction monitoringbased assays and (ii) the value of the oxidization step for unbiased monitoring of methionine-containing proteotypic peptides. Furthermore, a statistical analysis indicated that neither total ApoE and ApoE4 levels nor the ApoE/ApoE4 ratio correlated with the diagnosis of AD. These findings reinforce the conclusions of previous studies in which plasma ApoE levels had no obvious clinical significance. Molecular & Cellular

Human Central Nervous System (CNS) ApoE Isoforms Are Increased by Age, Differentially Altered by Amyloidosis, and Relative Amounts Reversed in the CNS Compared with Plasma

“…Astrocyte-derived apoE is a separate pool from liver-derived apoE in the periphery (22) and is found at concentrations of ϳ5-10 g/ml in CSF and 50 g/ml in plasma (23). Peripheral apoE measured in plasma has previously been shown to decrease with APOE-⑀4 carrier status (23)(24)(25)(26). In the CNS, ELISA-based methods for estimating the relative or absolute amounts of apoE allelic isoforms have yielded conflicting results, possibly due to isoform-specific antibody preference and inherent variability of the assay.…”

The clinical utility of mass spectrometry based protein assays

“…In a similar fashion, a LC-MS assay for DcR3 and GDF15 was demonstrated to match reported immunoassays results but with higher resolution at low protein concentrations than antibody-based strategies and with the possibility of multiplexing [86]. Likewise, in recent years there has been a significant amount of published data on the quantitation of apolipoproteins using LC-MS [87][88][89][90][91][92][93]. Apolipoproteins are associated with the progression of cardiovascular and metabolic diseases, with apolipoprotein B100 and apolipoprotein A1 the primary components of low density lipoproteins (LDL) and high density lipoproteins (HDL), respectively.…”

Evaluation of hydrophilic interaction chromatography (HILIC) versus C18 reversed-phase chromatography for targeted quantification of peptides by mass spectrometry

“…In order to evaluate the influence of matrix amount on signal, a labeled peptide (LGPLV*EQGR) was spiked at the same concentration in 100 L of water, serum or urine, respectively. LGPLV*EQGR peptide corresponds to the reporter peptide of the apolipoprotein E [40]. Spiked samples were submitted to enzymatic digestion and reconstituted in 200 L. The reconstituted samples were diluted with solvent (water or acetonitrile depending of chromatographic mode) before LC-MS/MS analysis.…”