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

Abstract

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-/upregulated 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.

Caspase-9 Is a Positive Regulator of Osteoblastic Cell Migration Identified by diaPASEF Proteomics

“…56 diaPASEF provides sensitive peptide detection and data completeness 56 and we have previously shown this strategy to achieve superior proteome coverage in chondrogenic micromass cultures compared to other commonly used LC-MS/MS-based proteomics workflows. 47 In the current study, Casp9 KO significantly affected, namely, proteins associated with cell migration/motility and DNA replication.…”

“…To map the changes in protein abundances associated with CASP-9 depletion, we used the diaPASEF approach that combines peptide separation using trapped ion mobility spectrometry and precursor m / z window cycling . diaPASEF provides sensitive peptide detection and data completeness and we have previously shown this strategy to achieve superior proteome coverage in chondrogenic micromass cultures compared to other commonly used LC-MS/MS-based proteomics workflows . In the current study, Casp9 KO significantly affected, namely, proteins associated with cell migration/motility and DNA replication.…”

“…After a literature search and screening of available databases, , these proteins have not been identified as CASP-9 substrates. Interestingly, four of these proteins (ADAM15, fibulin-1, BST-2, and ABHD2) were found previously to be upregulated in micromass cultures treated with CASP-9 inhibitor by proteomic screen . Therefore, we further focused our attention on these four proteins.…”

“…Consistent with this hypothesis, a proteomic screen of chondrogenic micromass cultures revealed higher ADAM15 levels after 7 days of treatment with CASP-9 inhibitor. 47 In MC3T3-E1 cells, however, prolonged treatment with CASP-9 inhibitor significantly reduces their viability, making longer exposure time intervals difficult to achieve. We thus hypothesize that ADAM15 may not be a direct target of CASP-9 but may rather be deregulated or cleaved by other proteases in response to longterm CASP-9 inhibition.…”

“…Interestingly, four of these proteins (ADAM15, fibulin-1, BST-2, and ABHD2) were found previously to be upregulated in micromass cultures treated with CASP-9 inhibitor by proteomic screen. 47 Therefore, we further focused our attention on these four proteins. BST-2 was not detected by immunoblotting (Supplementary Data 12) and fibulin-1 has been recently identified as a substrate of CASP-3, 50 a downstream molecule of CASP-9 in proteolytic cascade, so these two proteins were excluded from further analyses.…”