title: Nanopatterned Extracellular Matrices Enable Cell-Based Assays with a Mass Spectrometric Readout.
abstract: Cell-based assays are finding wider use in evaluating compounds in primary screens for drug development, yet it is still challenging to measure enzymatic activities as an end point in a cell-based assay. This paper reports a strategy that combines state-of-the-art cantilever free polymer pen lithography (PPL) with self-assembled monolayer laser desorption-ionization (SAMDI) mass spectrometry to guide cell localization and measure cellular enzymatic activities. Experiments are conducted with a 384 spot array, in which each spot is composed of ∼400 nanoarrays and each array has a 10 × 10 arrangement of 750 nm features that present extracellular matrix (ECM) proteins surrounded by an immobilized phosphopeptide. Cells attach to the individual nanoarrays, where they can be cultured and treated with small molecules, after which the media is removed and the cells are lysed. Phosphatase enzymes in the proximal lysate can then act on the immobilized phosphopeptide substrate to convert it to the dephosphorylated form. After the lysate is removed, the array is analyzed by SAMDI mass spectrometry to identify the extent of dephosphorylation and, therefore, the amount of enzyme activity in the cell. This novel approach of using nanopatterning to mediate cell adhesion and SAMDI to record enzyme activities in the proximal lysate will enable a broad range of cellular assays for applications in drug discovery and research not possible with conventional strategies.
authors.forename: Maria D
authors.initials: MD
authors.lastname: Cabezas
authors.forename: Chad A
authors.identifier: 0000-0002-6634-7627
authors.initials: CA
authors.lastname: Mirkin
authors.affiliation: Department of Cell and Molecular Biology, Feinberg School of Medicine , 303 East Chicago Avenue, Chicago, Illinois 60611, United States.
authors.forename: Milan
authors.identifier: 0000-0002-4964-796X
authors.initials: M
authors.lastname: Mrksich
mesh_terms: D004353:Drug Evaluation, Preclinical / Q000295:instrumentation / Q000379:methods*; D057075:Enzyme Assays / Q000295:instrumentation / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004867:Equipment Design; D016326:Extracellular Matrix Proteins / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D049329:Nanostructures / Q000737:chemistry*; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors; D046888:Tissue Array Analysis / Q000295:instrumentation / Q000379:methods*
chemical_list: D004791:Enzyme Inhibitors; D016326:Extracellular Matrix Proteins; D055767:Immobilized Proteins; D010744:Phosphoric Monoester Hydrolases
keywords: SAMDI mass spectrometry; Self-assembled monolayer; phosphatase inhibitor; polymer pen nanolithography