Proteomic Analysis of Chinese Hamster Ovary Cells

Abstract

In order to complement the recent genomic sequencing of Chinese hamster ovary (CHO) cells, proteomic analysis was performed on CHO including the cellular proteome, secretome, and glycoproteome using tandem mass spectrometry (MS/MS) of multiple fractions obtained from gel electrophoresis, multi-dimensional liquid chromatography, and solid phase extraction of glycopeptides (SPEG). From the 120 different mass spectrometry analyses generating 682,097 MS/MS spectra, 93,548 unique peptide sequences were identified with at most a 0.02 false discovery rate (FDR). A total of 6164 grouped proteins were identified from both glycoproteome and proteome analysis, representing an 8-fold increase in the number of proteins currently identified in the CHO proteome. Furthermore, this is the first proteomic study done using CHO genome exclusively which provides for more accurate identification of proteins. From this analysis, the CHO codon frequency was determined and found to be distinct from humans, which will facilitate expression of human proteins in CHO cells. Analysis of the combined proteomic and mRNA data sets indicated the enrichment of a number of pathways including protein processing and apoptosis but depletion of proteins involved in steroid hormone and glycosphingolipid metabolism. 504 of the detected proteins included N-acetylation modifications and 1292 different proteins were observed to be N-glycosylated. This first large-scale proteomic analysis will enhance the knowledge base about CHO capabilities for recombinant expression and provide information useful in cell engineering efforts aimed at modifying CHO cellular functions.

Proteomic differences in recombinant CHO cells producing two similar antibody fragments

“…Several criteria were used to filter the data before exporting the Progenesis output files to Proteome Discoverer 1.4 (Thermo Fisher Scientific) for protein identification: peptide features with adjusted ANOVA P ‐value ≤0.05 between experimental groups, mass features with charge states from +1 to +3, and the number of isotopes was set to 3 or less. All MS/MS spectra were exported from Progenesis software as an mgf file and searched against CHO‐specific protein sequence databases, using a combination of the translated NCBI genomic database (Baycin‐Hizal et al, 2012) containing 24,927 entries (fasta file downloaded January 2014) and the expressed cDNA database (BB‐CHO) (Meleady et al, 2012) containing 14,627 entries, through Proteome Discoverer 1.4 and the search algorithms Mascot and SequestHT. The search parameters used for all searches on Proteome Discoverer 1.4 were as follows: precursor mass tolerance set to 20 ppm, fragment mass tolerance set to 0.6 Da; up to two missed cleavages were allowed, carbamidomethylation set as a fixed modification, and methionine oxidation set as a variable modification.…”

Nuclear Localization of Integrin Cytoplasmic Domain-associated Protein-1 (ICAP1) Influences β1 Integrin Activation and Recruits Krev/Interaction Trapped-1 (KRIT1) to the Nucleus

“…The preceding experiments all relied on expression of fluorophore-tagged ICAP1 in CHO cells, a line that apparently lacks endogenous ICAP1 and KRIT1 (45). We therefore assessed ICAP1 nuclear localization in EA.hy926 cells, a widely used cell line generated by fusing human umbilical vein endothelial cells with the lung carcinoma A549.…”

Genomic landscapes of Chinese hamster ovary cell lines as revealed by the Cricetulus griseus draft genome

“…Meanwhile, efforts to engineer mouse cells have greatly benefited from numerous genomic tools and technologies, owing in large part to the availability of the Mus musculus reference genome sequence. Genomic resources are also becoming available for CHO cells, such as the CHO-K1 genome 5 , expressed sequence tag 6,7 and bacterial artificial chromosome (BAC) libraries 8 , and compendia of proteomic [9][10][11] and transcriptomic data 7,[12][13][14][15][16] . However, much like how murine cell line data are routinely studied in the context of the Mus musculus reference genome, there is a need for a standard reference for all CHO cell lines to contextualize all of these valuable genomic resources.…”