Proteomics

Dataset Information

3

Identification of Autocrine Growth Factors Secreted by CHO cells for Applications in Single Cell Cloning Media


ABSTRACT: 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.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo sapiens   Cricetulus griseus   Mus musculus  

TISSUE(S): Cho Cell

DISEASE(S): Not Available

SUBMITTER: U MING LIM  

LAB HEAD: Say Kong Ng

PROVIDER: PXD000214 | Pride | 2020-01-22

REPOSITORIES: Pride

Dataset's files

Source:
Action DRS
CHO_D2345_Mudpit-02.prot.zip Other
Human_D2345_Mudpit-01.prot.zip Other
Mouse_D2345_Mudpit-01.prot.zip Other
SCX4_D2B1F1.zip Other
SCX4_D2B1F2.zip Other
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Publications

Identification of autocrine growth factors secreted by CHO cells for applications in single-cell cloning media.

Lim U Ming UM   Yap Miranda Gek Sim MG   Lim Yoon Pin YP   Goh Lin-Tang LT   Ng Say Kong SK  

Journal of proteome research 20130625 7


Chinese hamster ovary (CHO) cell lines are widely used for the expression of therapeutic recombinant proteins, including monoclonal antibodies and other biologics. For manufacturing, cells derived from a single-cell clone are typically used to ensure product consistency. Presently, fetal bovine serum (FBS) is commonly used to support low cell density cultures to obtain clonal cell populations because cells grow slowly, or even do not survive at low cell densities in protein-free media. However,  ...[more]

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