Analysis of mature miRNA levels in the context of CHO cell proliferation
Ontology highlight
ABSTRACT: Mature microRNA transcripts were analyzed in 9 CHO cell lines growing at different proliferation rates. The goal was to identify microRNAs that correlate positively or negatively with CHO proliferation rates. A common reference design was chosen, where all samples were hybridized against a pool of all RNA samples.
Project description:To identify low abundance autocrine growth factors in CHO cell conditioned media, we utilized a label-free shotgun proteomics approach. CHO cell conditioned media were harvested from fed batch bioreactors and concentrated using methanol/chlorofrom precipitation. Proteins in the samples were then subjected to proteolysis with trypsin, and then subjected to primary fractionation using a SCX column, followed by RP liquid chromatography MS (LC-MS) with a LTQ Orbitrap Velos instrument using the data dependent acquisition (DDA) method. The MS system was set up and run with a method which enabled fast acquisitions of high quality peptide precursor and fragment ion data, with the desired precursor mass accuracy of ±5 ppm. For the LTQ Orbitrap Velos MS, the data-dependent MS/MS analytical workflow in positive ion mode was used. Each precursor survey scan (m/z: 300 to 1800) by the Orbitrap mass analyzer (resolution = 60,000 FWHM) was linked to 10 MS/MS events using the 2D ion trap CID approach, with dynamic ion exclusion set at 60 s. This value was determined based on the observed mean peptide chromatographic peak width. All other instrument parameters were set up according to the manufacturer’s suggested values for complex peptide samples. The nano-ESI source was fitted with a 30-µm stainless steel nano-bore emitter (Thermo Fisher Scientific) with 1.7 kV applied near the tip. Raw data files from the LTQ Orbitrap Velos MS were processed using the Proteome Discoverer 1.3 software (Thermo Fisher Scientific). The LC-MS data were searched against the human (Homo sapiens; UniProtKb, updated in August 2012, 45 848 entries), mouse (Mus musculus; UniProtKb, updated in August 2012, 31 528 entries) and chinese hamster (Cricetulus griceus; UniprotKb, updated in August 2012, 24 609 entries) protein databases using the Sequest search engine for the LTQ Orbitrap Velos LC-MS data, assuming tryptic digestion with precursor ions to fall within 10 ppm of projected m/z values and a fragment ion mass tolerance of 0.5 m/z. The specified search parameters were carbamidomethylation of cysteine as fixed modification, oxidation of methionine as dynamic modification and a maximum of two missed cleavage events. Reverse database searching resulted in a specific false discovery rate (FDR) of 1% at the peptide and protein level.
Project description:mature microRNA transcripts were analyzed in 5 CHO cell lines growing at different proliferation rates. The goal was to identify microRNAs that correlate positively or negatively with CHO proliferation rates. A common reference design was chosen, where all samples were hybridized against a pool of all RNA samples.
Project description:The reduction of cell culture temperature (i.e. temperature-shift) is a method used to extend the viability of some commercial cell culture processes and improve product quality. We utilised LC-MS/MS to profile the proteome in temperature shifted cells as well as those a normal physiological temperature.
Project description:Sodium butyrate (NaBu) is well-known for its capacity to hinder cellular growth and act as a histone deacetylase inhibitor. It is commonly employed in the cultivation of recombinant Chinese hamster ovary (CHO) cell cultures to boost the production of specific proteins, such as antibodies. In this investigation, two types of CHO cell lines, namely K1 and DG44, along with their respective mAb-producing lines, K1-Pr and DG44_Pr, were cultured with or without NaBu. To analyze the proteome, a SWATH-based profiling method was utilized. The outcomes were assessed using Spectronaut 17, while STRING and Gene Ontology pathway analyses were performed using Cytoscape. The analysis confirms the known effects of NaBu on mAbs production, adding information on redox homeostasis of the cells.
Project description:A comprehensive Chinese hamster ovary (CHO)-cell specific spectral library, containing extensive information of high-quality spectral ions covering more than 10k CHO cell proteins, was constructed to provide confident and reproducible protein identification and quantification in data-independent SWATH-MS analysis. The applicability of CHO spectral library was tested in the analyses of different CHO samples including whole cell lysate, harvested cell culture fluids, and downstream processing samples. The portability of CHO spectral library was also demonstrated in the processing and analyses of SWATH-MS data sets collected from multiple LC-MS instrumental setups and various CHO cell lines.
Project description:CHK-Q cell line was established from primary culture of Chinese hamster kidney tissues by repeated passages. DNA microarray analysis for chasing the variation in gene expression profile was performed for CHK cells during the culture process leading to immortalization and adaptation to serum-free and suspension conditions.
Project description:Oligopeptidases Prep and OOP are enzymes located in mitochondria and chloroplasts responsible for the cleavage of long peptides (8 to 65 aas) down to short peptides (3 to 7 aas), which in turn are degraded to free amino acids by aminopeptidases such as M1 and M17. In this study, Arabidopsis thaliana knockout mutants of the genes for these enzymes were studied by proteomics using a TMT (tandem mass tags) isobaric tags quantification strategy combined with HiRIEF LC-MS on a Q-Exactive Mass Spectrometer.
Project description:A 3rd generation Wye3aHamster microarray chip was used to carry out a differential expression microarray study of a panel of 30 slow- and fast-growing production cell lines, identifying a priority list of 240 transcripts (110 Up; 130 Down) that passed a statisical filter (ANOVA p-value <0.05, 1.3 fold-change) when the samples were grouped into fast versus slow and pairwise compared and a minimum Pearson Correlation Coefficent (PCC) of >0.7 when growth rate was used as a continuous variable (i.e. included genes have to satisfy BOTH criteria, not just one). This yielded a list of 240 genes. The 240-member genelist yielded 223 Annotated IDs, 203 of which are Unique and all of which are associated with contributing to a high rate of growth in production CHO cell lines. Prospective samples were isolated from 2 distinct groups of clones (slow growers and fast growers), all chosen from the same project (transfection of product transgene into parent line) generated at Pfizer Inc., Bioprocess R&D, Andover, MA, USA. Each group comprised 5 distinct clones, all of which had comparable Qp characteristics. The clones were passaged for 3 or 4 passages over 3-4 days to stabilise the phenotype and freeze stocks. Samples for array hybridization were generated in batch shake flask culture (60ml working volume) with AS1 medium and without feeds or temperature shift. Samples were collected at a single time point (72hrs - mid/late log) and each clone was grown in triplicate flasks, yielding a total of 30 samples for collection and processing. Two bioprocess-relevant variables, viable cell density (VCD) and viability were measured. From the VCD, the growth rate (h-1) was calculated.
Project description:Pyk2 is a multidomain non-receptor tyrosine kinase that undergoes a complex, multi-stage activation process. Ca2+-flux induces conformational rearrangements that relieve autoinhibitory FERM domain interactions. The kinase domain phosphorylates a key linker residue to recruit Src kinase. Pyk2 and Src mutually phosphorylate activation loop residues to confer full activation. While the mechanisms of autoinhibition are established, the conformational dynamics associated with autophosphorylation and Src recruitment remain unclear. Here, we employ hydrogen/deuterium exchange mass spectrometry (HDX-MS) to map the conformational changes associated with Src-mediated activation segment phosphorylation in a Pyk2 construct encompassing FERM and kinase domains (residues 20-692). Results reveal increased dynamics at regulatory interfaces spanning FERM and kinase domains. Phosphorylation of the activation segment stabilizes two antiparallel beta strands linking activation and catalytic loops. Increased dynamics of the C-terminal end of the activation loop propagate to the F-helix, explaining how phosphorylation prevents reversion to the autoinhibitory FERM interaction. HDX-guided site-directed mutagenesis and kinase activity profiling establish a mechanism for phosphorylation-induced active site sculpting to confer high activity.
Project description:C-mannosylation in addition to N- and O-glycosylation is a further but less well studied type of protein glycosylation taking place in the endoplasmic reticulum (ER) characterised by the modification of tryptophan residues with a single mannose effecting protein folding, secretion and/or function. Motivated by an interest in the functional role of C-mannosylation for early developmental processes, we aimed for the functional inactivation of the C mannosyltransferases DPY19L1 and DPY19L3, respectively, and excised parts of their coding sequence from the genome of the hiPSC line CBiPSC2 by applying CRISPR Cas9. To determine the effect of the genomic deletions in DPY19L1 or DPY19L3 on C-mannosylation, TSR2 and TSR3 of human thrombospondin 1 (THBS1) was recombinantly expressed in WT and KO hiPSCs followed by purification an LC-MS analysis. The results are in accordance to our previous findings that DPY19L1 is mainly acting on W1 and W2 whereas DPY19L3 acts on W3 of WxxWxxWxxC motifs of TSRs and proves the functional inactivation of the respective C-mannosyltransferases in the hiPSCs model. A secretome analysis of C mannosyltransferase deficient hiPSCs revealed that secretion of numerous proteins was reduced in the mutants including ADAMTS16, which was previously reported to be essential for optic fissure fusion in zebrafish. In order to analyse C-mannosylation of ADAMTS16, its TSR1 was recombinantly expressed in CHO-K1 WT, DPY19L1 KO and DPY19L3 KO cells, purified and analysed by LC MS analysis. The results revealed that the WSDWSSWSPC motif of TSR1 of ADAMTS16 can be C-mannosylated at all three tryptophan residues. Deletion of DPY19L1 prevented C-mannosylation of the two first tryptophans whereas C-mannosylation of the third tryptophan was not detected in the DPY19L3-mutant.