Valentina Golino, Antonio Malincolinco, Vicky Caponigro, Eduardo Sommella, Pietro Campiglia

Department of Pharmacy, University of Salerno, Via Giovanni Paolo II 132, 84084, Fisciano (SA) Italy

Patient-Derived Organoids (PDOs) closely replicate in vivo epithelial homeostasis, making them valuable in vitro models for studying diseases such as ColoRectal Cancer (CRC). CRC-derived organoids can be generated from patient biopsies, allowing for the study of tumor evolution, heterogeneity, drug testing, and personalized treatments [1]. In this study, proteomic variations in CRC-PDOs were analyzed using label-free quantitative (LFQ) proteomics with Data-Dependent Acquisition (DDA) and Data-Independent Acquisition (DIA) strategies [2], employing nano Liquid Chromatography coupled with High-Resolution Mass Spectrometry (nLC-HRMS). A bottom-up proteomic approach was used, digesting proteins into peptides for analysis by mass spectrometry. Sample preparation included in-solution digestion (ISD) with disulfide bond reduction, thiol alkylation, and trypsin digestion. Data were processed using Proteome Discoverer 2.5.0.400 for DDA and DIA-NN 1.9 for DIA. 

Univariate analysis was performed to identify proteins exhibiting statistically significant changes in expression in response to drug treatments, evaluated at two distinct concentrations. Multivariate analysis, including Principal Component Analysis (PCA), revealed how drug type and concentration influenced protein expression profiles, contributing to the variance in the dataset [3, 4]. Enrichment analysis of the significant proteins highlighted the molecular pathways impacted by the treatments, providing insights into the drug action mechanisms.

Acknowledgements: This work was supported by project PIR01_00032 ¬ BIO OPEN LAB ¬ BOL “CUP” J37E19000050007, project CIR01_00032—BOL “BIO Open Lab—Rafforzamento del capitale umano” and project PNC0000001 D34 Health—Digital Driven Diagnostics, prognostics and therapeutics for sustainable Health care “CUP” B53C22006090001. PNRR CN3, National Centre for Gene Therapy and Drugs Based on RNA Technology.

Keywords: Proteomics, Omics, LC-MS, Chemometrics.

References

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2. Krasny L., Huang PH. Mol Omics. 17 (2021), pag. 29–42. DOI: 10.1039/D0MO00088A 

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4. Bro R., Smilde A.K., Anal Methods. 6 (2014), pag. 2812–31. DOI: 10.1039/C3AY41907J