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Comparative analysis of DDA- and DIA-PASEF methods for proteomics analysis of human valve interstitial cells and osteoblasts during osteogenic differentiation

ABSTRACT: Shotgun proteomics is one of the key "omics" methods, the methodology of which is rapidly developing. Mass spectrometers of the TimsToF series (Bruker) with ion mobility are one of the young technological platforms for shotgun proteomics in which both data dependent (DDA) and data independent acquisition (DIA) proteomics methods might be performed. However, only a few comparisons of the effectiveness of DDA and DIA proteomics on TimsToF have been published, carried out mainly on the test samples. From the other hand, peculiarities of osteogenic differentiation of human valve interstitial cells (VICs) are fruitful therapeutic target for calcific aortic valve disease (CAVD) treatment. Still, there is no data whether pathological osteogenic differentiation of VICs similar to normal ossification. Combining this technical and biological tasks we performed comparative proteomics analysis of osteogenic differentiation of human VICs and osteoblasts by DIA- and DDA-PASEF proteomics on TimsToF Pro.

INSTRUMENT(S): timsTOF Pro

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Valve Cell, Osteoblast

DISEASE(S): Aortic Valve Stenosis

SUBMITTER: Arseniy Lobov

LAB HEAD: Anna B. Malashicheva

PROVIDER: PXD036566 | Pride | 2024-11-12

REPOSITORIES: Pride

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Project description:The treatment of bone defects caused by infection, trauma or neoplasms remains a clinical challenge. Autologous bone transplantation is limited by availability, donor site morbidity and surgical risk factors. This has given rise to stromal/stem-cell based therapy. Bone marrow derived stromal cells (BMSCs) have been studied to a large extent and show high regenerative potential but their use is limited by availability, donor site morbidity and the relatively low cell yield as they represent only <0.1% of cell harvested from bone marrow aspirate. At the same time, they are the closest mesenchymal stromal cells for bone tissue engineering given their tissue origin and, unlike other mesenchymal stromal cells, can support the formation of hematopoietic marrow. Adipose tissue derived stromal cells (ASCs) as part of the stromal vascular fraction of adipose tissue can as well undergo osteogenic differentiation but can be additionally isolated in a sufficient quantity from lipoaspirate after liposuction of abundant subcutaneous fat tissue. Here, it has been shown that there are no major differences in regard to proliferation or differentiation capacity of ASCs derived from subcutaneous fat of different anatomical regions. It has been shown that BMSCs are more prone to senescence during expansion and passage than ASCs and that ageing impacts proliferative capabilities of BMSCs more than that of ASCs while it has also been reported that osteogenic differentiation capacity is least impacted by age. Multiple studies have compared the characteristics of these two mesenchymal stromal cells in regard to bone tissue engineering in vitro. Most studies point to inferior extracellular matrix mineralization and lower expression of key osteogenic transcription markers like Runx2 in osteogenic differentiated ASCs compared to BMSCs. On the other hand, a study by Rath et al. found contrary results using particular culturing conditions like 3D bioglass scaffolds. An intraindividual comparison of human MSCs of three donors cultured on decellularized porcine bone confirmed superior osteogenic capacity of BMSCs compared to ASCs. In contrast to BMSCs, ASCs were not able to induce heterogenic ossification in a mouse model. In a sheep tibia defect model application of BMSCs resulted in a significantly higher amount of newly formed bone tissue. Importantly, Osteogenic differentiated ASCs do not support the formation of a hematopoietic marrow. Proteomics enables large-scale analysis of proteins present in a cell type and can be used to identify differentially regulated key proteins in a comparative approach. A comparative proteomic analysis of BMSCs and ASCs by Roche et al. in 2009 identified 556 proteins with 78% of these not being differentially regulated between these two cell populations, regarded as high similarity. Another comparative proteomic study of 2016 by Jeon et al. found 90 differentially regulated proteins out of 3000 total identified proteins. Both studies do not specify a number of different tissue donors and in part using cell lines. Looking for differences upon osteogenic differentiation, transcriptomic comparison of osteogenic differentiated porcine ASCs and BMSCs has been performed, resulting in 21 differentially expressed genes after 21 days of osteogenic culture conditions. Still, it remains unanswered, which are the key distinctive features of osteogenic differentiated ASCs and BMSCs at protein level that might help address the abovementioned weaknesses of ASCs in bone tissue engineering/regeneration for translational research. To overcome this need, an intraindividual comparative DIA based proteomic analysis of osteogenic differentiated human BMSC and ASCs was performed in this study.

			Action	DRS
	DDA_results.zip	Other
	DDA_spec_library_results.zip	Other
	DIA-ML_results.zip	Other
	DIA_results.zip	Other
	checksum.txt	Txt

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Comparative analysis of DDA- and DIA-PASEF methods for proteomics analysis of human valve interstitial cells and osteoblasts during osteogenic differentiation

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