Utilization and evaluation of CHO‐specific sequence databases for mass spectrometry based proteomics

Abstract

Recently released sequence information on Chinese hamster ovary (CHO) cells promises to not only facilitate our understanding of these industrially important cell factories through direct analysis of the sequence, but also to enhance existing methodologies and allow new tools to be developed. In this article we demonstrate the utilization of CHO specific sequence information to improve mass spectrometry (MS) based proteomic identification. The use of various CHO specific databases enabled the identification of 282 additional proteins, thus increasing the total number of identified proteins by 40-50%, depending on the sample source and methods used. In addition, a considerable portion of those proteins that were identified previously based on inter-species sequence homology were now identified by a larger number of peptides matched, thus increasing the confidence of identification. The new sequence information offers improved interpretation of proteomic analyses and will, in the years to come, prove vital to unraveling the CHO proteome.

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.…”

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.…”

Acute exposure to organic and inorganic sources of copper: Differential response in intestinal cell lines

“…Label‐free LC‐MS/MS was carried out on an Ultimate 3000 nanoLC system coupled to an LTQ Orbitrap XL mass spectrometer as previously described (Meleady et al., 2012). Peptide identification through Proteome Discoverer 2.1 using the search algorithms MASCOT (version 2.3) and SEQUEST HT was used to search against the UniProtKB‐SwissProt fasta database with 20 274 proteins (taxonomy: Homo sapien) using the following parameters: (a) MS/MS mass tolerance set at 0.5 Da, (b) peptide mass tolerance set to 20 ppm, (c) carbamidomethylation set as a fixed modification, (d) up to two missed cleavages were allowed, and (e) methionine oxidation set as a variable modification.…”