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

Allelic polymorphism of the apolipoprotein E (ApoE) gene (ApoE ε2, ApoE ε3 and ApoE ε4 alleles) gives rise to three protein isoforms (ApoE2, ApoE3 and ApoE4) that differ by 1 or 2 amino acids. Inheritance of the ApoE ε4 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 ε4 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 ε3ε4 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 case-control 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 monitoring-based 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.Apolipoprotein E (ApoE) is a 299-amino acid protein associated with lipoproteins in the plasma and the cerebrospinal fluid (1). The three major genetic variants of ApoE in the general population isoforms (E2, E3, and E4, encoded by the ε2, ε3 and ε4 alleles, respectively) differ by a single amino acid: E2 (cys112, cys158), E3 (cys112, arg158), and E4 (arg112, arg158). Among the different susceptibility genes associated with the risk of late-onset Alzheimer''s disease (AD)¹ and other neurological conditions, ApoE has been identified as a strong genetic determinant (1). Moreover, the risk of AD is strongly correlated with ApoE4 allele because the presence of one or two copies of the allele increases risk of late-onset AD by about three or 12 times, respectively. Furthermore, the presence of one or two copies of ApoE4 allele correlates with an earlier age of onset by about 10–20 years with regard to noncarriers in patients with late-onset disease (2–4).The diagnostic value of an ApoE assay in AD is however subject to debate. Indeed, supranormal (5), subnormal (6), and nondiscriminating (7–10) plasma concentrations of ApoE in AD cohorts have been reported in many studies based on immunoassay techniques. We wondered whether this ambiguity might be resolved by applying quantitative mass spectrometry (MS). Indeed, the combination of standardized, stable isotope dilution with targeted MS in selected reaction monitoring (SRM) mode is now widely accepted as a valuable alternative to immunoassays for accurate protein quantification (11–17) and may constitute the eagerly awaited bridge between the discovery and verification phases for candidate biomarker panels (18–22). A range of pilot studies have evaluated the sensitivity that can be achieved by SRM monitoring (23, 24); when tracking protein in a whole plasma hydrolysate, limits of quantification (LOQs) in the low μg/ml range are frequently reported (12). However, LOQs in the low nanogram/ml range or the detection of proteins expressed with a low copy number per cell require either immuno-enrichment of the target protein or fractionation of proteotypic peptides by cation exchange (24, 25) or off-gel electrophoresis (26). A generic strategy based on stable isotope standards and capture by antipeptide antibodies (referred to as SISCAPA) is a promising alternative to more traditional enrichment strategies. Numerous reports have already demonstrated the ability of single or multiplexed immuno-SRM to achieve limits of quantification of 1 nanogram/ml or below when starting from less than 100 μl of biofluid (27). Precision, reproducibility and robustness are also key parameters in the development of SRM-based assay platforms dedicated to the clinical verification phase. Indeed, accuracy may even be a secondary consideration at this point, because the main objective is to discriminate between specific and nondiscriminating biomarker candidates within the clinical panel. Various pilot studies of the intra- and interclass performances of SRM monitoring have demonstrated acceptable coefficients of variation and levels of imprecision (below 20%) and, importantly, have pinpointed the main pitfalls that can distort assay results (28–33). Indeed, performance levels and reliability of an assay can be drastically compromised by intraclass variance within the proteotypic peptide fraction, which is caused by chemical modifications of certain amino acid side chains. In particular, peptides containing methionine residues are usually not recommended for SRM-based assays, because the sulfur atom is prone to oxidation (34–36) both in vivo (37, 38) and during sample handling or storage (giving rise to methionine sulfoxide). Unfortunately, peptides containing one or more methionine residues are sometimes the only ones available. By way of an example, 17% of the 625 proteins identified by only one peptide in Human Plasma PeptideAtlas database contain at least one methionine. Hence, quantification of this type of proteotypic peptide is challenging because the ratio between nonoxidized and mono-oxidized sulfoxide forms of heavy-labeled standards also changes during storage. To circumvent this limitation, complete oxidization of the methionine-containing peptide population with chemical oxidant has been attempted but was not sufficiently robust or reproducible over several weeks in the context of large clinical study (39, 40).Here, we report on the absolute quantification of total ApoE and ApoE4 isoform peptides by liquid chromatography (LC)-SRM targeted MS in a case-control study (n = 669). Because ApoE4-specific trypsin peptide possesses a single methionine residue (LGADMEDVR), oxidation was performed immediately after enzymatic digestion. The completeness of was carefully checked. We continuously assessed the overall performance of the assay by introducing quality control samples throughout the clinical cohort study, in order to ensure the validity of statistical comparisons between healthy controls and groups of AD patients.