The main objective of this Phase I project is to develop quantitative Mass Spectrometric Immunoassays for beta-2-microglobulin, cystatin C, transferrin, transthyretin, and retinol binding protein. These five proteins were recently assayed via qualitative mass spectrometric immunoassays in 1,000 healthy cohorts, detecting (without providing concentration information) numerous protein variant. Because sandwich-based immunoassays such as ELISAs cannot specifically quantify the subtle protein modifications, in this project we propose to develop quantitative mass spectrometric immunoassays for these proteins and their isoforms. An internal reference standard will be carefully chosen, and a step-by-step standard curve quantitative mass spectrometric immunoassay development will be undertaken. The precision (intra-assay and inter-assay), linearity, sensitivity, and specificity of the quantitative mass spectrometric immunoassays will be determined. As a final validation step, the quantitative MSIA assays will be evaluated and benchmarked against existing ELISA assays. The quantitative mass spectrometric immunoassays proposed here will offer unique and straightforward means of quantifying protein variants, and when applied to disease cohorts, can provide valuable insight into the role of these variants in the onset of the disease, progression, and response to therapy.
Public Health Relevance: The development of quantitative Mass Spectrometric Immunoassays for beta-2- microglobulin, cystatin C, transferrin, transthyretin, and retinol binding protein will enable routine quantitative monitoring of these proteins and the changes in their modifications patterns that might be relevant to disease. With the new assays in hand, we will be better equipped to study the effects of these proteins' modifications in pathological processes and evaluate their potential as new indicators of the onset and progression of diseases, and response to therapy.
Public Health Relevance Statement: Project Narrative The development of quantitative Mass Spectrometric Immunoassays for beta-2- microglobulin, cystatin C, transferrin, transthyretin, and retinol binding protein will enable routine quantitative monitoring of these proteins and the changes in their modifications patterns that might be relevant to disease. With the new assays in hand, we will be better equipped to study the effects of these proteins' modifications in pathological processes and evaluate their potential as new indicators of the onset and progression of diseases, and response to therapy.
NIH Spending Category: Biotechnology
Project Terms: Animals; Antibodies; Assay; Benchmarking; Best Practice Analysis; Bioassay; Biologic Assays; Biological Assay; Blood Plasma; Characteristics; Cohort Studies; Collection; Concurrent Studies; Data; Detection; Development; Discipline; Disease; Disease Progression; Disorder; ELISA; Enzyme-Linked Immunosorbent Assay; Exhibits; Frequencies (time pattern); Frequency; General Population; General Public; Goals; Grant; Granulocyte/Pollen-Binding Protein; Hand; Human; Human, General; Immunoassay; Individual; Investigation; Isoforms; Man (Taxonomy); Man, Modern; Modification; Monitor; Non-Human Protein; Pathologic Processes; Pathological Processes; Pattern; Phase; Plasma; Plasma Proteins; Population; Post-Translational Modifications; Post-Translational Protein Processing; Posttranslational Modifications; Prealbumin; Proalbumin; Protein Isoforms; Protein Modification; Protein Modification, Post-Translational; Protein Processing, Post-Translational; Protein Processing, Posttranslational; Protein/Amino Acid Biochemistry, Post-Translational Modification; Proteins; Proteomics; Reference Standards; Reticuloendothelial System, Serum, Plasma; Retinoid Binding Proteins; Retinol Binding Proteins; Retinol-Binding Protein 1; Role; Sampling; Sensitivity and Specificity; Serum, Plasma; Siderophilin; Specificity; Structural Protein; Thymotaxin; Time; Transferrin; Transthyretin; Validation; Variant; Variation; assay development; base; beta-2 Microglobulin; cohort; cross reactivity; cystatin C; disease diagnosis; disease/disorder; gene product; insight; post gamma-globulins; post-gamma-protein; public health relevance; response; social role
