CEEPC/IPM/CMSC - Abstrakt prezentace

(CEEPC/IPM/CMSC 2022 - FrP-16)
High-Coverage Proteome Analysis of Rabbit Heart Samples Using 2D Conventional-Flow LC-MS

Rudolf Kupcik 1 *, Marie Vajrychova 1, Olga Lencova 2, Martin Sterba 2

  1. Biomedical Research Centre, University Hospital Hradec Kralove, CZ
  2. Department of Pharmacology, Faculty of Medicine in Hradec Kralove, Charles University, CZ

Abstrakt

Introduction
Anthracyclines, such as daunorubicin (DAU), are effective class of chemotherapeutic agents, however they are responsible for cumulative dose-dependent cardiotoxicity. Global proteomic approaches can help provide insight into its development. Due to high dynamic range of tissue proteomes, 2D liquid chromatography (LC) coupled to mass spectrometry (MS) can significantly enhance proteome coverage. However, long-term analysis can be negatively affected by instability of electrospray during nano LC-MS. Here, we present an optimized 2D conventional-flow LC-MS (68 μL/min) with aim to show its efficacy in proteomic analysis of rabbit hearts compared to nano LC-MS.
Methods
Samples of rabbit myocardium with or without DAU treatment (3mg/kg, weekly, 10 doses) were homogenized and lysed. Proteins were digested to peptides and derivatized by TMT isobaric labels. 2D peptide separation was realized using high-pH C18 sample fractionation followed by separation of collected fractions using conventional-flow (68 μL/min) and nano LC-MS (250 nL/min).
Results
Optimized 2D conventional-flow LC-MS was able to quantify more than 4000 proteins in TMT-labeled myocardial samples. The results showed that severe anthracycline cardiotoxicity phenotype was associated with marked global protein dysregulation, that involved profound remodeling of cardiomyocyte sarcomere, structural proteins and extracellular matrix. Pro-inflammatory proteins, proteasome subunits and multiple antioxidant proteins were markedly up-regulated, whereas the levels of mitochondrial proteins were declined compared to control myocardium.
Conclusion
2D conventional-flow LC-MS analysis was able to get high proteome coverage and comprehensive mapping of proteome changes in rabbit heart samples.

* Korespondující autor: rudolf.kupcik@fnhk.cz

Poděkování:

This research was funded by the InoMed project (Reg. No. 659 CZ.02.1.01/0.0/0.0/18_069/0010046) co-funded by the European Union and the state budget. Supported by MH CZ - DRO (UHHK, 00179906).


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