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Quantification of total ApoE and ApoE4 isoform by targeted mass spectrometry across a clinical cohort (n=800): a case study for SRM-based assay validation and methionine-containing proteotypic peptide
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communication par affiche. National audience. Cerebrospinal fluid and plasma apoE levels have been proposed as diagnostic biomarkers to discriminate patients with Alzheimer disease (AD) against normal ederly. However, controversy remains about the clinical significance of plasma apoE levels due to inconsistency across the correlations between those patients, which might arise from different ELISA protocols and/or size and demographic cohorts. Coupling between liquid chromatography and targeted mass spectrometry, in the so-called selected reaction-monitoring mode (LC-SRM), has been shown to be an effective and orthogonal alternative to immuno-enzymatic tests for absolute protein quantification in biofluids. In the present study, we have developed SRM-based assays of total ApoE and ApoE4 isoform in plasma to address the lack of consensus regarding the possible association between ApoE and ApoE4 genotype and AD. AQUA standardization was carried out with the heavy forms of proteotypic sequence LGPLVEQGR tracking total ApoE and of allele specific sequence LGADMEDVR monitoring ApoE4 isoform, respectively. Robustness and precision of the workflow have been extensively evaluated over a one-month period, with 12 technical replicates per weak carried out by two experimentalists. Statistics on the replicates show CV's better than 10% and a lower limit of quantification of 1 microgram/mL for the less sensitive peptide sequence LGADMEDVCGR specific of ApoE4 allele. The validated SRM-based assay was then applied to the absolute quantification of total ApoE and ApoE4 isoform across a clinical cohort of 800 patients.
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