Human apolipoprotein e resequencing by proteomic analysis and its application to serotyping

Area of Science:

• Proteomics
• Clinical Diagnostics
• Biochemistry

Background:

• Apolipoprotein E (ApoE) typing is crucial for understanding associations with Alzheimer's disease and familial dyslipidemia.
• Current ApoE typing relies on genetic testing (APOE genotyping).
• ApoE protein levels are clinically measured using immunoassays.

Purpose of the Study:

• To develop and validate a novel ApoE serotyping method using mass spectrometry.
• To enable ApoE typing directly from serum protein sequences, bypassing the need for DNA analysis.

Main Methods:

• Combined ApoE immunoassay with Orbitrap mass spectrometry for proteomic analysis.
• Performed protein resequencing on trace amounts of serum to determine ApoE types.
• Analyzed tryptic fragments of ApoE, leveraging mass differences in mutant proteins (e.g., ApoE2, ApoE4).

Main Results:

• Achieved 91.6% mean coverage of the full-length ApoE amino acid sequence.
• Successfully covered key residues (112 and 158) for accurate serotyping.
• Demonstrated exact correspondence between serotyping results and APOE genotyping for all tested combinations, including heterozygotes.

Conclusions:

• The novel ApoE serotyping method requires no stable isotope-labeled peptides or genome analysis.
• This technique can utilize residual blood from routine clinical samples, facilitating retrospective studies.
• The method is applicable to subjects lacking DNA and holds potential for detecting rare ApoE mutations.