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An optimized procedure for on-tissue localized protein digestion and quantification using hydrogel discs and isobaric mass tags: analysis of cardiac myxoma

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Abstract

An optimized workflow for multiplexed and spatially localized on-tissue quantitative protein analysis is here presented. The method is based on the use of an enzyme delivery platform, a polymeric hydrogel disc, allowing for a localized digestion directly onto the tissue surface coupled with an isobaric mass tag strategy for peptide labeling and relative quantification. The digestion occurs within such hydrogels, followed by peptide solvent extraction and identification by liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-MS/MS). Since this is a histology-directed on-tissue analysis, multiple hydrogels were placed onto morphologically and spatially different regions of interest (ROIs) within the tissue surface, e.g., cardiac myxoma tumor vascularized region and the adjacent hypocellular area. After a microwave digestion step (2 min), enzymatically cleaved peptides were labeled using TMT reagents with isobaric mass tags, enabling analysis of multiple samples per experiment. Thus, N = 8 hydrogel-digested samples from cardiac myxoma serial tissue sections (N = 4 from the vascularized ROIs and N = 4 from the adjacent hypocellular areas) were processed and then combined before a single LC-MS/MS analysis. Regulated proteins from both cardiac myxoma regions were assayed in a single experiment.

The workflow for histology-guided on-tissue localized protein digestion followed by isobaric mass tagging and LC-MS/MS analysis for proteins quantification is here summarized

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Acknowledgements

Funds from MIUR, Programma Operativo Nazionale, iCARE project, grant no. ICARE PON03PE_0009_2 and Prof. A. Procopio, Department of Health Sciences, University of Magna Graecia, Catanzaro, Italy, for the technical support on the microwave reaction are gratefully acknowledged.

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Authors and Affiliations

  1. Research Center for Advanced Biochemistry and Molecular Biology, Department of Experimental and Clinical Medicine, Magna Graecia University of Catanzaro, Campus “S. Venuta”, Viale Europa, Loc. Germaneto, 88100, Catanzaro, Italy

    Domenico Taverna, Caterina Gabriele, Giovanni Cuda & Marco Gaspari

  2. Department of Health Science, Magna Graecia University of Catanzaro, Viale Europa, 88100, Catanzaro, Italy

    Chiara Mignogna & Giuseppe Donato

  3. Cardiothoracic Surgery Unit, Sant’Anna Hospital, Via Pio X, 111, 88100, Catanzaro, Italy

    Gianluca Santise

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  1. Domenico Taverna
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Correspondence to Domenico Taverna.

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The study was approved by the Internal Ethic Committee of Sant’Anna Hospital, Catanzaro, Italy.

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Taverna, D., Mignogna, C., Gabriele, C. et al. An optimized procedure for on-tissue localized protein digestion and quantification using hydrogel discs and isobaric mass tags: analysis of cardiac myxoma. Anal Bioanal Chem 409, 2919–2930 (2017). https://doi.org/10.1007/s00216-017-0237-6

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  • DOI: https://doi.org/10.1007/s00216-017-0237-6

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