Serotyping in heterozygous combinations.

<p>Figure shows representative ion chromatograms of the wild-type (E3/E3) and all heterozygous combinations (E2/E3, E3/E4, E2/E4). Tryptic peptide polymorphisms correspond to each ApoE isoform. As described under “ApoE serotyping” in the <b>Materials and Methods</b>, the correlation between ApoE genotypes and isoforms enables determination of the genotype from the blood ApoE isoform combination.</p

Comparison of <i>APOE</i> genotypes with results of APOE protein resequencing and serotyping.

<p>The serotypes agreed with the genotypes for all enrolled subjects.</p><p>n = number of subject; SD = standard deviation; N.D. = not determined.</p

Apolipoprotein E resequencing and its application to serotyping.

<p>(<b>A</b>) ApoE resequencing. Figure shows a representative result of wild-type ApoE amino acid sequence determination (sequence coverage = 93.6%, excluding the 18-residue signal peptide) using Orbitrap LC-MS/MS. Black highlighting denotes the determined sequence. Amino acid residues C112 and R158, which demonstrate polymorphism in ApoE2 (C158) and ApoE4 (R112), are circled. Amino acids are represented by their one-letter codes. (<b>B</b>) Tryptic peptide polymorphisms and ion chromatograms. Mutations in amino acid residues 112 and 158, which were covered by protein resequencing, cause peptide fragment polymorphisms. The R158C mutation (ApoE2) results in the cLAVYQAGAR peptide, where the C112R mutation (ApoE4) yields the LGADMEDVR peptide. Figure shows representative chromatograms for the doubly charged ions extracted from subjects with E2/E3 and E3/E4 heterozygous combinations. The calculated and observed monoisotopic masses for each peptide are indicated. (<b>C</b>) Corresponding MS/MS spectra for each peptide in (<b>B</b>). Polymorphic peptide sequences from subjects with heterozygous combinations were confirmed by MS/MS. The b- and y-ions are labeled. In (<b>B</b>) and (<b>C</b>), lower-case “c” represents alkylated cysteine residues. C. mass = calculated mass; O. mass = observed mass; Da = dalton.</p