Dataset Information

Analysis of proteotype, cell growth and metabolic changes induced by caspase-9 inhibition in chondrogenic cells

ABSTRACT: Caspase-9 is the major apical caspase responsible for triggering the intrinsic apoptotic pathway. Our pilot 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. In order to search for the complex molecular background of the effect, proteomics analysis after caspase-9 inhibition was performed. A selective fluoromethylketone inhibitor was applied to inhibit caspase-9 in the chondrogenic cultures. In this PRIDE project we present three LC-MS datasets: i) diaPASEF data obtained using timsTOF Pro LC-MS system; ii) iTRAQ-2DLC-MS3 dataset; and iii) conventional LC-DIA-MS dataset, both measured on Orbitrap Lumos. In this project we demonstrate superiority of the diaPASEF method offering better proteome coverage.

INSTRUMENT(S): Orbitrap Fusion Lumos, timsTOF Pro

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Pavel Bouchal

LAB HEAD: Pavel Bouchal

PROVIDER: PXD034025 | Pride | 2023-04-03

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	3778_Lumos1_110IS_SPS-MS3-iTRAQ_02_1.raw	Raw
	3778_Lumos1_110IS_SPS-MS3-iTRAQ_14-17_1.raw	Raw
	3778_Lumos1_110IS_SPS-MS3-iTRAQ_18-25_1.raw	Raw
	3778_Lumos1_180IS_SPS-MS3-iTRAQ_03_1.raw	Raw
	3778_Lumos1_180IS_SPS-MS3-iTRAQ_04_1.raw	Raw

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Publications

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

Lapcik Petr P Vesela Barbora B Potesil David D Dadakova Katerina K Zapletalova Martina M Benes Petr P Bouchal Pavel P Matalova Eva E

Proteomics 20230407 11

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, iden ...[more]

PMID: 36960851

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Project description:Data independent acquisition (DIA or DIA/SWATH ) mass spectrometry has emerged as a primary measurement strategy in the field of quantitative proteomics. diaPASEF is a recent adaptation that leverages trapped ion mobility spectrometry (TIMS) to improve selectivity and increase sensitivity. The complex fragmentation spectra generated by co-isolation of peptides in DIA mode are most typically analyzed with reference to prior knowledge in the form of spectral libraries. The best established method for generating libraries uses data dependent acquisition (DDA) mode, or DIA mode if appropriately deconvoluted, often including offline fractionation to increase depth of coverage,to create spectral libraries. More recently strategies for spectral library generation based on gas phase fractionation (GPF), where a representative sample is injected serially using narrow window DIA methods designed to cover different slices of the precursor space, have been introduced and performed comparably to deep offline fractionation-based libraries for DIA data analysis. Here, we investigated whether an analogous GPF-based library building approach that accounts for the ion mobility (IM) dimension is useful for the analysis of diaPASEF data and can remove the need for offline fractionation. To enable a rapid library development approach for diaPASEF we designed a GPF acquisition scheme covering the majority of multiply charged precursors in the m/z vs 1/K0 space requiring 7 injections of a representative sample and compared this with libraries generated by direct deconvolution-based analysis of diaPASEF data or by deep offline fractionation and ddaPASEF. . We found that the GPF based library outperformed library generation by direct deconvolution of the diaPASEF data, and performed comparably to deep offline fractionation libraries, when analysing diaPASEF data acquired from 200ng of commercial HeLa digest. With the ion mobility integrated GPF scheme we establish a pragmatic approach to rapid and comprehensive library generation for the analysis of diaPASEF data.