BACKGROUND
since the introduction of human tissue plasminogen activator  as the first recombinant therapeutic protein product from mammalian cells to get approval in  <dig>  approximately 60–70% of all recombinant proteins are being produced in mammalian cell lines  <cit> , predominantly in chinese hamster ovary  cells.
the increasing demands for biologicals necessitates improved productivity from cho cells and the methods currently applied include optimization of culture conditions , improved reactor designs, medium formulation and cell engineering.
despite this, however, many aspects of cho cell biology remain poorly understood.
many culture processes operate a biphasic culture whereby cells are grown at 37°c to maximise biomass and then the culture is shifted to a lower temperature  to encourage protein production while maintaining a longer and more viable stationary/production phase  <cit> .
low temperature cultivation of mammalian cells is an established method to maintain cell viability while at the same time inhibiting growth; this inhibition is associated with g1-phase cell cycle arrest which has been positively correlated with increased productivity  <cit> .
however, the effect of low temperature on recombinant protein productivity can be product, clone or cell line specific  <cit> .
increased levels of mrna coding for the recombinant protein, either due to enhanced transcription or increased mrna stability of the recombinant gene of interest, have also been observed during low temperature culture and also correlate with improved recombinant protein productivity  <cit> .
post-translational modifications, particularly glycosylation, which are important for biological activity of recombinant proteins, are maintained or even improved at low temperature  <cit> .
in addition to affecting productivity, low temperature culture has been shown to reduce cellular metabolism , waste accumulation  and apoptosis  <cit> .
the molecular mechanisms regulating sub-physiological temperature response of cells in culture are still poorly understood, despite a number of recent transcriptomic and proteomic profiling studies  <cit> .
it is generally accepted that global cap-dependent protein translation decreases at sub-physiological temperatures, however ires mediated cap-independent protein translation is active  <cit> .
more than  <dig> proteins including cold-inducible rna binding protein , rna binding motif protein  <dig> , protein disulphide isomerase , nuclear localization sequence binding protein , vimentin, phosphoglycerate kinase and heat shock cognate  <dig> kda protein have been reported as being up regulated during low temperature culture  <cit> .
of these proteins, crip and rbm <dig> have been studied extensively  <cit> .
both cirp and rbm <dig> are involved in modulation of transcription and translation by functioning as rna chaperones  <cit> .
crip improves recombinant protein productivity and can arrest cell growth in a cell line-specific manner whereas rbm <dig> facilitates cap-independent protein synthesis at low temperature  <cit> .
rbm <dig> is also reported to interact with and partially block the activity of mirnas at low temperature  <cit> .
altered mirna expression following temperature shift has recently been reported and may be an important component of regulating protein expression and cell behavior at low temperature  <cit> .
in this study, we have used two-dimensional difference gel electrophoresis  to investigate the proteomic changes associated with the shift to low temperature in a biphasic batch culture of suspension-adapted cho-k <dig> cells.
in this manner we wished to gain insights into the effects of low temperature culture on mammalian cells, with potential relevance to mammalian cell-based recombinant protein production.
RESULTS
cell culture
suspension-adapted cho-k <dig> cells were seeded at  <dig> ×  <dig> cells/ml in spinner flasks and maintained at 37°c for  <dig> hrs or for  <dig> hrs at 37°c followed by a temperature shift to 31°c for a further  <dig> hrs.
<dig> hrs was chosen for this temperature shift because it represents the mid-exponential phase of cell growth.
31°c was selected for the temperature shift as it represents the average temperature employed by others performing similar studies, i.e.
28–33°c  <cit> .
as can be seen in figure  <dig> the cells that were temperature shifted immediately ceased logarithmic growth and did not exceed a peak viable cell density of  <dig>  ×  <dig> ±  <dig>  cells/ml, whereas the cells maintained in standard culture at 37°c continued in logarithmic growth for a further  <dig> hrs and achieved a peak viable density of  <dig>  ×  <dig> ±  <dig>  cells/ml.
the cells that were temperature shifted displayed a steady if slightly increasing viable cell density throughout, whereas the cells cultured at 37°c had entered the late stationary/death phase of the growth cycle by  <dig> hrs .
cultures were sampled at  <dig> hrs and  <dig> hrs for protein extraction from  <dig> independent experiments.
differential analysis of proteins
2d-dige protein expression maps  for the biological triplicate samples were generated for both temperature shifted and standard cultures for cho-k <dig> cells at  <dig> hrs and  <dig> hrs.
the  <dig> hrs pems were then compared to that at  <dig> hrs using the biological variance analysis  module of decyder software.
this was aided by the use of a pooled internal standard which facilitated spot matching and relative spot quantitation while minimizing the gel-to-gel variations.
spots originating from dust particles and gel impurities in each gel image were checked and removed manually to improve the quantitation and matching process.
landmarks, which aid the gel-to-gel spot matching process, were also defined in the gel to increase the accuracy of the matching algorithm.
manual checks were carried out to allow for cases where spots were detected more than once , these spots were then merged back to improve spot matching.
after spot matching and filtering,  <dig> and  <dig> spots were detected for the temperature shifted and standard culture experiments respectively.
spots that were  <dig>  fold up/down regulated at  <dig> hrs compared to  <dig> hrs with a t-test score of ≤  <dig>  and present on all gels were considered differentially expressed .
a total of  <dig> de spots  were observed in the temperature shift experiment and  <dig> de spots  were observed in the standard culture experiment .
spot identification
following spot picking and tryptic digestion, protein identification was carried out using maldi-tof ms. all identified proteins had an expectation value of  <dig>  or better.
expectation value for proteins was determined by ettan maldi-tof pro evaluation software  using the profound database search engine for peptide mass fingerprints.
an expectation value of  <dig>  or better means that there is ≤ 1% chance that the identification is random.
although many spots resulted in high quality spectra, they were not identified which may be due to insufficient amounts of protein, modification and/or variation of the amino acid sequences in cho proteins or the paucity of cho proteins in the protein databases.
despite this  <dig> proteins were identified  and the representative spectra of  <dig> of these proteins  are presented in figure  <dig>  following identification of the  <dig> spots, all identified proteins were compared for differential expression in both temperature shifted and standard culture experiments to identify temperature-dependent and temperature-independent protein changes .
for example, ganab, a glycan-processing enzyme that is involved in glycoprotein quality control in the endoplasmic reticulum  <cit> , was  <dig>  fold up regulated at  <dig> hrs compared to  <dig> hrs following temperature shift while it was unchanged when cells were maintained at 37°c.
activator of  <dig> kda heat shock protein atpase homolog  <dig>  however, was differentially expressed in both experiments , hence could be considered a temperature-independent protein change.
a number of the identified proteins including vimentin and gapdh have been previously identified as differentially expressed following temperature shift .
the identified differentially expressed proteins were also compared between both temperature shifted and standard cultures at  <dig> hrs .
three proteins, actb, aldh and dpysl <dig> were identified at more than one location on the gel possibly indicating some form of post-translational modification , of which ptms in aldh and dpysl <dig> were specific to the temperature shift to 31°c.
the identified proteins could be linked to a broad range of biological functions including structural, metabolic, differentiation, secretory pathways, translation, transcription, protein binding, signal transduction, cell adhesion and apoptosis.
validation of results by western blotting analysis
two proteins, vimentin and hnrpc, were selected from the list of differentially expressed identified proteins for validation by western blotting.
rbm <dig> is already known to be induced following shift to low temperature and was included as a positive control.
α-tubulin was used as a loading control.
western blot analysis reflected a similar pattern of expression to those observed using 2d-dige .
vimentin was  <dig>  fold up regulated following temperature shift and  <dig>  up regulated in standard culture from 2d-dige analysis.
densitometry analysis of western blot data revealed that vimentin was  <dig>  fold up regulated following temperature shift and  <dig>  fold down regulated in standard culture.
hnrpc was  <dig>  fold down regulated following temperature shift and  <dig>  fold down regulated in standard culture from 2d-dige analysis, while from densitometry analysis of western blot data, it was  <dig>  fold down regulated at low temperature and  <dig>  fold down regulated in standard culture.
discussion
the incorporation of a temperature reduction step is commonly employed during production cell culture in the biopharmaceutical industry.
this temperature shift is used as a means of simultaneously inducing growth arrest and extending long term culture viability thus increasing recombinant protein productivity and yield  <cit> .
to investigate the mechanisms regulating the effects of low temperature in cho-k <dig> cells we employed 2d-dige followed by maldi-tof mass spectrometry.
when suspension-adapted cho-k <dig> cells were cultured at 37°c for  <dig> hrs and then shifted to low temperature , cell growth was immediately arrested compared to cells maintained in standard cultures  .
following the temperature shift, the cells were observed to maintain a steady viable cell density whereas the cells at 37°c began entering the death phase by  <dig> hrs of culture.
while the viabilities of both cultures were comparable throughout the  <dig> hrs of culture this changed towards the end of the experiment for the cells maintained at 37°c .
it is possible that the high percentage of serum  in the culture medium protected both cultures from apoptotic cell death, therefore maintaining the high viabilities recorded.
serum is known to protect cells against apoptosis in suspension culture  <cit> .
following 2d-dige analysis to compare the differences in global protein expression at  <dig> hrs and  <dig> hrs of culture,  <dig> de spots were observed in the temperature shifted cells whereas  <dig> de spots were observed for the cells maintained in standard culture.
it is apparent from this data that cells at the end of a temperature shifted batch culture are more similar to healthy exponentially growing cells than cells which have achieved late stationary/death phase at 37°c.
reduced cellular metabolism at low temperature could be responsible for the smaller number of differentially expressed proteins observed after temperature shift in comparison to the cells grown at 37°c.
it has been observed that protein synthesis rates are reduced at low temperature due to a general inhibition of cap-dependent protein synthesis.
however some proteins, e.g.
rbm <dig>  are translated under cold stress via an ires mediated cap-independent mechanism  <cit> .
this type of protein translation initiation requires both eukaryotic initiation factors  and message-specific cellular ires trans-acting factors   <cit> .
eukaryotic translation initiation factor 4a, isoform  <dig> , an rna helicase enzyme, is one of the critical translational initiation factors that are required in ires mediated cap-independent protein translation  <cit> .
eif4a is  <dig>  fold up regulated following temperature shift.
this indicates that eif4a may play an important role in facilitating translation in this system.
eif4a has been shown to be up regulated in g <dig> arrested cho cells  <cit> , which may also indicate a role for this protein in the observed growth arrest.
of the  <dig> spots picked , a total of  <dig> proteins were successfully identified .
of these,  <dig> proteins  were specifically altered following temperature shift .
two of these proteins  were present at more than one location in the gel indicating a low-temperature-specific protein modification.
conversely,  <dig> proteins were differentially regulated specifically in standard culture, of which one protein, actb, was present at two different positions in the gel.
grouping of the identified proteins by biological function indicated that proteins involved in metabolism were the dominant group affected in both temperature shifted and standard cultures , followed by structural proteins .
the indication that low temperature affects metabolism and structure is in keeping with other studies  <cit> .
however, the number of proteins identified here is insufficient for a comprehensive analysis of the main biological functions affected in this system, as we may be observing a bias towards the identification of the more highly expressed metabolic and structural proteins within the cell such as gapdh and vimentin.
the most obvious effect following reduction of culture temperature is the immediate decrease in growth observed.
a number of growth-related proteins were identified as being differentially expressed in a temperature-specific manner, including hnrpc, rrn <dig> and lgals <dig>  hnrpc is a nuclear pre-mrna binding protein that enhances the translation of c-myc mrna by interacting with an ires in the 5' utr and thus impacting proliferation  <cit> .
silencing of hnrp c1/c <dig> protein expression by sirna has also been shown to result in inhibition of cell proliferation  <cit> .
these results are in keeping with our finding that hnrpc is  <dig>  fold down regulated following reduction of the culture temperature.
hnrpc also interacts with iress in the 5'utr of 'upstream of n-ras'  and affects cell proliferation through iress, including the pitslre ires, which is activated at mitosis  <cit> .
this is important because during mitosis, similar to low temperature culture inductions, protein synthesis is rapidly and severely repressed possibly due to inhibition of global cap-dependent translation  <cit> .
the fact that hnrpc and eif4a are both differentially expressed in this experiment clearly indicates the importance of ires-based regulation of the translational machinery.
rrn <dig> protein levels were found to be  <dig>  fold down regulated at the end of the temperature shifted culture which is unsurprising as its activity is known to be reduced in stationary cells  <cit> .
lgals <dig>  has been shown to act as a negative autocrine growth factor that regulates cell proliferation  <cit>  which correlates with the  <dig>  fold up regulation of lgals <dig> observed in the temperature shifted samples and growth data.
another common feature of cells that are cultured at reduced temperatures is that they display delayed apoptosis compared to cells cultured at 37°c  <cit> .
it has been proposed that the reduction in apoptosis is as a result of the reduced cellular metabolism at low temperatures  <cit> .
however, others have reported the induction of anti-apoptotic proteins  following culture at reduced temperature  <cit> .
bcl- <dig> was not identified as a de protein in this study but a number of apoptosis-associated proteins were identified including importin-α, nadh dehydrogenase  fe-s protein  <dig>  and annexin a <dig>  importin-α, a nuclear import receptor, facilitates trafficking of the growth promoting ras effector, rassf <dig>  into the nucleus  <cit> .
over expression of importin-α also results in significant increases in p <dig> transcript levels and apoptosis, implicating it in the nuclear import of p <dig>  <cit> .
the temperature-specific  <dig>  fold down regulation of importin-α levels therefore may have an impact on both proliferation and protection against apoptosis.
unlike importin-α, the other two apoptosis-related proteins annexin a <dig> and ndusf <dig> were both up regulated , and are positive regulators of apoptosis.
annexin a <dig> is selectively translocated from the nucleus to the cytosol during apoptosis and is recognized as an early marker of apoptotic cell death  <cit> .
ndufs <dig> is part of the mitochondrial complex i. inhibition of ndufs <dig> expression by sirna has been shown to provide resistance to ifn-β/retinoic acid-induced apoptosis  <cit> .
the data presented indicates that by the end of the temperature shifted culture the cells were beginning to initiate apoptosis.
this is in agreement with other findings that despite the delay in apoptosis, once initiated, the rate and manner of cell death is similar to that observed in standard culture at 37°c  <cit> .
by comparison, cells at  <dig> hrs in standard culture were already in early death phase .
this is likely due to a variety of reasons including nutrient limitation, oxygen depletion and waste accumulation at  <dig> hrs in standard culture.
a number of apoptosis related proteins, including prohibitin , aldehyde dehydrogenase family  <dig>  subfamily a <dig>  and alpha-enolase  were found to be differentially regulated in this system .
over expression of phb inhibits cell proliferation by arresting cells in g0/g <dig> phase and has also been shown to increase cell survival  <cit> .
aldh1a <dig> is a member of aldehyde dehydrogenase family that can oxidize a wide variety of aldehydes  to the corresponding carboxylic acids .
down regulation of aldh1a <dig> has been correlated with the increased susceptibility of cells towards apoptosis  <cit> .
eno <dig> catalyses the formation of phosphoenolpyruvate from 2-phosphoglycerate.
the over expression of eno <dig> has been shown to inhibits cell growth and induce apoptosis in neuroblastoma cells  <cit> .
phb and aldh1a <dig> were  <dig>  and  <dig>  fold down regulated respectively, while eno <dig> was  <dig>  fold up regulated at  <dig> hrs at 37°c in comparison to culture at  <dig> hrs.
these observations are in keeping with the reduced viability observed at  <dig> hrs in standard culture in comparison to temperature shifted cultures.
a number of cytoskeletal proteins including vimentin, profilin, beta actin  and capping protein  muscle z-line beta were differentially expressed following temperature shift and/or in standard culture .
vimentin, profilin and beta-actin were also observed to be differentially expressed by baik et.
al.
<cit> .
vimentin is one of the major cytoskeletal proteins and its increased expression has previously been shown in the heat-resistant phenotypes of cho cells  <cit> .
vimentin was  <dig>  fold up regulated following temperature shift to 31°c and was unchanged at standard culture.
profilin is a small ubiquitous actin monomer sequestering protein that is required for normal actin polymerization in response to thermal stress  <cit> , and was  <dig>  fold up regulated following temperature shift.
however profilin was unchanged at standard culture.
actb is involved in many biological functions in cell such as regulating cell shape and growth.
increased expression of actb has been shown to provide resistance against apoptosis  <cit> .
actb was  <dig>  fold down regulated at  <dig> hrs at 37°c and was not significantly altered following temperature shift.
capzb binds to one end of actin filaments, regulates actin polymerization including the number and length of actin filaments, and strengthens the actin cytoskeleton in the cytoplasm  <cit> .
capzb was  <dig>  fold down regulated at  <dig> hrs at 37°c, whereas no change was observed following temperature shift.
although the role of vimentin and profilin in regulating low temperature specific phenotypes is unclear at this point, the reduced expression of actb and capzb could possibly be associated with increased apoptosis in standard culture  <cit> .
cellular metabolism is generally reduced in low culture temperature  <cit>  but this does not apply to every enzyme and pathway.
gapdh, a multi-functional glycolytic enzyme, was almost 2-fold up regulated following temperature shift.
similar results have been reported in other published data  <cit>  .
in contrast metabolic enzymes involved in energy generation  were down regulated in dying cells  which indicates reduced energy generation in standard culture at 37°c.
this may be due to depletion of nutrients in the media at  <dig> hrs of culture.
out of  <dig> identified proteins,  <dig> proteins  were differentially expressed in both the temperature shifted cells and those maintained at 37°c .
in most cases the regulation between  <dig> hrs and  <dig> hrs was similar in the temperature shifted and standard cultures indicating that these are culture-dependent changes.
as with the de proteins identified from the temperature shift, these proteins cover a range of functions including stress response e.g.
ahsa <dig>  of the  <dig> identified proteins,  <dig> were found to be differentially expressed between temperature shifted and non-shifted cultures at  <dig> hrs .
three proteins, actb, aldh and dpysl <dig>  were identified from multiple spots.
actb was present at two different pis and molecular weights in the gel containing samples from standard culture.
aldh and dpysl <dig> were identified at two different pis on gels from the temperature shifted culture suggesting potential low temperature-induced post translational modifications.
aldh is a nad+-dependent enzyme that can protect against hyperoxia-induced cell death through reduction of ros, activation of erk/mapk, and pi3k-akt cell survival signalling pathways  <cit> .
analysis of the aldh spots revealed  <dig>  and  <dig>  fold up regulation following temperature shift and no change in the cultures maintained at 37°c.
the role of this protein in low temperature response is unknown.
dpysl <dig> promotes microtubule formation by binding to tubulin heterodimers  <cit> .
different variants, possibly phosphorylated, of dpysl <dig> have been reported previously  <cit> .
in this study, dpysl <dig> was  <dig>  fold up regulated following temperature shift whereas it was  <dig>  fold up regulated in the cultures maintained at 37°c.
however, the second spot identified as dpysl <dig> was specifically  <dig>  fold down regulated at 31°c.
the functional association of dpysl <dig> with low temperature response is unclear.
in-silico phosphoproteome analysis predicted that both aldh and dpysl <dig> contain phosphorylation sites and hence the multiple spots may indicate that they are phosphorylated.
a previous study also identified two proteins as being phosphorylated at tyrosine residues in cho cells cultured at low temperature  <cit>  suggesting that active cell signalling occurs in response to temperature shifts.
CONCLUSIONS
in conclusion, the present study has identified a number of temperature-specific as well as temperature-independent changes in protein expression that occur during batch suspension culture.
a number of the protein changes can be linked to the growth inhibition observed subsequent to temperature shift.
further studies targeting the proteins identified in this study using cho-k <dig> cells producing a recombinant protein product will improve the understanding of the cold shock response and may be useful in identifying candidate targets to re-engineer cho cells to produce recombinant therapeutic proteins more efficiently.

 since the introduction of human tissue plasminogen activator as the first recombinant therapeutic protein product from mammalian cells to get approval in approximately 60–70% of all recombinant proteins are being produced in mammalian cell lines, predominantly in chinese hamster ovary cells. the increasing demands for biologicals necessitates improved productivity from cho cells and the methods currently applied include optimization of culture conditions, improved reactor designs, medium formulation and cell engineering. despite this, however, many aspects of cho cell biology remain poorly understood. many culture processes operate a biphasic culture whereby cells are grown at 37°c to maximise biomass and then the culture is shifted to a lower temperature to encourage protein production while maintaining a longer and more viable stationary/production phase. low temperature cultivation of mammalian cells is an established method to maintain cell viability while at the same time inhibiting growth; this inhibition is associated with g1-phase cell cycle arrest which has been positively correlated with increased productivity. however, the effect of low temperature on recombinant protein productivity can be product, clone or cell line specific. increased levels of mrna coding for the recombinant protein, either due to enhanced transcription or increased mrna stability of the recombinant gene of interest, have also been observed during low temperature culture and also correlate with improved recombinant protein productivity. post-translational modifications, particularly glycosylation, which are important for biological activity of recombinant proteins, are maintained or even improved at low temperature. in addition to affecting productivity, low temperature culture has been shown to reduce cellular metabolism, waste accumulation and apoptosis. the molecular mechanisms regulating sub-physiological temperature response of cells in culture are still poorly understood, despite a number of recent transcriptomic and proteomic profiling studies. it is generally accepted that global cap-dependent protein translation decreases at sub-physiological temperatures, however ires mediated cap-independent protein translation is active. more than proteins including cold-inducible rna binding protein, rna binding motif protein, protein disulphide isomerase, nuclear localization sequence binding protein, vimentin, phosphoglycerate kinase and heat shock cognate kda protein have been reported as being up regulated during low temperature culture. of these proteins, crip and rbm have been studied extensively. both cirp and rbm are involved in modulation of transcription and translation by functioning as rna chaperones. crip improves recombinant protein productivity and can arrest cell growth in a cell line-specific manner whereas rbm facilitates cap-independent protein synthesis at low temperature. rbm is also reported to interact with and partially block the activity of mirnas at low temperature. altered mirna expression following temperature shift has recently been reported and may be an important component of regulating protein expression and cell behavior at low temperature. in this study, we have used two-dimensional difference gel electrophoresis to investigate the proteomic changes associated with the shift to low temperature in a biphasic batch culture of suspension-adapted cho-k cells. in this manner we wished to gain insights into the effects of low temperature culture on mammalian cells, with potential relevance to mammalian cell-based recombinant protein production.  cell culture suspension-adapted cho-k cells were seeded at × cells/ml in spinner flasks and maintained at 37°c for hrs or for hrs at 37°c followed by a temperature shift to 31°c for a further hrs. hrs was chosen for this temperature shift because it represents the mid-exponential phase of cell growth. 31°c was selected for the temperature shift as it represents the average temperature employed by others performing similar studies, i.e. 28–33°c. as can be seen in figure the cells that were temperature shifted immediately ceased logarithmic growth and did not exceed a peak viable cell density of × ± cells/ml, whereas the cells maintained in standard culture at 37°c continued in logarithmic growth for a further hrs and achieved a peak viable density of × ± cells/ml. the cells that were temperature shifted displayed a steady if slightly increasing viable cell density throughout, whereas the cells cultured at 37°c had entered the late stationary/death phase of the growth cycle by hrs. cultures were sampled at hrs and hrs for protein extraction from independent experiments. differential analysis of proteins 2d-dige protein expression maps for the biological triplicate samples were generated for both temperature shifted and standard cultures for cho-k cells at hrs and hrs. the hrs pems were then compared to that at hrs using the biological variance analysis module of decyder software. this was aided by the use of a pooled internal standard which facilitated spot matching and relative spot quantitation while minimizing the gel-to-gel variations. spots originating from dust particles and gel impurities in each gel image were checked and removed manually to improve the quantitation and matching process. landmarks, which aid the gel-to-gel spot matching process, were also defined in the gel to increase the accuracy of the matching algorithm. manual checks were carried out to allow for cases where spots were detected more than once, these spots were then merged back to improve spot matching. after spot matching and filtering, and spots were detected for the temperature shifted and standard culture experiments respectively. spots that were fold up/down regulated at hrs compared to hrs with a t-test score of ≤ and present on all gels were considered differentially expressed. a total of de spots were observed in the temperature shift experiment and de spots were observed in the standard culture experiment. spot identification following spot picking and tryptic digestion, protein identification was carried out using maldi-tof ms. all identified proteins had an expectation value of or better. expectation value for proteins was determined by ettan maldi-tof pro evaluation software using the profound database search engine for peptide mass fingerprints. an expectation value of or better means that there is ≤ 1% chance that the identification is random. although many spots resulted in high quality spectra, they were not identified which may be due to insufficient amounts of protein, modification and/or variation of the amino acid sequences in cho proteins or the paucity of cho proteins in the protein databases. despite this proteins were identified and the representative spectra of of these proteins are presented in figure following identification of the spots, all identified proteins were compared for differential expression in both temperature shifted and standard culture experiments to identify temperature-dependent and temperature-independent protein changes. for example, ganab, a glycan-processing enzyme that is involved in glycoprotein quality control in the endoplasmic reticulum, was fold up regulated at hrs compared to hrs following temperature shift while it was unchanged when cells were maintained at 37°c. activator of kda heat shock protein atpase homolog however, was differentially expressed in both experiments, hence could be considered a temperature-independent protein change. a number of the identified proteins including vimentin and gapdh have been previously identified as differentially expressed following temperature shift. the identified differentially expressed proteins were also compared between both temperature shifted and standard cultures at hrs. three proteins, actb, aldh and dpysl were identified at more than one location on the gel possibly indicating some form of post-translational modification, of which ptms in aldh and dpysl were specific to the temperature shift to 31°c. the identified proteins could be linked to a broad range of biological functions including structural, metabolic, differentiation, secretory pathways, translation, transcription, protein binding, signal transduction, cell adhesion and apoptosis. validation of results by western blotting analysis two proteins, vimentin and hnrpc, were selected from the list of differentially expressed identified proteins for validation by western blotting. rbm is already known to be induced following shift to low temperature and was included as a positive control. α-tubulin was used as a loading control. western blot analysis reflected a similar pattern of expression to those observed using 2d-dige. vimentin was fold up regulated following temperature shift and up regulated in standard culture from 2d-dige analysis. densitometry analysis of western blot data revealed that vimentin was fold up regulated following temperature shift and fold down regulated in standard culture. hnrpc was fold down regulated following temperature shift and fold down regulated in standard culture from 2d-dige analysis, while from densitometry analysis of western blot data, it was fold down regulated at low temperature and fold down regulated in standard culture. discussion the incorporation of a temperature reduction step is commonly employed during production cell culture in the biopharmaceutical industry. this temperature shift is used as a means of simultaneously inducing growth arrest and extending long term culture viability thus increasing recombinant protein productivity and yield. to investigate the mechanisms regulating the effects of low temperature in cho-k cells we employed 2d-dige followed by maldi-tof mass spectrometry. when suspension-adapted cho-k cells were cultured at 37°c for hrs and then shifted to low temperature, cell growth was immediately arrested compared to cells maintained in standard cultures. following the temperature shift, the cells were observed to maintain a steady viable cell density whereas the cells at 37°c began entering the death phase by hrs of culture. while the viabilities of both cultures were comparable throughout the hrs of culture this changed towards the end of the experiment for the cells maintained at 37°c. it is possible that the high percentage of serum in the culture medium protected both cultures from apoptotic cell death, therefore maintaining the high viabilities recorded. serum is known to protect cells against apoptosis in suspension culture. following 2d-dige analysis to compare the differences in global protein expression at hrs and hrs of culture, de spots were observed in the temperature shifted cells whereas de spots were observed for the cells maintained in standard culture. it is apparent from this data that cells at the end of a temperature shifted batch culture are more similar to healthy exponentially growing cells than cells which have achieved late stationary/death phase at 37°c. reduced cellular metabolism at low temperature could be responsible for the smaller number of differentially expressed proteins observed after temperature shift in comparison to the cells grown at 37°c. it has been observed that protein synthesis rates are reduced at low temperature due to a general inhibition of cap-dependent protein synthesis. however some proteins, e.g. rbm are translated under cold stress via an ires mediated cap-independent mechanism. this type of protein translation initiation requires both eukaryotic initiation factors and message-specific cellular ires trans-acting factors. eukaryotic translation initiation factor 4a, isoform, an rna helicase enzyme, is one of the critical translational initiation factors that are required in ires mediated cap-independent protein translation. eif4a is fold up regulated following temperature shift. this indicates that eif4a may play an important role in facilitating translation in this system. eif4a has been shown to be up regulated in g arrested cho cells, which may also indicate a role for this protein in the observed growth arrest. of the spots picked, a total of proteins were successfully identified. of these, proteins were specifically altered following temperature shift. two of these proteins were present at more than one location in the gel indicating a low-temperature-specific protein modification. conversely, proteins were differentially regulated specifically in standard culture, of which one protein, actb, was present at two different positions in the gel. grouping of the identified proteins by biological function indicated that proteins involved in metabolism were the dominant group affected in both temperature shifted and standard cultures, followed by structural proteins. the indication that low temperature affects metabolism and structure is in keeping with other studies. however, the number of proteins identified here is insufficient for a comprehensive analysis of the main biological functions affected in this system, as we may be observing a bias towards the identification of the more highly expressed metabolic and structural proteins within the cell such as gapdh and vimentin. the most obvious effect following reduction of culture temperature is the immediate decrease in growth observed. a number of growth-related proteins were identified as being differentially expressed in a temperature-specific manner, including hnrpc, rrn and lgals hnrpc is a nuclear pre-mrna binding protein that enhances the translation of c-myc mrna by interacting with an ires in the 5' utr and thus impacting proliferation. silencing of hnrp c1/c protein expression by sirna has also been shown to result in inhibition of cell proliferation. these results are in keeping with our finding that hnrpc is fold down regulated following reduction of the culture temperature. hnrpc also interacts with iress in the 5'utr of'upstream of n-ras' and affects cell proliferation through iress, including the pitslre ires, which is activated at mitosis. this is important because during mitosis, similar to low temperature culture inductions, protein synthesis is rapidly and severely repressed possibly due to inhibition of global cap-dependent translation. the fact that hnrpc and eif4a are both differentially expressed in this experiment clearly indicates the importance of ires-based regulation of the translational machinery. rrn protein levels were found to be fold down regulated at the end of the temperature shifted culture which is unsurprising as its activity is known to be reduced in stationary cells. lgals has been shown to act as a negative autocrine growth factor that regulates cell proliferation which correlates with the fold up regulation of lgals observed in the temperature shifted samples and growth data. another common feature of cells that are cultured at reduced temperatures is that they display delayed apoptosis compared to cells cultured at 37°c. it has been proposed that the reduction in apoptosis is as a result of the reduced cellular metabolism at low temperatures. however, others have reported the induction of anti-apoptotic proteins following culture at reduced temperature. bcl- was not identified as a de protein in this study but a number of apoptosis-associated proteins were identified including importin-α, nadh dehydrogenase fe-s protein and annexin a importin-α, a nuclear import receptor, facilitates trafficking of the growth promoting ras effector, rassf into the nucleus. over expression of importin-α also results in significant increases in p transcript levels and apoptosis, implicating it in the nuclear import of p. the temperature-specific fold down regulation of importin-α levels therefore may have an impact on both proliferation and protection against apoptosis. unlike importin-α, the other two apoptosis-related proteins annexin a and ndusf were both up regulated, and are positive regulators of apoptosis. annexin a is selectively translocated from the nucleus to the cytosol during apoptosis and is recognized as an early marker of apoptotic cell death. ndufs is part of the mitochondrial complex i. inhibition of ndufs expression by sirna has been shown to provide resistance to ifn-β/retinoic acid-induced apoptosis. the data presented indicates that by the end of the temperature shifted culture the cells were beginning to initiate apoptosis. this is in agreement with other findings that despite the delay in apoptosis, once initiated, the rate and manner of cell death is similar to that observed in standard culture at 37°c. by comparison, cells at hrs in standard culture were already in early death phase. this is likely due to a variety of reasons including nutrient limitation, oxygen depletion and waste accumulation at hrs in standard culture. a number of apoptosis related proteins, including prohibitin, aldehyde dehydrogenase family subfamily a and alpha-enolase were found to be differentially regulated in this system. over expression of phb inhibits cell proliferation by arresting cells in g0/g phase and has also been shown to increase cell survival. aldh1a is a member of aldehyde dehydrogenase family that can oxidize a wide variety of aldehydes to the corresponding carboxylic acids. down regulation of aldh1a has been correlated with the increased susceptibility of cells towards apoptosis. eno catalyses the formation of phosphoenolpyruvate from 2-phosphoglycerate. the over expression of eno has been shown to inhibits cell growth and induce apoptosis in neuroblastoma cells. phb and aldh1a were and fold down regulated respectively, while eno was fold up regulated at hrs at 37°c in comparison to culture at hrs. these observations are in keeping with the reduced viability observed at hrs in standard culture in comparison to temperature shifted cultures. a number of cytoskeletal proteins including vimentin, profilin, beta actin and capping protein muscle z-line beta were differentially expressed following temperature shift and/or in standard culture. vimentin, profilin and beta-actin were also observed to be differentially expressed by baik et. al.. vimentin is one of the major cytoskeletal proteins and its increased expression has previously been shown in the heat-resistant phenotypes of cho cells. vimentin was fold up regulated following temperature shift to 31°c and was unchanged at standard culture. profilin is a small ubiquitous actin monomer sequestering protein that is required for normal actin polymerization in response to thermal stress, and was fold up regulated following temperature shift. however profilin was unchanged at standard culture. actb is involved in many biological functions in cell such as regulating cell shape and growth. increased expression of actb has been shown to provide resistance against apoptosis. actb was fold down regulated at hrs at 37°c and was not significantly altered following temperature shift. capzb binds to one end of actin filaments, regulates actin polymerization including the number and length of actin filaments, and strengthens the actin cytoskeleton in the cytoplasm. capzb was fold down regulated at hrs at 37°c, whereas no change was observed following temperature shift. although the role of vimentin and profilin in regulating low temperature specific phenotypes is unclear at this point, the reduced expression of actb and capzb could possibly be associated with increased apoptosis in standard culture. cellular metabolism is generally reduced in low culture temperature but this does not apply to every enzyme and pathway. gapdh, a multi-functional glycolytic enzyme, was almost 2-fold up regulated following temperature shift. similar results have been reported in other published data. in contrast metabolic enzymes involved in energy generation were down regulated in dying cells which indicates reduced energy generation in standard culture at 37°c. this may be due to depletion of nutrients in the media at hrs of culture. out of identified proteins, proteins were differentially expressed in both the temperature shifted cells and those maintained at 37°c. in most cases the regulation between hrs and hrs was similar in the temperature shifted and standard cultures indicating that these are culture-dependent changes. as with the de proteins identified from the temperature shift, these proteins cover a range of functions including stress response e.g. ahsa of the identified proteins, were found to be differentially expressed between temperature shifted and non-shifted cultures at hrs. three proteins, actb, aldh and dpysl were identified from multiple spots. actb was present at two different pis and molecular weights in the gel containing samples from standard culture. aldh and dpysl were identified at two different pis on gels from the temperature shifted culture suggesting potential low temperature-induced post translational modifications. aldh is a nad+-dependent enzyme that can protect against hyperoxia-induced cell death through reduction of ros, activation of erk/mapk, and pi3k-akt cell survival signalling pathways. analysis of the aldh spots revealed and fold up regulation following temperature shift and no change in the cultures maintained at 37°c. the role of this protein in low temperature response is unknown. dpysl promotes microtubule formation by binding to tubulin heterodimers. different variants, possibly phosphorylated, of dpysl have been reported previously. in this study, dpysl was fold up regulated following temperature shift whereas it was fold up regulated in the cultures maintained at 37°c. however, the second spot identified as dpysl was specifically fold down regulated at 31°c. the functional association of dpysl with low temperature response is unclear. in-silico phosphoproteome analysis predicted that both aldh and dpysl contain phosphorylation sites and hence the multiple spots may indicate that they are phosphorylated. a previous study also identified two proteins as being phosphorylated at tyrosine residues in cho cells cultured at low temperature suggesting that active cell signalling occurs in response to temperature shifts.  in conclusion, the present study has identified a number of temperature-specific as well as temperature-independent changes in protein expression that occur during batch suspension culture. a number of the protein changes can be linked to the growth inhibition observed subsequent to temperature shift. further studies targeting the proteins identified in this study using cho-k cells producing a recombinant protein product will improve the understanding of the cold shock response and may be useful in identifying candidate targets to re-engineer cho cells to produce recombinant therapeutic proteins more efficiently. 
to ensure maximal productivity of recombinant proteins during production culture it is typical to encourage an initial phase of rapid cell proliferation to achieve high biomass followed by a stationary phase where cellular energies are directed towards production of rp. of the proteins identified by maldi-tof ms, were specifically differentially expressed upon reduction of the culture temperature and were found related to a variety of cellular functions such as regulation of growth, cap-independent translation, apoptosis, the cytoskeleton and glycoprotein quality control. these results indicate the extent of the temperature response in cho-k cells and suggest a number of key regulatory proteins and pathways that are involved in modulating the response of cells to mild hypothermia. during many such biphasic cultures, the initial phase of rapid cell growth at 37°c is followed by a growth arrest phase induced through reduction of the culture temperature. low temperature induced growth arrest is associated with many positive phenotypes including increased productivity, sustained viability and an extended production phase, although the mechanisms regulating these phenotypes during mild hypothermia are poorly understood. regulation of these identified proteins and pathways could be useful for future approaches to engineer cho cells for improved recombinant protein production. in this study differential protein expression in suspension cho-k cells was investigated following a reduction of the culture temperature from 37°c to 31°c in comparison to standard batch culture maintained at 37°c using 2d-dige and mass spectrometry.
 to ensure maximal productivity of recombinant proteins during production culture it is typical to encourage an initial phase of rapid cell proliferation to achieve high biomass followed by a stationary phase where cellular energies are directed towards production of rp. during many such biphasic cultures, the initial phase of rapid cell growth at 37°c is followed by a growth arrest phase induced through reduction of the culture temperature. low temperature induced growth arrest is associated with many positive phenotypes including increased productivity, sustained viability and an extended production phase, although the mechanisms regulating these phenotypes during mild hypothermia are poorly understood.  in this study differential protein expression in suspension cho-k cells was investigated following a reduction of the culture temperature from 37°c to 31°c in comparison to standard batch culture maintained at 37°c using 2d-dige and mass spectrometry. there is only limited proteomic analysis of suspension-grown cho cells describing a direct comparison of temperature shifted versus non-temperature shifted cultures using 2d-dige. this investigation has enabled the identification of temperature-dependent as well as temperature-independent proteomic changes. proteins were observed as differentially expressed following temperature shift, of which were up regulated. of the proteins identified by maldi-tof ms, were specifically differentially expressed upon reduction of the culture temperature and were found related to a variety of cellular functions such as regulation of growth, cap-independent translation, apoptosis, the cytoskeleton and glycoprotein quality control.  these results indicate the extent of the temperature response in cho-k cells and suggest a number of key regulatory proteins and pathways that are involved in modulating the response of cells to mild hypothermia. regulation of these identified proteins and pathways could be useful for future approaches to engineer cho cells for improved recombinant protein production. 