MS-based ligand binding assays with speed, sensitivity, and specificity

Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX, USA.
Proteomics (Impact Factor: 3.81). 11/2012; 12(21). DOI: 10.1002/pmic.201200151
Source: PubMed


Immunoassays are widely used in biochemical/clinical laboratories owing to their simplicity, speed, and sensitivity. We combined self-assembled monolayer-based immunoassays with MALDI-TOF MS to show that high-fidelity surface preparations with a novel matrix deposition/crystallization technique permits quantitative analysis of monolayer-bound antigens at picomolar detection limits. Calibration curves for intact proteins are possible over a broad concentration range and improved specificity of MS-immunoassays is highlighted by simultaneous label-free quantitation of ligand-bound protein complexes.

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    ABSTRACT: Immunoassays are employed in academia and the healthcare and biotech industries for high-throughput, quantitative screens of biomolecules. We have developed monolayer-based immunoassays for MALDI-TOF MS. To improve parallelization, we adapted the workflow to photolithography-generated arrays. Our work shows Parylene-CTM coatings provide excellent "solvent pinning" for reagents and biofluids, enabling sensitive MS detection of immobilized components. With a unique MALDI-matrix crystallization technique we show routine interassay RSD <10% at picomolar concentrations and highlight platform compatibility for relative and label-free quantitation applications. Parylene-arrays provide high sample densities and promise screening throughputs exceeding 1000 samples/h with modern liquid-handlers and MALDI-TOF systems.
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