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

Abstract

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.

Robustness and Ruggedness of Isoelectric Focusing and Superficially Porous Liquid Chromatography with Fourier Transform Mass Spectrometry

“…Plus, while the observed linear dynamic range is in general agreement with the reported ∼4000 dynamic range of our instrument; it is important to note that, under max injection time conditions, the intensity data is extrapolated from known equations in order to arrive at quantitative trends similar to that of a triple quadrupole mass spectrometer. 54,34 Overall, the work highlights baseline figures of merits associated with the IEF-SPLC-FTMS workflow that could be improved upon in diverse ways. For example, future investigations should continue to explore protein pI, size, amount, and PTM pK a impacts on the detection of distinct modification clases (e.g., citrullination which are early indicators of neurodegenerative diseases 55−57 ).…”

“…Before axis normalization ∼95% of observed proteoforms exhibited an inter-replicate RT precision from the mean of ±30 s. This reduced to ±12 s after normalization of the RT axis (Figure 3D), which is consistent with RT reproducibility previously shown for 1D SPLC-FTMS. 34 The observed w pI precision from the inter-replicate mean was ±0.33 prior to axis normalization, which reduced to ±0.21 after (Figure 3E). However, further evaluation revealed that the precision estimate was generally independent of species mass but dependent on intensity (Figure S4).…”

“…28−33 Superficially porous reversed-phase LC (SPLC) and FTMS also provides high peak capacity, large quantitative dynamic range, and good detection limits for intact proteins studies. 34 Zhang et al previously showed when SPLC-FTMS was integrated after a first dimension separation by solutionbased "offgel" IEF, the workflow effectively separated complex mixtures (e.g., heart tissues and cerebrospinal fluid (CSF)) by their isoelectric point (pI) at increments of ∼0.33 ΔpI/cm. 19 In comparison to other multidimensional platforms that utilized other pI chromatography strategies, the offgel approach provides advantages in improved pI resolution/ separation, liquid phase recovery, and low influence of offgel reagents on downstream orthogonal chromatographic separation.…”

Surface Preparation Strategies for Improved Parallelization and Reproducible MALDI-TOF MS Ligand Binding Assays

“…TLT/VIC was performed as previously described. 10,11 MALDI matrix, α-cyano-4hydroxycinnamic acid (CHCA), was sublimed at ∼25 ng/mm 2 surface density and VIC performed with methanol. Samples were analyzed with a Voyager DE-Pro MALDI-TOF MS (Applied Biosystems, Inc., Foster City, CA).…”

“…The combination of controlled analyte density on the surface with homogeneous matrix/analyte cocrystallization by TLT/VIC enabled assay precisions similar to those observed for photometric immunoassays and supports label-free quantitation via end-point calibration curves. 10 The previous work also demonstrated quantitative detection of analyte from complex matrices including biofluids. Additionally, combining enrichment of target analyte with antibodies and MALDI-TOF yields key molecular mass information, providing a high degree of assay selectivity for resolving related or complexed species.…”