A proteomic approach towards understanding crypoprotective action of Me2SO on the CHO cell proteome

Abstract

Chinese hamster ovary (CHO) cell lines are the most widely used in vitro cells for research and production of recombinant proteins such as rhGH, tPA, and erythropoietin. We aimed to investigate changes in protein profiles after cryopreservation using 2D-DIGE MALDI-TOF MS and network pathway analysis. The proteome changes that occur in CHO cells between freshly prepared cells and cryopreserved cells with and without Me2SO were compared to determine the key proteins and pathways altered during recovery from cryopreservation. A total of 54 proteins were identified and successfully matched to 37 peptide mass fingerprints (PMF). 14 protein spots showed an increase while 23 showed decrease abundance in the Me2SO free group compared to the control. The proteins with increased abundance included vimentin, heat shock protein 60 kDa, mitochondrial, heat shock 70 kDa protein 9, protein disulfide-isomerase A3, voltage-dependent anion-selective channel protein 2. Those with a decrease in abundance were myotubularin, glutathione peroxidase, enolase, phospho glyceromutase, chloride intracellular channel protein 1. The main canonical functional pathway affected involved the unfolded protein response, aldosterone Signaling in Epithelial Cells, 14-3-3-mediated signaling. 2D-DIGE MALDI TOF mass spectrometry and network pathway analysis revealed the differential proteome expression of FreeStyle CHO cells after cryopreservation with and without 5% Me2SOto involve pathways related to post-translational modification, protein folding and cell death and survival (score = 56, 22 focus molecules). This study revealed, for the first time to our knowledge the proteins and their regulated pathways involved in the cryoprotective action of 5% Me2SO. The use of 5% Me2SO as a cryoprotectant maintained the CHO cell proteome in the cryopreserved cells, similar to that of fresh CHO cells.