Measurement of blood protease kinetic parameters with self-assembled monolayer ligand binding assays and label-free MALDI-TOF MS

Steven M. Patrie, Michael J. Roth, Daniel A. Plymire, Erica Maresh, Junmei Zhang

Research output: Contribution to journalArticle

6 Citations (Scopus)

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 LBAs 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 nonspecific adsorption of abundant proteins present at ∼100 000× the concentration of the endogenous enzyme. In the first protocol, in-solution digestion of the ADAMTS-13 substrate (vWFh) was performed with immunoaffinity 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 time, a ∼16× improvement over other MS-based LBA formats. Implementation of a vacuum-sublimed MALDI matrix provided good MALDI-TOF MS intra- 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.

Original languageEnglish (US)
Pages (from-to)10597-10604
Number of pages8
JournalAnalytical Chemistry
Volume85
Issue number21
DOIs
StatePublished - Nov 5 2013

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Self assembled monolayers
Kinetic parameters
Labels
Assays
Blood
Peptide Hydrolases
Ligands
Plasmas
Substrates
Enzymes
Liquid chromatography
Metalloproteases
Biotechnology
Surface chemistry
Vacuum
Adsorption
Kinetics
Proteins

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Measurement of blood protease kinetic parameters with self-assembled monolayer ligand binding assays and label-free MALDI-TOF MS. / Patrie, Steven M.; Roth, Michael J.; Plymire, Daniel A.; Maresh, Erica; Zhang, Junmei.

In: Analytical Chemistry, Vol. 85, No. 21, 05.11.2013, p. 10597-10604.

Research output: Contribution to journalArticle

Patrie, Steven M. ; Roth, Michael J. ; Plymire, Daniel A. ; Maresh, Erica ; Zhang, Junmei. / Measurement of blood protease kinetic parameters with self-assembled monolayer ligand binding assays and label-free MALDI-TOF MS. In: Analytical Chemistry. 2013 ; Vol. 85, No. 21. pp. 10597-10604.
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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 LBAs 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 nonspecific adsorption of abundant proteins present at ∼100 000× the concentration of the endogenous enzyme. In the first protocol, in-solution digestion of the ADAMTS-13 substrate (vWFh) was performed with immunoaffinity 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 time, a ∼16× improvement over other MS-based LBA formats. Implementation of a vacuum-sublimed MALDI matrix provided good MALDI-TOF MS intra- 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.",
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