A two-dimensional protein map of Chinese hamster ovary cells

Champion, Kathleen M.; Arnott, David; Henzel, William J.; Hermes, Sam M.; Weikert, Stefanie; Stults, John T.; Vanderlaan, Martin; Krummen, Lynne A.

doi:10.1002/(sici)1522-2683(19990101)20:4/5<994::aid-elps994>3.0.co;2-n

Cited by 40 publications

(14 citation statements)

References 37 publications

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“…3, Table 1). Among the major proteins (*0.2% of integral intensity) with decreased quantity in S phase, the protein p58 (58 kDa, pI 4.8) appears to be tubulin according to the published 2-D PAGE data [21,22]. Among the *1200 proteins analyzed, nine proteins were downregulated by two-to threefold in S phase compared to the profiles in G0 (Table 1).…”

Section: Resultsmentioning

confidence: 86%

Protein profiles of the Chinese hamster ovary cells in the resting and proliferating stages

Naryzhny

Lee

2001

Electrophoresis

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Identification and characterization of the proteins that regulate the transition from the resting stage (G0) through G1 to S phase of the cell cycle are of central importance to understand the control of cell proliferation and chromosome replication. Unlike in lower organisms, where relatively small numbers of key factors are involved in this process, the factors involved in the same control mechanisms in mammalian systems are much more complex. Furthermore, accumulating lines of evidence now suggest that the nuclear matrix and chromatin organization also play an essential role for the cell cycle control in mammalian cells. To gain a better understanding of the overall dynamics and changes of the protein factors in the context of matrix/chromatin organization, we examined the protein profiles of the Chinese hamster ovary (CHO) cells in different cell cycle compartments. The methods used in this study included subcellular fractionations (cytosol, nuclear extraction, chromatin, and nuclear matrix), two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), silver staining, and immunoblotting. As expected, significant changes of protein profiles were observed when cells entered into proliferating stages from G0. Among approximately 1200 protein spots analyzed by 2-D PAGE, at least 12 showed marked increase or decrease at this transitional period. Further cell-cycle progression from G1 to S phase showed less dramatic changes of overall protein protile. However, the profile of certain proteins showed rather dramatic changes of their subcellular localization during this transitional period. In particular, the levels of proliferating cell nuclear antigen (PCNA) in the nuclear matrix and chromatin dramatically increased in mid-G1 and in the beginning of S phase, respectively, while the overall PCNA level was relatively constant throughout the cell cycle.

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Section: Resultsmentioning

confidence: 86%

Protein profiles of the Chinese hamster ovary cells in the resting and proliferating stages

Naryzhny

Lee

2001

Electrophoresis

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“…N-Terminal microsequencing was carried out as previously described [23]. The proteins on 2-DE gel were electroblotted onto a PVDF membrane (Mini Problot) at constant voltage 24 V for 1 h. The membrane was washed with water, then stained with 0.1% Coomassie Brilliant Blue R-250 for 1 min and destained until the background became clear.…”

Section: N-terminal Sequencing Analysismentioning

confidence: 99%

Proteomic analysis of apoptosis initiation induced byall-trans retinoic acid in human acute promyelocytic leukemia cells

et al. 2001

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The irreversible destiny of apoptosis in its early stage might play a critical role in the apoptosis of human acute promyelocytic leukemia (APL) cell line induced by all-trans retinoic acid (ATRA). To characterize protein alterations during the apoptosis-initiation phase and to understand the metabolic status at that time, we investigated the protein profiles in the apoptosis-initiation phase of APL cell line HL-60 by proteomic analysis. ATRA-withdrawal was conducted to demonstrate that there was committed initiation phase of apoptosis triggered by 10(-6) M ATRA at day 3. Only after that time point, ATRA-treated cells irreversibly went to apoptosis. Also at that time point, the positive regulators of apoptosis such as STAT3 increased at protein level, whereas negative regulators (Bcl-2 and p-STAT3) decreased. In addition, caspase-3 also increased after that time. Furthermore, comparative proteomic analysis was utilized to examine the protein expression profiles during the initiation stage of apoptosis. Our results showed 12 upregulated and 7 downregulated proteins experiencing twofold alteration, including key regulators of signal transduction such as G-proteins and nucleic receptors, proteins related with metabolism, oxidation and reduction, proteins associated with the nucleus and cytoskeleton-related proteins. Some of them could be positive modulators to trigger apoptosis, whereas others could contribute to intracellular defense against apoptosis induced by exogenous triggers. The results above suggest that there is a subtle balance between apoptosis and the intracellular defense against apoptosis. Once the balance is disturbed, cells would irreversibly initiate to undergo the execution of apoptosis.

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“…2-DE was performed essentially as described in [33] and is summarized here. Approximately 40 mg protein were separated on analytical gels.…”

Section: Two-dimensional Polyacrylamide Gel Electrophoresismentioning

confidence: 99%

“…Protein spots were excised from preparatively loaded gels and digestions were performed as described previously [33]. MALDI-TOF mass spectra were acquired in reflectron mode on a PE Biosystems, (Foster City, CA, USA) Voyager DE STR instrument equipped with delayed extraction.…”

Section: In Situ Digestion Of Proteins and Maldi-tof-msmentioning

confidence: 99%

Similarity of theEscherichia coli proteome upon completion of different biopharmaceutical fermentation processes

Champion¹,

Nishihara²,

Joly

et al. 2001

Proteomics

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A comprehensive view of the physiological state of Escherichia coli cells at the completion of fermentation processes for biopharmaceutical production was attained via two-dimensional gel electrophoretic analysis of cellular proteins. For high cell density fermentations in which phosphate is depleted to induce recombinant protein expression from the alkaline phosphatase promoter, proteome analysis confirms that phosphate limitation occurs. Known phosphate starvation inducible proteins are observed at high levels; these include the periplasmic phosphate binding protein and the periplasmic phosphonate binding protein. The phn (EcoK) locus of these E. coli K-12 strains remains cryptic, as demonstrated by failure to grow with phosphonate as the sole phosphorus source. Proteome analysis also provided evidence that cells utilize alternative carbon and energy sources during these fermentation processes. To address regulatory issues in the biopharmaceutical industry, comparative electrophoretic analyses were conducted on a qualitative basis for four different fermentation processes. Using this approach, the protein profiles for these processes were found to be highly similar, with the vast majority (85-90%) of proteins detected in all profiles. The observed similarity in proteomes suggests that multiproduct host cell protein immunoassays are a feasible means of quantifying host-derived polypeptides from a variety of biopharmaceutical fermentation processes.

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A two-dimensional protein map of Chinese hamster ovary cells

Cited by 40 publications

References 37 publications

Protein profiles of the Chinese hamster ovary cells in the resting and proliferating stages

Protein profiles of the Chinese hamster ovary cells in the resting and proliferating stages

Proteomic analysis of apoptosis initiation induced byall-trans retinoic acid in human acute promyelocytic leukemia cells

Similarity of theEscherichia coli proteome upon completion of different biopharmaceutical fermentation processes

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