Mass Spectrometric Immunoassay of Apolipoproteins

DESCRIPTION (provided by applicant): Proposed in this Fast Track Phase I/Phase II proposal is the development of mass spectrometric immunoassays (MSIA) targeting apolipoproteins in human blood. Assays, utilizing antibody-derivatized affinity pipettes and MALDI-TOF mass spectrometry, will be developed for Apo-A, -C, -D, -E, -H and -J. These assays will have general use in studies investigating the quantitative modulation of, and point mutations and posttranslational modifications present in, the apolipoproteins. Phase I research will address the Specific Aim of optimizing affinity pipette chemistries for apolipoproteins. By reaching the Phase I milestone of high reproducibility, low non-specific binding qualitative assays (for the apolipoproteins when analyzed from small volumes (< 50 uL) of human blood), we will proceed into Phase II research. Phase II objectives include: 1) The use of enzymatically-active MALDI-TOF MS targets in conjunction with MSIA for structural characterization of the apolipoproteins, 2) The incorporation of quantitative methodologies into the assays, and 3) The development of a MSIA assay for common ApoE phenotypes. The final objective of the program is the development of kits and low-cost analytical platforms for the high-throughput analysis of apolipoproteins. Upon supply to the public, the approach will find initial applications in proteomic investigations linking protein variants with disease and potential long-term applications as phenotyping assay for use in clinical/diagnostic or drug administration applications.