Orsolya Tünde Kovács 1 , Eszter Tóth 2 , Olivér Ozohanics 3 , László Drahos 2 , Edit Buzás 1 , György Nagy 1,4
1 Department of Genetics, Cell-and Immunobiology, Semmelweis University, Budapest
2 Research Center for Natural Sciences, Hungarian Academy of Sciences, Budapest
3 Department of Medical Biochemistry, Semmelweis University, Budapest
4 Buda Hospital of the Hospitaller, Budapest
Introduction: Osteoclasts have essential role in certain rheumatological inflammatory disorders, however the detailed pathomechaism of these diseases is not yet fully understood. In this regard the differentiation of osteoclasts from human blood derived monocytes is an extremely important process. Nevertheless we have lack of data about the molecular changes during osteoclast differentiation, and the proteome of osteoclasts is still unknown.
Aims: Our aims were to optimize a sample preparation and analytical method using nanoHPLC-MS/MS for the proteomic analysis of osteoclasts and their differentiation. After optimizing the methods using healthy samples, we planned to identify and compare the expressed proteins of monocytes, preosteclasts and osteoclasts; and to determine the differences in osteoclasts proteome of healty population and diseased individuals.
Method: First, we collect blood samples from healthy donors and isolate monocytes by magnetic separation. After this, preosteoclasts and osteoclasts are differentiated from monocytes in vitro using specific growth factors. Next, monocytes, preosteoclast and osteoclast samples are lysed, proteins are reduced, alkylated and digested with trypsin. After C18 clean-up, peptides are separated with nanoHPLC and analyzed with tandem mass spectrometry. The evaulation of raw data is performed using different proteomic databases and softwares.
Results: We have successfully optimized the proteomic sample preparation and analysis for maximizing the number of identified proteins and minimizing false positive results. We have determined the proteome of monocytes, preosteoclast, as well as osteoclast samples. Based on our preliminary results there are significant differences among the expressed proteins during osteoclast differentiation.
Conclusion: The developed sample preparation protocol, the applied chromatographic and mass spectrometric conditions, and the properly adjusted evaluation methods are fully appropriate for the proteomic analysis of monocytes, preosteoclasts and osteoclasts. The recently identified proteins, and the determined dynamic proteomic changes during osteoclast differentiation may promote the better understanding of the molecular pathomechanism of rheumatological diseases.
Doctoral School: Molecular Medicine
Program: Basis of Human Molecular Genetics and Gene Diagnostics
Supervisor: György Nagy