Proteomic workflows for deep phenotypic profiling of 3D organotypic liver models

Author:

Koutsilieri Stefania1,Mickols Evgeniya12,Végvári Ákos3,Lauschke Volker M.145ORCID

Affiliation:

1. Department of Physiology and Pharmacology Karolinska Institutet Stockholm Sweden

2. Department of Pharmacy Uppsala University Uppsala Sweden

3. Division of Chemistry I Department of Medical Biochemistry and Biophysics Karolinska Institutet Stockholm Sweden

4. Dr Margarete Fischer‐Bosch Institute of Clinical Pharmacology Stuttgart Germany

5. University of Tübingen Tübingen Germany

Abstract

AbstractOrganotypic human tissue models constitute promising systems to facilitate drug discovery and development. They allow to maintain native cellular phenotypes and functions, which enables long‐term pharmacokinetic and toxicity studies, as well as phenotypic screening. To trace relevant phenotypic changes back to specific targets or signaling pathways, comprehensive proteomic profiling is the gold‐standard. A multitude of proteomic workflows have been applied on 3D tissue models to quantify their molecular phenotypes; however, their impact on analytical results and biological conclusions in this context has not been evaluated. The performance of twelve mass spectrometry‐based global proteomic workflows that differed in the amount of cellular input, lysis protocols and quantification methods was compared for the analysis of primary human liver spheroids. Results differed majorly between protocols in the total number and subcellular compartment bias of identified proteins, which is particularly relevant for the reliable quantification of transporters and drug metabolizing enzymes. Using a model of metabolic dysfunction‐associated steatotic liver disease, we furthermore show that critical disease pathways are robustly identified using a standardized high throughput‐compatible workflow based on thermal lysis, even using only individual spheroids (1500 cells) as input. The results increase the applicability of proteomic profiling to phenotypic screens in organotypic microtissues and provide a scalable platform for deep phenotyping from limited biological material.

Funder

Vetenskapsrådet

Knut och Alice Wallenbergs Stiftelse

Innovative Medicines Initiative

Publisher

Wiley

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