Raimund Hoffrogge

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

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. Biotechnol. Bioeng. 2012; 109:1386–1394.

2-DE Proteomic Profiling of Neuronal Stem Cells

Proteomics has become a powerful tool in neuroscience studies. Although numerous human neural stem cells are available for research purposes since many years, there exists only limited information on proteomic data from stable neural stem cell lines. Profiling and functional proteome studies of neuronal stem cells will help to describe the protein inventory as well as protein activity and interactions, subcellular localization and posttranslational modifications. The proteomic analysis of neuronal differentiation processes will elucidate the complex events leading to the generation of different phenotypes via distinctive developmental programs that control self-renewal, differentiation, and plasticity. Using the ReNcell VM197 model, a cell line derived from human fetal ventral mesencephalon stem cells, we studied the protein inventory of the stem cells by 2-DE gel electrophoresis and mass spectrometric protein identification and constructed a 2-DE protein map consisting of more than 400 identified protein spots. This proteome reference database constitutes the basis for further investigations of differential protein expression during differentiation. A profiling of the neuronal differentiation-associated changes displayed the large rearrangement of the proteome during this process, and the proteomic techniques proved to be a valuable tool for the elucidation of neuronal differentiation process and for target protein screening.

The DNA methylation landscape of Chinese hamster ovary (CHO) DP-12 cells

Chinese hamster ovary (CHO) cells represent the most commonly used production cell line for therapeutic proteins. By recent genome and transcriptome sequencing a basis was created for future investigations of genotype-phenotype relationships and for improvement of CHO cell productivity and product quality. In this context information is missing about DNA cytosine methylation as a crucial epigenetic modification and an important element in mammalian genome regulation and development. Here, we present the first DNA methylation map of a CHO cell line in single-base resolution that was generated by whole genome bisulfite sequencing combined with gene expression analysis by CHO microarrays. We show CHO DP-12 cells to exhibit global hypomethylation compared to a majority of mammalian methylomes and hypermethylation of CpG-dense regions at gene promoters called CpG islands. We also observed partially methylated domains that cover 62% of the CHO DP-12 cell genome and contain functional clusters of genes. Gene expression analysis showed these clusters to be either highly or weakly expressed with regard to CHO-specific characteristics and hence proves DNA methylation in CHO cells to be an important link between genomics and transcriptomics.

Interferon-β modulates protein synthesis in the central nervous system

Interferon-beta (IFN-beta), acting canonically via the modulation of transcription, affects neocortical pyramidal neurons. By use of 2-D differential gel electrophoresis and subsequent mass spectrometry we identified IFN-beta regulated proteins in the central nervous system. These proteins are involved in cytoskeleton assembly, protein transport and nucleotide metabolism and, as such, serve regenerative and protective functions. Electrophysiologically, IFN-beta mediated protein synthesis is essential for part of the excitatory neuronal effects, as revealed under blockade of protein biosynthesis. This study presents novel effects of IFN-beta in the central nervous system and begins to unravel the mechanism behind the known excitability changes in neurons.

2D-DIGE screening of high-productive CHO cells under glucose limitation--basic changes in the proteome equipment and hints for epigenetic effects.

CHO derivates (Chinese hamster ovary) belong to the most important mammalian cells for industrial recombinant protein production. Many efforts have been made to improve productivity and stability of CHO cells in bioreactor processes. Here, we followed up one barely understood phenomenon observed with process optimizations: a significantly increased cell-specific productivity in late phases of glucose-limited perfusion cultivations, when glucose (and lactate) reserves are exhausted. Our aim was to elucidate the cellular activities connected to the metabolic shift from glucose surplus to glucose limitation phase. With 2D-DIGE, we compared three stages in a perfusion culture of CHO cells: the initial growth with high glucose concentration and low lactate production, the second phase with glucose going to limitation and high lactate level, and finally the state of glucose limitation and also low lactate concentration but increased cell-specific productivity. With our proteomic approach we were able to demonstrate consequences of glucose limitation for the protein expression machinery which also could play a role for a higher recombinant protein production. Most interestingly, we detected epigenetic effects on the level of proteins involved in histone modification (HDAC1/-2, SET, RBBP7, DDX5). Together with shifts in the protein inventory of energy metabolism, cytoskeleton and protein expression, a picture emerges of basic changes in the cellular equipment under long-term glucose limitation of CHO cells.

CellViCAM--Cell viability classification for animal cell cultures using dark field micrographs.

Online monitoring of cell density and cell viability is a challenging but essential task to control and optimize biotechnical processes and is of particular interest for the growing field of animal cell cultures. For this purpose, we introduce an optical approach for automated cell detection and viability classification of suspended mammalian cells. Our proposed system CellViCAM is capable of evaluating dark field micrographs by means of several image processing and supervised machine learning techniques without the use of any dyes or fluorescent labeling. Using a human cell line as the reference culture, an efficient cell detection procedure has been established also enabling a cell density estimation. Furthermore, a comprehensive but reagent-free viability analysis, based on a semi-automatic training data generation, has been developed. By means of an extensive validation dataset we can show that the CellViCAM approach can be considered as an equivalent to staining-based methods and moreover, how it provides a technical platform for a more differentiated cell state classification into living, necrotic, early and late apoptosis.

Neuroproteomics in stem cell differentiation.

The term “proteome” is used to describe the entire complement of proteins in a given organism or in a system at a given time. Proteome analysis in neuroscience, also called “neuroproteomics” or “neuromics” is in its initial stage, and shows a deficit of studies in the context of brain development. It is the main objective of this review to illustrate the potential of neuroproteomics as a tool to unravel the differentiation of neural stem or progenitor cells to terminally differentiated neurons. Experimental results regarding the rat striatal progenitor model cell line ST14A are presented to illustrate the large rearrangements of the proteome during the differentiation process of neural progenitor cells and their modification by neurotrophic factors like the glial cell line‐derived neurotrophic factor (GDNF). Thereby native stem cells and cells transfected with GDNF gene were investigated at the proliferative state and at seven time points up to 72 h after induction of differentiation. In addition, the immortalized human fetal midbrain stem cell line ReNcell VM was analyzed in order to detect stem cell differentiation associated changes of the protein profile. This review gives also an outlook on technical improvements and perspectives of application of neural stem cell proteomics.

Surface Plasmon Resonance Spectroscopy (SPR) Interaction Studies of the Circadian‐Controlled Tomato LHCa4*1 (CAB 11) Protein with Its Promoter

Feedback regulation is an important biochemical mechanism which is also able to direct the circadian timing at the transcriptional level. Independent investigations highlighted a conserved ca. 10 nucleotide motif present in many circadian regulated Lhc genes. Two of such nucleotide motifs exist within 119 nucleotides of the Lhca4*1 promoter from tomato. This promoter fragment was used as a bait in a yeast one hybrid screen and interestingly a clone encoding with sequence identity to the LHCa4*1 protein was isolated as an interaction partner. The LHCa4*1 protein was heterologous expressed and binding to the 119 bp promoter fragment was demonstrated by surface plasmon resonance spectroscopy (SPR, Biacore). This result allows to postulate an autoregulatory feedback loop involved in expression of the Lhca4*1 gene.

Process boundaries of irreversible scCO2 -assisted phase separation in biphasic whole-cell biocatalysis

The formation of stable emulsions in biphasic biotransformations catalyzed by microbial cells turned out to be a major hurdle for industrial implementation. Recently, a cost‐effective and efficient downstream processing approach, using supercritical carbon dioxide (scCO2) for both irreversible emulsion destabilization (enabling complete phase separation within minutes of emulsion treatment) and product purification via extraction has been proposed by Brandenbusch et al. (2010). One of the key factors for a further development and scale‐up of the approach is the understanding of the mechanism underlying scCO2‐assisted phase separation. A systematic approach was applied within this work to investigate the various factors influencing phase separation during scCO2 treatment (that is pressure, exposure of the cells to CO2, and changes of cell surface properties). It was shown that cell toxification and cell disrupture are not responsible for emulsion destabilization. Proteins from the aqueous phase partially adsorb to cells present at the aqueous‐organic interface, causing hydrophobic cell surface characteristics, and thus contribute to emulsion stabilization. By investigating the change in cell‐surface hydrophobicity of these cells during CO2 treatment, it was found that a combination of catastrophic phase inversion and desorption of proteins from the cell surface is responsible for irreversible scCO2 mediated phase separation. These findings are essential for the definition of process windows for scCO2‐assisted phase separation in biphasic whole‐cell biocatalysis. Biotechnol. Bioeng. 2015;112: 2316–2323.

Label-free protein quantification of sodium butyrate treated CHO cells by ESI-UHR-TOF-MS

Effects of butyrate on CHO producer cells are contradictory, promoting productivity and at the same time repressing proliferation. Though in previous omics studies the background of butyrate impact on producer cells has been investigated, the knowledge about the mechanism is still very limited. As previous proteomic results on this field are mainly based on 2DE-gels, we conducted a label-free MS quantification, based on fast high resolution ESI-MS and a straight forward software solution, to gain insight in shifted cellular processes of CHO cells 25h after butyrate treatment. 118 proteins or subunits with significantly altered abundances were identified suggesting changes in carbohydrate, protein metabolic and cell cycle processes. Effects of butyrate on the nucleosome assembly as a known direct epigenetic influence on HDAC activity turned out to be unexpectedly fast and persistent, as confirmed by Western blots of histone-H4 acetylation. Contradictory to increased cell specific productivity, most elements of protein metabolism exhibited decreased levels after butyrate treatment. In comparison to published results some overlap of our label free MS data could be observed but also apparently diverging findings, showing the need for complementary omics techniques for a holistic view on cellular processes such as response to butyrate.