Dynamics of lipids and metabolites during the cell cycle of Chlamydomonas reinhardtii.

Metabolites and lipids are the final products of enzymatic processes, distinguishing the different cellular functions and activities of single cells or whole tissues. Understanding these cellular functions within a well-established model system requires a systemic collection of molecular and physiological information. In the current report, the green alga Chlamydomonas reinhardtii was selected to establish a comprehensive workflow for the detailed multi-omics analysis of a synchronously growing cell culture system. After implementation and benchmarking of the synchronous cell culture, a two-phase extraction method was adopted for the analysis of proteins, lipids, metabolites and starch from a single sample aliquot of as little as 10-15 million Chlamydomonas cells. In a proof of concept study, primary metabolites and lipids were sampled throughout the diurnal cell cycle. The results of these time-resolved measurements showed that single compounds were not only coordinated with each other in different pathways, but that these complex metabolic signatures have the potential to be used as biomarkers of various cellular processes. Taken together, the developed workflow, including the synchronized growth of the photoautotrophic cell culture, in combination with comprehensive extraction methods and detailed metabolic phenotyping has the potential for use in in-depth analysis of complex cellular processes, providing essential information for the understanding of complex biological systems.

Application of two-dimensional gel-based mass spectrometry to functionally dissect resistance to targeted cancer therapy.

PURPOSE: The majority of gastric cancers are diagnosed at advanced stages, characterized by robust therapy resistance. The oncoprotein hypoxia-inducible factor 1 (HIF-1) is associated with therapy resistance, partly via activation of the DNA damage response. We have noted a robust ability of gastric cancer cells to functionally compensate the loss of HIF-1 in vitro. The purpose of this study was to identify molecular pathways that underlie this compensation. EXPERIMENTAL DESIGN: We performed 2DE to compare the nuclear proteome of wild-type and HIF-1-deficient gastric cancer cells. Differently expressed protein spots were identified via MS). After bioinformatic evaluation, functional validation of selected identified pathways was performed. RESULTS: 2DE displayed a total of 2523 protein spots, from which 87 were identified as regulated by HIF-1. Seventy of the identified spots were different proteins and 17 were isoforms. Bioinformatic analyses revealed that a significant amount of the identified proteins were related to cellular survival pathways. Specifically, members of the proteasome pathway were found upregulated upon loss of HIF-1. Combined inhibition of HIF-1 and the proteasome inflicted significant DNA damage, supporting the hypothesis that the proteasome is of functional importance to compensate the loss of HIF-1. CONCLUSIONS AND CLINICAL RELEVANCE: Our data show robust and functional changes of the nuclear proteome upon inactivation of the HIF-1 oncoprotein in gastric cancer cells. We propose that 2DE-MS represents a useful tool to functionally dissect resistance mechanisms to targeted therapy and to identify novel targets for antiproliferative combination therapy.

PROTEOMER: A workflow-optimized laboratory information management system for 2-D electrophoresis-centered proteomics.

In recent years proteomics became increasingly important to functional genomics. Although a large amount of data is generated by high throughput large-scale techniques, a connection of these mostly heterogeneous data from different analytical platforms and of different experiments is limited. Data mining procedures and algorithms are often insufficient to extract meaningful results from large datasets and therefore limit the exploitation of the generated biological information. In our proteomic core facility, which almost exclusively focuses on 2-DE/MS-based proteomics, we developed a proteomic database custom tailored to our needs aiming at connecting MS protein identification information to 2-DE derived protein expression profiles. The tools developed should not only enable an automatic evaluation of single experiments, but also link multiple 2-DE experiments with MS-data on different levels and thereby helping to create a comprehensive network of our proteomics data. Therefore the key feature of our "PROTEOMER" database is its high cross-referencing capacity, enabling integration of a wide range of experimental data. To illustrate the workflow and utility of the system, two practical examples are provided to demonstrate that proper data cross-referencing can transform information into biological knowledge.

Proteomic shifts in embryonic stem cells with gene dose modifications suggest the presence of balancer proteins in protein regulatory networks.

Large numbers of protein expression changes are usually observed in mouse models for neurodegenerative diseases, even when only a single gene was mutated in each case. To study the effect of gene dose alterations on the cellular proteome, we carried out a proteomic investigation on murine embryonic stem cells that either overexpressed individual genes or displayed aneuploidy over a genomic region encompassing 14 genes. The number of variant proteins detected per cell line ranged between 70 and 110, and did not correlate with the number of modified genes. In cell lines with single gene mutations, up and down-regulated proteins were always in balance in comparison to parental cell lines regarding number as well as concentration of differentially expressed proteins. In contrast, dose alteration of 14 genes resulted in an unequal number of up and down-regulated proteins, though the balance was kept at the level of protein concentration. We propose that the observed protein changes might partially be explained by a proteomic network response. Hence, we hypothesize the existence of a class of "balancer" proteins within the proteomic network, defined as proteins that buffer or cushion a system, and thus oppose multiple system disturbances. Through database queries and resilience analysis of the protein interaction network, we found that potential balancer proteins are of high cellular abundance, possess a low number of direct interaction partners, and show great allelic variation. Moreover, balancer proteins contribute more heavily to the network entropy, and thus are of high importance in terms of system resilience. We propose that the "elasticity" of the proteomic regulatory network mediated by balancer proteins may compensate for changes that occur under diseased conditions.

High MS-compatibility of silver nitrate-stained protein spots from 2-DE gels using ZipPlates and AnchorChips for successful protein identification.

The availability of easy-to-handle, sensitive, and cost-effective protein staining protocols for 2-DE, in conjunction with a high compatibility for subsequent MS analysis, is still a prerequisite for successful proteome research. In this article we describe a quick and easy-to-use methodological protocol based on sensitive, homogeneous, and MS-compatible silver nitrate protein staining, in combination with an in-gel digestion, employing the Millipore 96-well ZipPlate system for peptide preparation. The improved quality and MS compatibility of the generated protein digests, as compared to the otherwise weakly MS-compatible silver nitrate staining, were evaluated on real tissue samples by analyzing 192 Coomassie-stained protein spots against their counterparts from a silver-stained 2-DE gel. Furthermore, the applicability of the experimental setup was evaluated and demonstrated by the analysis of a large-scale MALDI-TOF MS experiment, in which we analyzed an additional ~1000 protein spots from 2-DE gels from mouse liver and mouse brain tissue.

Study of posttranslational modifications in lenticular alphaA-Crystallin of mice using proteomic analysis techniques.

In the present work the complexity in the 2D-gel protein pattern of murin lenticular alphaA-Crystallin was analyzed. An in depth study of the different protein isoforms was done combining different proteomic tools. Lens proteins of four different ages, from embryo to 100-week-old mice, were separated by large 2D-PAGE, revealing an increase in the number and intensity of the spots of alphaA-Crystallin during the process of aging. For further analyses the oldest mice were chosen. Comparison and evaluation of two different staining methods proved Imidazole-Zinc to be a good alternative to the generally used Coomassie stain. The characterization of the different alphaA-Crystallin protein species was done using nanoLC-ESI-MS/MS (liquid chromatography electrospray ionisation tandem mass spectrometry). Data interpretation was done by database searching, manual validation and a new MS/MS-interpretation tool for posttranslational modifications--the PTM-Explorer. Using this way, eight different phosphorylation sites were identified and localized; the identification of four of them was not published so far. Furthermore, quantitative N-terminal acetylation of alphaA-Crystallin and variable C-terminal truncation was observed, also not published in this extent yet. The results of the mass spectrometric analysis were validated by immunoblotting experiments using two different alphaA-Crystallin specific antibodies. In addition, a fluorescent phospho-specific stain was used to detect the protein spots including phosphorylation groups. Re-separation 2D-PAGE was done to round off the present study and explain the appearance of some of the protein spots in the gel as artifacts of the 2D-PAGE separation.

No adaptation in the amplitude modulation domain in trained listeners.

The present study shows that on average, exposure to a 15 min, 5 kHz tone modulated sinusoidally in amplitude at 16 Hz with a 100% depth does not affect significantly amplitude modulation (AM) detection thresholds measured between 4 and 64 Hz when listeners are extensively trained to the AM detection task, with and without adaptor before data collection. These results are compatible with previous work given that a clear 6-dB adaptation effect was observed during the first pilot trials. However, the results reveal that adaptation effects are not robust, and suggest that the mechanisms underlying adaptation to AM must be reevaluated.

Identification of phosphorylation and acetylation sites in alphaA-crystallin of the eye lens ( mus musculus) after two-dimensional gel electrophoresis.

Posttranslational modifications are of great interest because of their relevance in biological systems as proteins are commonly activated or deactivated by phosphorylation, glycation and acetylation [1, 2]. During eye lens aging the number of the alphaA-crystallin isoproteins increases. This could be observed by the use of 2D-PAGE (two-dimensional gel electrophoresis). The number of alphaA-crystallin spots in the gel increased during eye lens aging. For further analysis the spots of 2D-PAGE were cut out and the identification of the proteins was done using nanoLC-ESI-MS/MS (liquid chromatography electrospray ionization tandem mass spectrometry). The created MS/MS-data were analyzed using the Sequest algorithm. Searches with different parameters were done to preferably get the complete sequence coverage and to identify posttranslational modifications of the alphaA-crystallin. The acetylated N-terminus of this protein could be detected. Furthermore, phosphorylation of serine 122 and 148 was identified in two different spots.

Genetic analysis of the mouse brain proteome.

Proteome analysis is a fundamental step in systematic functional genomics. Here we have resolved 8,767 proteins from the mouse brain proteome by large-gel two-dimensional electrophoresis. We detected 1,324 polymorphic proteins from the European collaborative interspecific backcross. Of these, we mapped 665 proteins genetically and identified 466 proteins by mass spectrometry. Qualitatively polymorphic proteins, to 96%, reflect changes in conformation and/or mass. Quantitatively polymorphic proteins show a high frequency (73%) of allele-specific transmission in codominant heterozygotes. Variations in protein isoforms and protein quantity often mapped to chromosomal positions different from that of the structural gene, indicating that single proteins may act as polygenic traits. Genetic analysis of proteomes may detect the types of polymorphism that are most relevant in disease-association studies.