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.

4 Reads
  • [Show abstract] [Hide abstract]
    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.
    No preview · Article · Dec 2012 · ACS Applied Materials & Interfaces
  • [Show abstract] [Hide abstract]
    ABSTRACT: We report novel ligand binding assay (LBA) surface modalities that permit plasma protease catalytic efficiency (kcat/km) determination by MALDI-TOF MS without the use of liquid chromatography or internal standards such as chemical- or metalized-labels. Two model LBA were constructed on planar self-assembled monolayers (SAMs) and used to evaluate the clinically-relevant metalloprotease ADAMTS-13 kinetics in plasma. The SAM chemistries were designed to improve biosampling efficiency by minimization of non-specific adsorption of abundant proteins present at ~100,000× the concentration of the endogenous enzyme. In the first protocol, the in-solution digestion of the ADAMTS-13 substrate (vWF) was performed with immuno-affinity enrichment of the reaction substrate and product to SAM-arrays. The second configuration examined protease kcat/km via a surface digestion modality where different substrates were covalently immobilized to the SAM at controlled surface density for optimized protease screens. The results show the MALDI-TOF MS LBA platforms provide limits of quantitation to ~1% protease activity (~60 pM enzyme concentration) in <1 h analysis times, an ~16× improvement over other MS-based LBA formats. Implementation of vacuum sublimed MALDI-matrix provided good MALDI-TOF MS intraday and interday repeatability, ~1.2% and ~6.6% RSD, respectively. Platform reliability permitted kcat/km determination without internal standards with observed values ~10× improved versus conventional fluorophoric assays. Application of the assays to 12 clinical plasma samples demonstrated proof of concept for clinical applications. Overall, this work demonstrates that rationally-designed surface chemistries for MALDI-TOF MS may serve as an alternative, label-free methodology with potential for a wide range of biotechnology applications related to targeted enzyme molecular diagnostics.
    No preview · Article · Oct 2013 · Analytical Chemistry
  • [Show abstract] [Hide abstract]
    ABSTRACT: Porcine liver carboxylesterase was captured using an immunoaffinity membrane, which was prepared by separating an anti-porcine esterase antibody using non-denaturing two-dimensional electrophoresis, followed by transfer to a polyvinylidene difluoride membrane and staining. The activity of this esterase was 0.008 units after it was captured in the tiny spaces (4 mm(2)) of this membrane and eluted by rinsing with 5 μL of aspartic acid solution. The molecular mass of the eluted esterase was m/z 61,885 according to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry after the purification of this enzyme from the porcine liver cytosol. The purified enzyme's activity was inhibited by 6,9-diamino-2-ethoxyacridine, and this inhibition was retained even after extracting the enzyme from the immunoaffinity membrane. These results indicate that micro-scale extraction and analysis of a carboxylesterase are possible when the enzyme is trapped using an immunoaffinity membrane and eluted with aspartic acid.
    No preview · Article · Mar 2014 · Applied biochemistry and biotechnology