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Cellular Assays with a Molecular Endpoint Measured by SAMDI Mass Spectrometry.

ABSTRACT: Cell-based, high-throughput screening (HTS) assays are increasingly important tools used in drug discovery, but frequently rely on readouts of gene expression or phenotypic changes and require development of specialized, labeled reporters. Here a cell-based, label-free assay compatible with HTS is introduced that can report quantitatively on enzyme activities by measuring mass changes of substrates with matrix-assisted laser desorption/ionization mass spectrometry. The assay uses self-assembled monolayers to culture cells on arrays presenting substrates, which serve as reporters for a desired enzyme activity. Each spot of cells is treated with a compound, cultured and lysed, enabling endogenous enzymes to act on the immobilized peptide substrate. It is demonstrated that the assay can measure protein tyrosine phosphatase (PTP) activity from as few as five cells and a screen is described that identifies a compound that reduces PTP activity in cell lysates. This approach offers a valuable addition to the methods available for cell-based screening.

SUBMITTER: Berns EJ

PROVIDER: S-EPMC4981186 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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Cellular Assays with a Molecular Endpoint Measured by SAMDI Mass Spectrometry.

Berns Eric J EJ Cabezas Maria D MD Mrksich Milan M

Small (Weinheim an der Bergstrasse, Germany) 20160530 28

Cell-based, high-throughput screening (HTS) assays are increasingly important tools used in drug discovery, but frequently rely on readouts of gene expression or phenotypic changes and require development of specialized, labeled reporters. Here a cell-based, label-free assay compatible with HTS is introduced that can report quantitatively on enzyme activities by measuring mass changes of substrates with matrix-assisted laser desorption/ionization mass spectrometry. The assay uses self-assembled ...[more]

PMID: 27240220

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Project description:BackgroundAmyloid-β 1-42 (Aβ1-42) peptide is a well-established cerebrospinal fluid (CSF) biomarker for Alzheimer's disease (AD). Reduced levels of Aβ1-42 are indicative of AD, but significant variation in the absolute concentrations of this analyte has been described for both healthy and diseased populations. Preanalytical factors such as storage tube type are reported to impact Aβ recovery and quantification accuracy. Using complementary immunological and mass spectrometry-based approaches, we identified and characterized preanalytical factors that influence measured concentrations of CSF Aβ peptides in stored samples.MethodsCSF from healthy control subjects and patients with AD was aliquoted into polypropylene tubes at volumes of 0.1 ml and 0.5 ml. CSF Aβ1-42 concentrations were initially measured by immunoassay; subsequent determinations of CSF Aβ1-42, Aβ1-40, Aβ1-38, Aβ1-37, and Aβ1-34 concentrations were made with an absolute quantitative mass spectrometry assay. In a second study, CSF from healthy control subjects and patients with dementia was denatured with guanidine hydrochloride (GuHCl) at different stages of the CSF collection and aliquoting process and then measured with the mass spectrometry assay.ResultsTwo distinct immunoassays demonstrated that CSF Aβ1-42 concentrations measured from 0.5-ml aliquots were higher than those from 0.1-ml aliquots. Tween-20 surfactant supplementation increased Aβ1-42 recovery but did not effectively resolve measured concentration differences associated with aliquot size. A CSF Aβ peptide mass spectrometry assay confirmed that Aβ peptide recovery was linked to sample volume. Unlike the immunoassay experiments, measured differences were consistently eliminated when aliquots were denatured in the original sample tube. Recovery from a panel of low-retention polypropylene tubes was assessed, and 1.5-ml Eppendorf LoBind® tubes were determined to be the least absorptive for Aβ1-42. A comparison of CSF collection and processing methods suggested that Aβ peptide recovery was improved by denaturing CSF earlier in the collection/aliquoting process and that the Aβ1-42/Aβ1-40 ratio was a useful method to reduce variability.ConclusionsAnalyte loss due to nonspecific sample tube adsorption is a significant preanalytical factor that can compromise the accuracy of CSF Aβ1-42 measurements. Sample denaturation during aliquoting increases recovery of Aβ peptides and improves measurement accuracy. The Aβ1-42/Aβ1-40 ratio can overcome some of the quantitative variability precipitated by preanalytical factors affecting recovery.

Dataset Information

Cellular Assays with a Molecular Endpoint Measured by SAMDI Mass Spectrometry.

Publications

Cellular Assays with a Molecular Endpoint Measured by SAMDI Mass Spectrometry.

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