Human apolipoprotein e resequencing by proteomic analysis and its application to serotyping.
BACKGROUND
Apolipoprotein E (ApoE) typing is considered important because of the association between ApoE and Alzheimer's disease and familial dyslipidemia and is currently performed by genetic testing (APOE genotyping). ApoE levels in plasma and serum are clinically determined by immunoassay.


METHODS
Combining an ApoE immunoassay reagent with proteomic analysis using an Orbitrap mass spectrometer, we attempted to resequence ApoE from trace amounts of serum for typing (serotyping). Most (24 of 33) ApoE mutant proteins registered to date with Online Mendelian Inheritance in Man, such as ApoE2 and ApoE4, involve lysine and arginine mutations. Digestion of mutant ApoE with trypsin will thus result in fragments that differ substantially from wild-type ApoE3 in terms of mass, making serotyping ideally suited to mass spectrometry analysis.


RESULTS
The mean coverage of the amino acid sequence of full-length ApoE was 91.6% in the protein resequence. Residues 112 and 158 (which are mutated in ApoE2 and ApoE4) were covered in all samples, and the protein sequences were used for serotyping. Serotypes including all heterozygous combinations (ApoE2/E3, E2/E4, E3/E4) corresponded exactly to the APOE genotyping results in each of the subjects.


CONCLUSIONS
Our novel ApoE serotyping method with protein resequencing requires no synthesis of stable isotope-labeled peptides or genome analysis. The method can use residual blood from samples collected for routine clinical tests, thus enabling retrospective studies with preserved body fluids. The test could be applied to samples from subjects whose DNA is unavailable. In future studies, we hope to demonstrate the capability of our method to detect rare ApoE mutations.
Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University, Chiba, Japan ; Division of Laboratory Medicine and Clinical Genetics, Chiba University Hospital, Chiba, Japan ; Clinical Proteomics Research Center, Chiba University Hospital, Chiba, Japan.
Motoi
M
Nishimura
Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University, Chiba, Japan ; Clinical Proteomics Research Center, Chiba University Hospital, Chiba, Japan.
Mamoru
M
Satoh
Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University, Chiba, Japan.
Satomi
S
Nishimura
Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University, Chiba, Japan.
Shoko
S
Kakinuma
Division of Laboratory Medicine and Clinical Genetics, Chiba University Hospital, Chiba, Japan.
Kenichi
K
Sato
Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University, Chiba, Japan ; Division of Laboratory Medicine and Clinical Genetics, Chiba University Hospital, Chiba, Japan.
Setsu
S
Sawai
Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University, Chiba, Japan.
Sachio
S
Tsuchida
Division of Laboratory Medicine and Clinical Genetics, Chiba University Hospital, Chiba, Japan.
Takeshi
T
Kazama
Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University, Chiba, Japan ; Division of Laboratory Medicine and Clinical Genetics, Chiba University Hospital, Chiba, Japan ; Clinical Proteomics Research Center, Chiba University Hospital, Chiba, Japan.
Kazuyuki
K
Matsushita
Chemical Analysis Center, Chiba University, Chiba, Japan.
Sayaka
S
Kado
Clinical Proteomics Research Center, Chiba University Hospital, Chiba, Japan ; Laboratory of Biomolecular Dynamics, Department of Physics, School of Science, Kitasato University, Sagamihara, Japan.
Yoshio
Y
Kodera
Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University, Chiba, Japan ; Division of Laboratory Medicine and Clinical Genetics, Chiba University Hospital, Chiba, Japan ; Clinical Proteomics Research Center, Chiba University Hospital, Chiba, Japan.
Fumio
F
Nomura
D000595:Amino Acid Sequence; D001057:Apolipoproteins E / Q000097:blood* / Q000737:chemistry*; D006801:Humans; D007118:Immunoassay; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D040901:Proteomics; D017421:Sequence Analysis; D012703:Serotyping
D001057:Apolipoproteins E