DiaPASEF proteotype analysis indicates changes in cell growth and metabolic switch induced by caspase-9 inhibition in chondrogenic cells

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Authors

LAPČÍK Petr VESELÁ Barbora POTĚŠIL David DADÁKOVÁ Kateřina ZAPLETALOVÁ Martina BENEŠ Petr BOUCHAL Pavel MATALOVÁ Eva

Year of publication 2023
Type Article in Periodical
Magazine / Source Proteomics
MU Faculty or unit

Faculty of Science

Citation
Web https://doi.org/10.1002/pmic.202200408
Doi http://dx.doi.org/10.1002/pmic.202200408
Keywords caspase-9; chondrogenesis; diaPASEF; micromass cultures; proteomics
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Description Caspase-9 is the major apical caspase responsible for triggering the intrinsic apoptotic pathway. Our previous study indicated that specific inhibition of caspase-9 caused microscopically evident alterations in appearance of the primary chondrogenic cultures which cannot be explained by decrease in apoptosis. To describe a complex molecular background of this effect, proteomics analysis of control and caspase-9 inhibitor-treated chondrogenic cultures were performed. Proteins were extracted, identified and quantified using LC-MS in both data dependent and data independent acquisition (DIA) mode. While directDIA analysis of diaPASEF data obtained using timsTOF Pro LC-MS system revealed 7849 protein groups (Q-value <0.01), a parallel analysis of iTRAQ-2DLC-MS3 and conventional DIA-MS data identified only 5146 and 4098 protein groups, respectively, showing diaPASEF a superior method for the study. The detailed analysis of diaPASEF data disclosed 236/551 significantly down-/up-regulated protein groups after caspase-9 inhibition, respectively (|log2FC|>0.58, Q value <0.05). Classification of downregulated proteins revealed changes in extracellular matrix organization, collagen metabolism, and muscle system processes. Moreover, deregulations suggest a switch from glycolytic to lipid based metabolism in the inhibited cells. No essential changes were found in the proteins involved in apoptosis. The data indicate new non-apoptotic participation of caspases in chondrocyte homeostasis with potential applications in cartilage pathophysiology.
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