Helmut Zengerling - Academia.edu (original) (raw)

Uploads

Papers by Helmut Zengerling

Research paper thumbnail of Serial Immobilized pH Gradient Isoelectric Focusing over pH 4–9

Springer Protocols Handbooks, 2009

ABSTRACT It has long been apparent that the separation capacity of conventional 18 cm IPGs as the... more ABSTRACT It has long been apparent that the separation capacity of conventional 18 cm IPGs as the first separation step in two dimensional polyacrylamide gel electro-phoresis (2D-PAGE) was theoretically incapable of separating the complexity that is to be expected by the protein species of a mammalian cell line or tissue (1). We first attempted to improve the resolution of 2D-PAGE by developing IPGs of 54 cm in length (2). While this strategy was successful, it required the laborintensive preparation and batchwise validation of own-caste IPGs for each experiment. Subsequently we found that qualitatively similar excellent results could be obtained by placing commercially available IPGs in series (end to end) during IEF. We submitted this method as a patent application on September 12, 2003 (WO2005026715, but since allowed to lapse in all countries but Germany) and published it in the scientific literature in August 2005 (3). The main advantage of this analytical strategy is that less sample is consumed per high resolution protein separation as compared to the alternative use of conventional combinations of individual narrow pH range IPGs for IEF. Thus, the system is well suited to the optimal analysis of samples that are limited in amount, as we have since demonstrated in a number of analytical investigations, including cases where samples from small human clinical biopsies were employed (4–6). Farhoud and colleagues independently published an essentially identical serial IEF method and device (7), also emphasizing the maximal resolution and yield of minimally consumed scarce samples. That paper was submitted for publication in July 2005, four months after the publication of our patent application.

Research paper thumbnail of Unexpected common mechanistic pathways for embryotoxicity of warfarin and lovastatin

Reproductive Toxicology, 2010

Novel molecular content for fast in vitro strategies in the context of safety tests concerning de... more Novel molecular content for fast in vitro strategies in the context of safety tests concerning developmental toxicity has a potential to substantially reduce animal experiments according to the "3R" concept (Reduce/Refine/Replace). Here we present and discuss data from a differential proteomic profiling of samples generated using embryonic stem cell drived in vitro models treated with a set of model substances. Among substance-dependent proteomic changes, potential surrogate markers were some isoforms of heat shock proteins and a component of the Ras pathway, present in several redundant isoforms due to posttranslational modifications. Both proteins are implicated in cell migration, cell survival, growth and embryonic development.

Research paper thumbnail of Protein Biomarkers for in Vitro Testing of Embryotoxicity

Journal of Proteome Research, 2010

There are new challenges for hazard and risk assessment in the chemical industry with regard to R... more There are new challenges for hazard and risk assessment in the chemical industry with regard to REACH legislation in Europe and related activities in the US and Japan, which require the development of novel in vitro models for the molecular characterization of drug-or chemical-related effects. In particular the investigation of developmental toxicity so far is regulated by guide lines requiring huge numbers of test animals. In the frame of a European FP6 project on reproductive toxicology (www.reprotect.eu) we prepared protein samples from mouse embryonic stem cells differentiated into contracting cardiomyocytes according to the validated embryonic stem cell test (EST) protocol, which had been exposed to toxic substances selected by an expert committee from different in vivo categories of embryotoxicity. Lysates were used to carry out the following investigations: (i) identify optimal dose range conditions in the EST that are suitable for (ii) performing a differential quantitative proteomic study of underlying molecular pathways, (iii) define classes of substances with similar proteomic patterns, (iv) relate these classes to the traditional in vivo categories of embryotoxicity with (v) the final goal to identify novel surrogate protein biomarkers for embryo toxicity. We found two distinct classes of toxic substances with clear pathway-related differences.

Research paper thumbnail of A Connection between the Mitochondrial Permeability Transition Pore, Autophagy, and Cerebral Amyloidogenesis

Journal of Proteome Research, 2008

In a drug reprofiling attempt, we explored novel neuroprotective properties of 4-azasteroids by s... more In a drug reprofiling attempt, we explored novel neuroprotective properties of 4-azasteroids by synthesizing chemical affinity tags capturing adenine nucleotide translocator-1, as a potential target. Dutasteride inhibits the mitochondrial transition pore and induces an increase of autophagosomal structures in human cell lines. In vivo, a surprising reduction of the beta-amyloid plaque load in a model for cerebral amyloidosis appears to connect release of neurotoxic peptides, mitochondrial apoptosis and autophagy.

Research paper thumbnail of Isoelectric focusing in serial immobilized pH gradient gels to improve protein separation in proteomic analysis

ELECTROPHORESIS, 2005

We previously demonstrated the separation of proteins by isoelectric focusing (IEF) over pH 4-8 i... more We previously demonstrated the separation of proteins by isoelectric focusing (IEF) over pH 4-8 immobilized pH gradients (IPGs) over 54 cm (Poland et al., Electrophoresis 2003, 24, 1271. Here we show that similar results can be conveniently achieved using commercially available IPGs of appropriate pH ranges positioned end-on-end in series during electrophoresis, which we term 'daisy chain IEF'. Proteins efficiently electrophorese from one IPG to another during IEF by traversing buffer-filled porous bridges between the serial IPGs. A variety of materials can function as bridges, including paper, polyacrylamide gels or even IPGs. The quality of two-dimensional (2-D) protein patterns is not apparently worse than that generated by conventional IEF using the same individual IPGs. A major advantage of this method is that sample is consumed efficiently, without the requirement for preliminary steps, such as chamber IEF. This advantage is pronounced when working with extremely limited sources of samples, such as with clinical biopsies or cellular subfractions. The present study was limited by the commercial availability of suitable pH gradients. Proteomics analyses could be further improved if commercial vendors would manufacture IPGs with suitable pH ranges to achieve high resolution (,100 cm) IEF separation of proteins in one electrophoretic step over the pH range 2-12. Electrophoresis 2005, 26, 3185-3190 3185

Research paper thumbnail of Comparative Profiling of the Mammalian Mitochondrial Proteome:  Multiple Aconitase-2 Isoforms Including N -formylkynurenine Modifications as Part of a Protein Biomarker Signature for Reactive Oxidative Species

Journal of Proteome Research, 2006

The activity of mitochondria induces, as a byproduct, a variety of post-translational modificatio... more The activity of mitochondria induces, as a byproduct, a variety of post-translational modifications in associated proteins, which have functional downstream consequences for processes such as apoptosis, autophagy, and plasticity; e.g., reactive oxygen species (ROS), which induce N-formyl-kynurenine from oxidized tryptophans in certain mitochondrial proteins which are localized in close spatial proximity to their source. This type of fast molecular changes has profound influence on cell death and survival with implications in a number of pathologies. The quantitative and differential analysis of bovine heart mitochondria by four 2D-PAGE methods, including 2D-PAGE with high-resolution IEF as first dimension, revealed that due to limited resolution, those methods employing blue native-, tricine-urea-, and 16-BAC-PAGE as the first dimension are less applicable for the differential quantitative analysis of redundant protein spots which might give insight into post-translational modifications that are relevant in age- and stress-related changes. Moreover, 2D-PAGE with high resolution IEF was able to resolve a surprisingly large number of membrane proteins from mitochondrial preparations. For aconitase-2, an enzyme playing an important role in mitochondrial aging, a more thorough molecular analysis of all separable isoforms was performed, leading to the identification of two particular N-formylkynurenine modifications. Next to protein redundancy, native protein-protein interactions, with the potential of relating certain post-translational modification patterns to distinct oligomeric states, e.g., oxidative phosphorylation super complexes, might provide novel and (patho-) physiologically relevant information. Among proteins identified, 14 new proteins (GenBank entries), previously not associated with mitochondria, were found.

Research paper thumbnail of Serial Immobilized pH Gradient Isoelectric Focusing over pH 4–9

Springer Protocols Handbooks, 2009

ABSTRACT It has long been apparent that the separation capacity of conventional 18 cm IPGs as the... more ABSTRACT It has long been apparent that the separation capacity of conventional 18 cm IPGs as the first separation step in two dimensional polyacrylamide gel electro-phoresis (2D-PAGE) was theoretically incapable of separating the complexity that is to be expected by the protein species of a mammalian cell line or tissue (1). We first attempted to improve the resolution of 2D-PAGE by developing IPGs of 54 cm in length (2). While this strategy was successful, it required the laborintensive preparation and batchwise validation of own-caste IPGs for each experiment. Subsequently we found that qualitatively similar excellent results could be obtained by placing commercially available IPGs in series (end to end) during IEF. We submitted this method as a patent application on September 12, 2003 (WO2005026715, but since allowed to lapse in all countries but Germany) and published it in the scientific literature in August 2005 (3). The main advantage of this analytical strategy is that less sample is consumed per high resolution protein separation as compared to the alternative use of conventional combinations of individual narrow pH range IPGs for IEF. Thus, the system is well suited to the optimal analysis of samples that are limited in amount, as we have since demonstrated in a number of analytical investigations, including cases where samples from small human clinical biopsies were employed (4–6). Farhoud and colleagues independently published an essentially identical serial IEF method and device (7), also emphasizing the maximal resolution and yield of minimally consumed scarce samples. That paper was submitted for publication in July 2005, four months after the publication of our patent application.

Research paper thumbnail of Unexpected common mechanistic pathways for embryotoxicity of warfarin and lovastatin

Reproductive Toxicology, 2010

Novel molecular content for fast in vitro strategies in the context of safety tests concerning de... more Novel molecular content for fast in vitro strategies in the context of safety tests concerning developmental toxicity has a potential to substantially reduce animal experiments according to the "3R" concept (Reduce/Refine/Replace). Here we present and discuss data from a differential proteomic profiling of samples generated using embryonic stem cell drived in vitro models treated with a set of model substances. Among substance-dependent proteomic changes, potential surrogate markers were some isoforms of heat shock proteins and a component of the Ras pathway, present in several redundant isoforms due to posttranslational modifications. Both proteins are implicated in cell migration, cell survival, growth and embryonic development.

Research paper thumbnail of Protein Biomarkers for in Vitro Testing of Embryotoxicity

Journal of Proteome Research, 2010

There are new challenges for hazard and risk assessment in the chemical industry with regard to R... more There are new challenges for hazard and risk assessment in the chemical industry with regard to REACH legislation in Europe and related activities in the US and Japan, which require the development of novel in vitro models for the molecular characterization of drug-or chemical-related effects. In particular the investigation of developmental toxicity so far is regulated by guide lines requiring huge numbers of test animals. In the frame of a European FP6 project on reproductive toxicology (www.reprotect.eu) we prepared protein samples from mouse embryonic stem cells differentiated into contracting cardiomyocytes according to the validated embryonic stem cell test (EST) protocol, which had been exposed to toxic substances selected by an expert committee from different in vivo categories of embryotoxicity. Lysates were used to carry out the following investigations: (i) identify optimal dose range conditions in the EST that are suitable for (ii) performing a differential quantitative proteomic study of underlying molecular pathways, (iii) define classes of substances with similar proteomic patterns, (iv) relate these classes to the traditional in vivo categories of embryotoxicity with (v) the final goal to identify novel surrogate protein biomarkers for embryo toxicity. We found two distinct classes of toxic substances with clear pathway-related differences.

Research paper thumbnail of A Connection between the Mitochondrial Permeability Transition Pore, Autophagy, and Cerebral Amyloidogenesis

Journal of Proteome Research, 2008

In a drug reprofiling attempt, we explored novel neuroprotective properties of 4-azasteroids by s... more In a drug reprofiling attempt, we explored novel neuroprotective properties of 4-azasteroids by synthesizing chemical affinity tags capturing adenine nucleotide translocator-1, as a potential target. Dutasteride inhibits the mitochondrial transition pore and induces an increase of autophagosomal structures in human cell lines. In vivo, a surprising reduction of the beta-amyloid plaque load in a model for cerebral amyloidosis appears to connect release of neurotoxic peptides, mitochondrial apoptosis and autophagy.

Research paper thumbnail of Isoelectric focusing in serial immobilized pH gradient gels to improve protein separation in proteomic analysis

ELECTROPHORESIS, 2005

We previously demonstrated the separation of proteins by isoelectric focusing (IEF) over pH 4-8 i... more We previously demonstrated the separation of proteins by isoelectric focusing (IEF) over pH 4-8 immobilized pH gradients (IPGs) over 54 cm (Poland et al., Electrophoresis 2003, 24, 1271. Here we show that similar results can be conveniently achieved using commercially available IPGs of appropriate pH ranges positioned end-on-end in series during electrophoresis, which we term 'daisy chain IEF'. Proteins efficiently electrophorese from one IPG to another during IEF by traversing buffer-filled porous bridges between the serial IPGs. A variety of materials can function as bridges, including paper, polyacrylamide gels or even IPGs. The quality of two-dimensional (2-D) protein patterns is not apparently worse than that generated by conventional IEF using the same individual IPGs. A major advantage of this method is that sample is consumed efficiently, without the requirement for preliminary steps, such as chamber IEF. This advantage is pronounced when working with extremely limited sources of samples, such as with clinical biopsies or cellular subfractions. The present study was limited by the commercial availability of suitable pH gradients. Proteomics analyses could be further improved if commercial vendors would manufacture IPGs with suitable pH ranges to achieve high resolution (,100 cm) IEF separation of proteins in one electrophoretic step over the pH range 2-12. Electrophoresis 2005, 26, 3185-3190 3185

Research paper thumbnail of Comparative Profiling of the Mammalian Mitochondrial Proteome:  Multiple Aconitase-2 Isoforms Including N -formylkynurenine Modifications as Part of a Protein Biomarker Signature for Reactive Oxidative Species

Journal of Proteome Research, 2006

The activity of mitochondria induces, as a byproduct, a variety of post-translational modificatio... more The activity of mitochondria induces, as a byproduct, a variety of post-translational modifications in associated proteins, which have functional downstream consequences for processes such as apoptosis, autophagy, and plasticity; e.g., reactive oxygen species (ROS), which induce N-formyl-kynurenine from oxidized tryptophans in certain mitochondrial proteins which are localized in close spatial proximity to their source. This type of fast molecular changes has profound influence on cell death and survival with implications in a number of pathologies. The quantitative and differential analysis of bovine heart mitochondria by four 2D-PAGE methods, including 2D-PAGE with high-resolution IEF as first dimension, revealed that due to limited resolution, those methods employing blue native-, tricine-urea-, and 16-BAC-PAGE as the first dimension are less applicable for the differential quantitative analysis of redundant protein spots which might give insight into post-translational modifications that are relevant in age- and stress-related changes. Moreover, 2D-PAGE with high resolution IEF was able to resolve a surprisingly large number of membrane proteins from mitochondrial preparations. For aconitase-2, an enzyme playing an important role in mitochondrial aging, a more thorough molecular analysis of all separable isoforms was performed, leading to the identification of two particular N-formylkynurenine modifications. Next to protein redundancy, native protein-protein interactions, with the potential of relating certain post-translational modification patterns to distinct oligomeric states, e.g., oxidative phosphorylation super complexes, might provide novel and (patho-) physiologically relevant information. Among proteins identified, 14 new proteins (GenBank entries), previously not associated with mitochondria, were found.