Apolipoprotein E (ApoE) serotyping allows clinicians to evaluate patient risk for the development of Alzheimer’s disease, related dementia, and familial dyslipidemia.1 Generally, this requires genetic analysis on preserved DNA. When DNA is unavailable, clinicians can use antibodies from ELISA kits to detect the ApoE4 isoform, which differs from wild-type ApoE3 by a single, mutated amino acid residue. Unfortunately, these kits only identify the presence or absence of ApoE4 to the exclusion of other ApoE types, and users must create standard curves to distinguish between homozygous and heterozygous ApoE4.
Proteomic analysis, which relies upon trypsin digestion to fractionate target proteins, may offer an alternative. Trypsin digestion functions by cleaving the peptide bond between the carboxyl group of an arginine (Arg) or lysine (Lys) residue and the amino group of the proximate residue. Since 24 of 33 identified mutant alleles carry those mutations on Arg or Lys residues, trypsin digestion produces fragments with mass values that unambiguously diverge from wild-type ApoE3. This, coupled with a clear correlation between genotype and ApoE isoform, may offer researchers and clinicians a fast, reliable method for serotyping.
Recently, Nishimura et al. used liquid chromatography–tandem mass spectrometry (LC-MS/MS) to type ApoE in trace amounts of human serum.2 To do this, the researchers collected 100 individual blood samples for direct DNA sequencing. Of these, the team chose 16 blinded samples for further analysis. They immunoprecipitated these samples and carbamidomethylated the proteins that remained in the pellet. After reductive alkylation, the researchers performed SDS-PAGE on a 10–20% continuous gradient gel. This produced 34 kDa bands that corresponded to ApoE. The researchers then used in-gel tryptic digestion and LC-MS/MS on LTQ Orbitrap XL technology (Thermo Scientific). Finally, they relied on Proteome Discoverer 1.2 (Thermo Scientific) for protein identification, with a false discovery rate of <1%.
The researchers achieved a mean sequence coverage of 91.6 ± 4.57% for the 16 blinded serum samples. They assert that this level of sequence coverage is tantamount to resequencing the protein of almost full-length ApoE. This is clinically relevant, as it allows unambiguous serotyping. In all 16 cases, the assigned serotype matched the actual genotype. The authors note that, for all samples, resequencing by LC-MS/MS covered polymorphic residues 112 and 158, which are mutated in ApoE2 and ApoE4.
Overall, Nishimura et al. determined that immunoprecipitation and protein resequencing by LC-MS/MS produces accurate serotyping from small serum samples (5 µL). The specific benefits of this method include that it uses commercially available reagents and does not require stable isotope-labeling or genome analysis. This method also allows clinicians to detect all heterozygous combinations, which is a novel advance in serotyping ApoE.3 The researchers propose that, based on the current sensitivity of MS technology, this method should also detect other mutant alleles, including those that are rare and/or not associated with Lys or Arg residues. Furthermore, the applications of this method include serotyping with blood remnants from clinical tests, the option to perform retrospective studies using preserved body fluids, and the potential to investigate the pathophysiological significance of the relationship between ApoE mutations and Lys/Arg residues.
1. Hauser, P.S., et al. (2011) “Apolipoprotein E: From lipid transport to neurobiology,” Progress in Lipid Research, 50 (pp. 62–74).
2. Nishimura, M., et al. (2014) “Human Apolipoprotein E Resequencing by Proteomic Analysis and Its Application to Serotyping,” PLOS ONE, 9(1) (p. e85356).
3. Wang, M., et al. (2012) “15N-labeled full-length apolipoprotein E4 as an internal standard for mass spectrometry quantification of apolipoprotein E isoforms,” Analytical Chemistry, 84 (pp. 8340–4).
Post Author: Melissa J. Mayer. Melissa is a freelance writer who specializes in science journalism. She possesses passion for and experience in the fields of proteomics, cellular/molecular biology, microbiology, biochemistry, and immunology. Melissa is also bilingual (Spanish) and holds a teaching certificate with a biology endorsement.