title: An optimized procedure for on-tissue localized protein digestion and quantification using hydrogel discs and isobaric mass tags: analysis of cardiac myxoma.
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. Graphical abstract 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.
authors.affiliation: 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. domenicotaverna@unicz.it.
authors.forename: Domenico
authors.initials: D
authors.lastname: Taverna
authors.affiliation: Department of Health Science, Magna Graecia University of Catanzaro, Viale Europa, 88100, Catanzaro, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Mignogna
authors.affiliation: 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.
authors.forename: Caterina
authors.initials: C
authors.lastname: Gabriele
authors.affiliation: Cardiothoracic Surgery Unit, Sant'Anna Hospital, Via Pio X, 111, 88100, Catanzaro, Italy.
authors.forename: Gianluca
authors.initials: G
authors.lastname: Santise
authors.affiliation: Department of Health Science, Magna Graecia University of Catanzaro, Viale Europa, 88100, Catanzaro, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Donato
authors.affiliation: 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.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Cuda
authors.affiliation: 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.
authors.forename: Marco
authors.initials: M
authors.lastname: Gaspari
mesh_terms: D014408:Biomarkers, Tumor / Q000032:analysis*; D002853:Chromatography, Liquid / Q000379:methods; D005260:Female; D006338:Heart Neoplasms / Q000737:chemistry* / Q000175:diagnosis; D006801:Humans; D020100:Hydrogels / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D008875:Middle Aged; D009232:Myxoma / Q000737:chemistry* / Q000175:diagnosis; D009363:Neoplasm Proteins / Q000032:analysis*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods; D046888:Tissue Array Analysis / Q000379:methods*
chemical_list: D014408:Biomarkers, Tumor; D020100:Hydrogels; D009363:Neoplasm Proteins
keywords: Histology-directed; Hydrogel; Isobaric tags; Myxoma; Quantitative proteomics; TMT