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Proteome-wide drug screening using mass spectrometric imaging of bead-arrays.

ABSTRACT: A fundamental challenge in the drug discovery process is to develop compounds with high efficacy and minimal side-effects. We describe a new approach to proteome-wide drug screening for detection of on- and off-target binding which combines the advantages of mass spectrometry with microarray technology. The method involves matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) of agarose micro-beads randomly arrayed at high-density in custom micro-well plates. Each bead carries a unique protein target and a corresponding photocleavable mass-tag for coding (PC-Mass-Tag). Compounds bound to specific protein beads and a photo-released coding PC-Mass-Tag are detected simultaneously using MALDI-MSI. As an initial demonstration of this approach, two kinase-targeted drugs, Dasatinib and Brigatinib (AP26113), were simultaneously screened against a model 50-member kinase-bead library. A MALDI-MSI scan performed at the equivalent density of 495,000 beads in the footprint of a microscope slide yielded 100% sensitivity for detecting known strong interactions with no false positives.

SUBMITTER: Zhou Y

PROVIDER: S-EPMC4872124 | biostudies-other | 2016 May

REPOSITORIES: biostudies-other

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Proteome-wide drug screening using mass spectrometric imaging of bead-arrays.

Zhou Ying Y Liu Ziying Z Rothschild Kenneth J KJ Lim Mark J MJ

Scientific reports 20160519

A fundamental challenge in the drug discovery process is to develop compounds with high efficacy and minimal side-effects. We describe a new approach to proteome-wide drug screening for detection of on- and off-target binding which combines the advantages of mass spectrometry with microarray technology. The method involves matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) of agarose micro-beads randomly arrayed at high-density in custom micro-well plates. Each be ...[more]

PMID: 27194112

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Project description:Background:Autoantibodies are produced when tolerance to self-antigens is broken and they can be mediators of tissue injury and systemic inflammation. They are excellent biomarkers because they are minimally invasive to screen and are highly abundant in serum due to limited proteolysis and slow clearance. Conventionally used methods of identifying autoantibodies in patient sera include indirect immunofluorescence, enzyme-linked immunoabsorbent assays (ELISAs) and protein microarrays. Here we present a novel proteome-wide immuno-mass spectrometric method to identify serum autoantibody targets. Methods:Serum samples from patients with inflammatory bowel disease (IBD) were analyzed by ELISA for the presence of autoantibodies to CUB and zona pellucida-like domain-containing protein 1 (CUZD1). Protein was extracted from the human pancreas as well as 16 other human tissues to make a complex tissue lysate protein mixture. Antibodies in patient sera were immobilized and purified on protein G magnetic beads and subsequently incubated with pancreatic lysate containing CUZD1 or the aforementioned complex tissue lysate. After extensive washing, antibody-bound protein antigens were trypsin-digested and identified using shotgun mass spectrometry. Results:The protocol was optimized for the immunoaffinity purification of autoantibody targets from tissue lysate, using CUZD1 from pancreatic lysate and anti-CUZD1 autoantibodies present in IBD patient serum as a proof-of-concept. Pancreatic secretory granule membrane major glycoprotein 2, whose autoantibodies are a known biomarker of Crohn's disease, was also immunoprecipitated from IBD patient serum, as an additional internal positive control. Conclusions:This study demonstrates the effectiveness of a proteomic approach to identify serum autoantibody targets, using immunoaffinity purification followed by tandem mass spectrometry. Our methodology is applicable for proteome-wide analysis of autoantibody targets in a wide variety of clinical settings.

Project description:Mapping of proteins involved in normal eye functions is a prerequisite to identify pathological changes during eye disease processes. We therefore analysed the proteome of human vitreous by applying in-depth proteomic screening technologies. For ethical reasons human vitreous samples were obtained by vitrectomy from "surrogate normal patients" with epiretinal gliosis that is considered to constitute only negligible pathological vitreoretinal changes. We applied different protein prefractionation strategies including liquid phase isoelectric focussing, 1D SDS gel electrophoresis and a combination of both and compared the number of identified proteins obtained by the respective method. Liquid phase isoelectric focussing followed by SDS gel electrophoresis increased the number of identified proteins by a factor of five compared to the analysis of crude unseparated human vitreous. Depending on the prefractionation method proteins were subjected to trypsin digestion either in-gel or in solution and the resulting peptides were analysed on a UPLC system coupled online to an LTQ Orbitrap XL mass spectrometer. The obtained mass spectra were searched against the SwissProt database using the Mascot search engine. Bioinformatics tools were used to annotate known biological functions to the detected proteins. Following this strategy we examined the vitreous proteomes of three individuals and identified 1111 unique proteins. Besides structural, transport and binding proteins, we detected 261 proteins with known enzymatic activity, 51 proteases, 35 protease inhibitors, 35 members of complement and coagulation cascades, 15 peptide hormones, 5 growth factors, 11 cytokines, 47 receptors, 30 proteins of visual perception, 91 proteins involved in apoptosis regulation and 265 proteins with signalling activity. This highly complex mixture strikingly differs from the human plasma proteome. Thus human vitreous fluid seems to be a unique body fluid. 262 unique proteins were detected which are present in all three patient samples indicating that these might represent the constitutive protein pattern of human vitreous. The presented catalogue of human vitreous proteins will enhance our understanding of physiological processes in the eye and provides the groundwork for future studies on pathological vitreous proteome changes.

Dataset Information

Proteome-wide drug screening using mass spectrometric imaging of bead-arrays.

Publications

Proteome-wide drug screening using mass spectrometric imaging of bead-arrays.

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