Heidrun Rhode - Academia.edu (original) (raw)

Papers by Heidrun Rhode

A multidimensional chromatographic method is presented which is adapted for fractionation of body... more A multidimensional chromatographic method is presented which is adapted for fractionation of body fluids for proteomic analysis and biomarker search. This method combines native size exclusion (SEC, first dimension, 1D), followed by anion exchange (AEC, 2D) and lectin affinity (LAC, 3D) chromatography. After serial 1D-fractionation, all further procedures are performed in microplate format. Thus, beginning with 2D-fractionation, parallelization and automation is achieved for separation, spectrophotometric readout, temporary storage, hit picking, medium exchange, digest, desalting, and finally storage within an autosampler of LC-MS. The central unit is an automated workstation enabling automated multichannel pipetting and robotic handling of microplates, reservoirs, and column arrays. The whole fractionation process, hit picking and analytics are controlled by adapted software packages. Some tools and tricks have been developed in order to improve sample preparation between fractiona...

Clinica Chimica Acta, Mar 1, 1999

ABSTRACT

Journal of Biomolecular Screening, 2007

How to effectively mix small volumes of liquids within microplate wells is a still underestimated... more How to effectively mix small volumes of liquids within microplate wells is a still underestimated and often neglected challenge. The method the authors introduce here relies on violent turbulent motion within a liquid caused by spotting an organic solvent drop onto its surface. The amount needed, less than 1 to 3 µL, is generally small enough not to alter bioactive molecules. Moreover, a solvent may be selected for its compatibility with assay components. The method was tested with layers of aqueous liquids that differ in pH and concentration of a pH-dependent dye, allowing mixing to be monitored optically. Rapid mixing was caused by spotting drops of alcohols, acetone, acetonitrile, and aqueous solutions of these, as long as the difference of surface tension between the drop and the uppermost layer of the bulk liquid surpassed 30 dynes/cm. Along with this difference, position and velocity of spotting, as well as viscosity and geometry of the bulk liquid volume, may influence the tu...

Journal of Immunological Methods, 1995

Using the enzyme activity inhibiting monoclonal antibody IB 10B8 against alkaline phosphatase of ... more Using the enzyme activity inhibiting monoclonal antibody IB 10B8 against alkaline phosphatase of calf intestine (AP), the interaction of a macromolecular antigen with the antibody was studied with different reaction conditions and with different conformations of the antigen, i.e. using (i) different pH values, (ii) different temperatures, (iii) different substrate saturation of the enzyme, (iv) different glycosylphosphatidyl-AP (GPI-AP) aggregates, and (v) membrane-bound species. In the case of antibody excess and negligible substrate consumption enzymic product formation proceeds according to [P] = a + b x t - c x exp(-d x t). By direct progress curve fitting and secondary data evaluation using nonlinear regression, omitting numerical derivation and graphic techniques, kinetic constants of the immune reaction have been estimated. The method does not require any artificial labelling nor any separation of bound and free entities. (i) Upon increasing pH from 9.8 to 11.0, the dissociation constant of the enzyme-antibody complex is increased strongly, mainly due to the decreasing association rate constant. (ii) A temperature increase from 25 degrees C to 37 degrees C produces a marked increase of both the association and dissociation rate constant. (iii) To differentiate between the interaction of the antibody with the free (E) and substrate-bound (ES) enzyme, experiments were done at different substrate concentrations. The results were fitted to a model allowing determination of association and dissociation rate constants of the free and substrate-bound enzyme. The inverse variation of association and dissociation rate constants caused by substrate binding produces a marked increase of the dissociation constant of the antibody-enzyme complex. The antibody-bound enzyme shows a nearly three-fold higher Km value and a six-fold lower catalytic constant as compared to the free enzyme. (iv) Investigations of the interaction of the antibody with anchorless AP, different hydrophobic aggregates of purified GPI-AP (fractions II-V). (v) Membrane-bound GPI-AP show that the epitopes of all species are fully accessible to the antibody and not cryptic. Surprisingly the insertion of the GPI-moiety into the membrane and the aggregation of the different GPI-AP fractions II-V seem to improve antibody binding. Such improvement of binding was not found in control experiments with Fab, indicating only for the bivalent antibody a stronger interaction with the multivalent antigen than with the monovalent antigen.

Biological Chemistry, 2016

Recent research implicated glycosylphosphatidylinositol-anchored proteins (GPI-AP) and GPI-specif... more Recent research implicated glycosylphosphatidylinositol-anchored proteins (GPI-AP) and GPI-specific phospholipase D (GPI-PLD) in the pathogenesis of fatty liver disease and hepatocellular carcinoma (HCC). Given that c-Myc is frequently amplified in HCC, we investigated their regulation in a c-Myc transgenic disease model of liver cancer and HCC patient samples. Whole genome scans defined 54 significantly regulated genes coding for GPI-AP of which 29 and 14 were repressed in expression in transgenic tumors and steatotic human hepatocyte cultures, respectively, to influence lipid-mediated signal transduction, extracellular matrix and immunity pathways. Analysis of gene specific promoter revealed >95% to carry c-Myc binding sites thus establishing a link between c-Myc activity and transcriptional response. Alike, serum GPI-PLD activity was increased 4-fold in transgenic mice; however its tissue activity was reduced by 70%. The associated repression of the serine/threonine phosphatase 2A (PP2A), i.e. a key player of c-Myc proteolysis, indicates co-ordinate responses aimed at impairing tissue GPI-PLD anti-proliferative activities. Translational research identified >4-fold increased GPI-PLD serum protein expression though enzyme activities were repressed by 60% in NASH and HCC patients. Taken collectively, c-Myc influences GPI-AP signaling transcriptionally and posttranslational and represses GPI-AP anti-proliferative signaling in tumors. The findings broaden the perspective of molecular targeted therapies and disease monitoring.

Proteomics. Clinical applications, 2016

Acute phase proteins (APPs) are highly conserved plasma proteins that are increasingly secreted b... more Acute phase proteins (APPs) are highly conserved plasma proteins that are increasingly secreted by the liver in response to a variety of injuries, independently of their location and cause. APPs favor the systemic regulation of defense, coagulation, proteolysis, and tissue repair. Various APPs have been applied as general diagnostic parameters for a long time. Through proteomic techniques, more and more APPs have been discovered to be differentially altered. Since they are not consistently explainable by a stereotypic hepatic expression of sets of APPs, most of these results have unfortunately been neglected or attributed to the nonspecificity of the acute phase reaction. Moreover, it appears that various extrahepatic tissues are also able to express APPs. These extrahepatic APPs show focally specific roles in tissue homeostasis and repair and are released primarily into interstitial and distal fluids. Since these focal proteins might leak into the circulatory system, mixtures of he...

Proteomics. Clinical applications, 2016

Several reasons have been put forward to explain the irreproducibility of proteomic biomarker sea... more Several reasons have been put forward to explain the irreproducibility of proteomic biomarker search. However, these reasons pertain to almost every part of biomarker search across the entire analytical workflow but are entirely experimental or methodological. However, in this article we point out that there is a further cause of such irreproducibility. This is not an additional methodological or experimental cause but arises directly from the biology of protein expression. It arises from the fact that disease changes the diversity within protein families. This cause of irreproducibility has been very little studied in relation to proteomic biomarker search. Gene expression is highly variable even in healthy people. Therefore, multiple proteoforms are also to be expected when gene expression is disrupted by disease, proteoforms that may be differently altered by pathology. In consequence, it is illogical to expect that the whole protein family produces a reliably usable biomarker. I...

Amyotrophic Lateral Sclerosis and Frontotemporal Degeneration, 2016

Neurofilaments are leading neurochemical biomarkers for amyotrophic lateral sclerosis (ALS). Here... more Neurofilaments are leading neurochemical biomarkers for amyotrophic lateral sclerosis (ALS). Here, we investigated the effect of preanalytical factors on neurofilament concentrations in cerebrospinal fluid (CSF) in a "reverse" round-robin with 15 centers across Europe/U.S. Samples from ALS and control patients (5/5 each center, n = 150) were analyzed for phosphorylated neurofilament heavy chain (pNfH) and neurofilament light chain (NfL) at two laboratories. CSF pNfH was increased (p < 0.05) in ALS in 10 out of 15 centers and NfL in 5 out of 12 centers. The coefficient of variation (CV%) of pNfH measurements between laboratories was 18.7 ± 19.1%. We calculated a diagnostic cut-off of >568.5 pg/mL for pNfH (sensitivity 78.7%, specificity 93.3%) and >1,431pg/mL for NfL (sensitivity 79.0%, specificity 86.4%). Values in ALS patients are already comparable between most centers, supporting eventual implementation into clinical routine. However, continuous quality control programs will be necessary for inclusion in the diagnostic work-up.

GBM Fall meeting Hamburg 2007, 2007

BMC veterinary research, Jan 25, 2015

Cumulating reports suggest that acute phase proteins (APPs) do not only play a role as systemic i... more Cumulating reports suggest that acute phase proteins (APPs) do not only play a role as systemic inflammatory mediators, but are also expressed in different tissues as local reaction to inflammatory stimuli. The present study aimed to evaluate presence and changes in luminal lung concentrations of the APPs haptoglobin (Hp), lipopolysaccharide binding protein (LBP), C-reactive protein (CRP), and lactoferrin (Lf) in calves with an acute respiratory disease experimentally induced by Chlamydia (C.) psittaci. Intra-bronchial inoculation of the pathogen resulted in a consistent respiratory illness. In venous blood of the infected calves (n = 13), concentrations of plasma proteins and serum LBP were assessed (i) before exposure and (ii) 8 times within 14 days after inoculation (dpi). Increasing clinical illness correlated significantly with increasing LBP-and decreasing albumin concentrations in blood, both verifying a systemic acute phase response. Broncho-alveolar lavage fluid (BALF) was ...

GBM Annual Fall meeting M�nster 2004, 2004

Analytical and bioanalytical chemistry, 2015

Triton X-100 has been widely used in many analytical and preparative protocols for a long time. N... more Triton X-100 has been widely used in many analytical and preparative protocols for a long time. Nevertheless, mass spectrometry, chromatographic separation, and spectrophotometric readout may be considerably hampered by this detergent due to signal suppression, complex formation, and high blank values, respectively. Additionally, Triton X-100 is not safe to remove prior to analytics. Here, microdialysis is introduced as a parallelizable, high-throughput method to clean samples from Triton X-100 with high efficacy and precision. To achieve this, we exploit the potential to considerably increase the critical micellar concentration of Triton X-100 by alteration of matrix properties. To that end, addition of several chaotropic compounds and organic solvents has been shown to increase the critical micellar concentration as well as the removal rate of the detergent. For application, matrix additives can be selected for analyte stability requirements out of a variety of compounds. Convenie...

PROTEOMICS - Clinical Applications, 2010

Aim of the study was to identify long-term differences of middle and high-molecular-weight serum ... more Aim of the study was to identify long-term differences of middle and high-molecular-weight serum constituents under high- and low-flux hemodialysis treatments. Thus, the entire predialytic serum proteomes had to be analyzed using identical hemodialysis membrane material but with different cut-off values. A cross-over study and a global native chromatographic proteomic approach were used to analyze serum compositions of 16 patients suffering from end-stage renal disease. No significant or reproducible differences were found between predialytic serum samples from high- and low-flux dialysis treatments using UV-absorbance and fluorescence spectrometry, PMF, or sequence tags. In contrast, there are characteristic differences in the predialytic serum composition of the patients considered and two control sets, which include samples obtained post-dialytically from patients and samples from healthy controls. Only a fraction of β(2)-microglobulin, an example of so-called middle molecules, exhibits the expected molecular weight. A small fraction was found with high molecular weight unaffected by any dialysis treatment. Moreover, immunoreactivity of fragments of β(2)-microglobulin, surprisingly, was also not affected by the cut-off of dialysis membranes. Thus, simply increasing the pore size of a hemodialysis membrane may not have any long-term effect on serum composition.

A multidimensional chromatographic method is presented which is adapted for fractionation of body... more A multidimensional chromatographic method is presented which is adapted for fractionation of body fluids for proteomic analysis and biomarker search. This method combines native size exclusion (SEC, first dimension, 1D), followed by anion exchange (AEC, 2D) and lectin affinity (LAC, 3D) chromatography. After serial 1D-fractionation, all further procedures are performed in microplate format. Thus, beginning with 2D-fractionation, parallelization and automation is achieved for separation, spectrophotometric readout, temporary storage, hit picking, medium exchange, digest, desalting, and finally storage within an autosampler of LC-MS. The central unit is an automated workstation enabling automated multichannel pipetting and robotic handling of microplates, reservoirs, and column arrays. The whole fractionation process, hit picking and analytics are controlled by adapted software packages. Some tools and tricks have been developed in order to improve sample preparation between fractiona...

Clinica Chimica Acta, Mar 1, 1999

ABSTRACT

Journal of Biomolecular Screening, 2007

How to effectively mix small volumes of liquids within microplate wells is a still underestimated... more How to effectively mix small volumes of liquids within microplate wells is a still underestimated and often neglected challenge. The method the authors introduce here relies on violent turbulent motion within a liquid caused by spotting an organic solvent drop onto its surface. The amount needed, less than 1 to 3 µL, is generally small enough not to alter bioactive molecules. Moreover, a solvent may be selected for its compatibility with assay components. The method was tested with layers of aqueous liquids that differ in pH and concentration of a pH-dependent dye, allowing mixing to be monitored optically. Rapid mixing was caused by spotting drops of alcohols, acetone, acetonitrile, and aqueous solutions of these, as long as the difference of surface tension between the drop and the uppermost layer of the bulk liquid surpassed 30 dynes/cm. Along with this difference, position and velocity of spotting, as well as viscosity and geometry of the bulk liquid volume, may influence the tu...

Journal of Immunological Methods, 1995

Using the enzyme activity inhibiting monoclonal antibody IB 10B8 against alkaline phosphatase of ... more Using the enzyme activity inhibiting monoclonal antibody IB 10B8 against alkaline phosphatase of calf intestine (AP), the interaction of a macromolecular antigen with the antibody was studied with different reaction conditions and with different conformations of the antigen, i.e. using (i) different pH values, (ii) different temperatures, (iii) different substrate saturation of the enzyme, (iv) different glycosylphosphatidyl-AP (GPI-AP) aggregates, and (v) membrane-bound species. In the case of antibody excess and negligible substrate consumption enzymic product formation proceeds according to [P] = a + b x t - c x exp(-d x t). By direct progress curve fitting and secondary data evaluation using nonlinear regression, omitting numerical derivation and graphic techniques, kinetic constants of the immune reaction have been estimated. The method does not require any artificial labelling nor any separation of bound and free entities. (i) Upon increasing pH from 9.8 to 11.0, the dissociation constant of the enzyme-antibody complex is increased strongly, mainly due to the decreasing association rate constant. (ii) A temperature increase from 25 degrees C to 37 degrees C produces a marked increase of both the association and dissociation rate constant. (iii) To differentiate between the interaction of the antibody with the free (E) and substrate-bound (ES) enzyme, experiments were done at different substrate concentrations. The results were fitted to a model allowing determination of association and dissociation rate constants of the free and substrate-bound enzyme. The inverse variation of association and dissociation rate constants caused by substrate binding produces a marked increase of the dissociation constant of the antibody-enzyme complex. The antibody-bound enzyme shows a nearly three-fold higher Km value and a six-fold lower catalytic constant as compared to the free enzyme. (iv) Investigations of the interaction of the antibody with anchorless AP, different hydrophobic aggregates of purified GPI-AP (fractions II-V). (v) Membrane-bound GPI-AP show that the epitopes of all species are fully accessible to the antibody and not cryptic. Surprisingly the insertion of the GPI-moiety into the membrane and the aggregation of the different GPI-AP fractions II-V seem to improve antibody binding. Such improvement of binding was not found in control experiments with Fab, indicating only for the bivalent antibody a stronger interaction with the multivalent antigen than with the monovalent antigen.

Biological Chemistry, 2016

Recent research implicated glycosylphosphatidylinositol-anchored proteins (GPI-AP) and GPI-specif... more Recent research implicated glycosylphosphatidylinositol-anchored proteins (GPI-AP) and GPI-specific phospholipase D (GPI-PLD) in the pathogenesis of fatty liver disease and hepatocellular carcinoma (HCC). Given that c-Myc is frequently amplified in HCC, we investigated their regulation in a c-Myc transgenic disease model of liver cancer and HCC patient samples. Whole genome scans defined 54 significantly regulated genes coding for GPI-AP of which 29 and 14 were repressed in expression in transgenic tumors and steatotic human hepatocyte cultures, respectively, to influence lipid-mediated signal transduction, extracellular matrix and immunity pathways. Analysis of gene specific promoter revealed >95% to carry c-Myc binding sites thus establishing a link between c-Myc activity and transcriptional response. Alike, serum GPI-PLD activity was increased 4-fold in transgenic mice; however its tissue activity was reduced by 70%. The associated repression of the serine/threonine phosphatase 2A (PP2A), i.e. a key player of c-Myc proteolysis, indicates co-ordinate responses aimed at impairing tissue GPI-PLD anti-proliferative activities. Translational research identified >4-fold increased GPI-PLD serum protein expression though enzyme activities were repressed by 60% in NASH and HCC patients. Taken collectively, c-Myc influences GPI-AP signaling transcriptionally and posttranslational and represses GPI-AP anti-proliferative signaling in tumors. The findings broaden the perspective of molecular targeted therapies and disease monitoring.

Proteomics. Clinical applications, 2016

Acute phase proteins (APPs) are highly conserved plasma proteins that are increasingly secreted b... more Acute phase proteins (APPs) are highly conserved plasma proteins that are increasingly secreted by the liver in response to a variety of injuries, independently of their location and cause. APPs favor the systemic regulation of defense, coagulation, proteolysis, and tissue repair. Various APPs have been applied as general diagnostic parameters for a long time. Through proteomic techniques, more and more APPs have been discovered to be differentially altered. Since they are not consistently explainable by a stereotypic hepatic expression of sets of APPs, most of these results have unfortunately been neglected or attributed to the nonspecificity of the acute phase reaction. Moreover, it appears that various extrahepatic tissues are also able to express APPs. These extrahepatic APPs show focally specific roles in tissue homeostasis and repair and are released primarily into interstitial and distal fluids. Since these focal proteins might leak into the circulatory system, mixtures of he...

Proteomics. Clinical applications, 2016

Several reasons have been put forward to explain the irreproducibility of proteomic biomarker sea... more Several reasons have been put forward to explain the irreproducibility of proteomic biomarker search. However, these reasons pertain to almost every part of biomarker search across the entire analytical workflow but are entirely experimental or methodological. However, in this article we point out that there is a further cause of such irreproducibility. This is not an additional methodological or experimental cause but arises directly from the biology of protein expression. It arises from the fact that disease changes the diversity within protein families. This cause of irreproducibility has been very little studied in relation to proteomic biomarker search. Gene expression is highly variable even in healthy people. Therefore, multiple proteoforms are also to be expected when gene expression is disrupted by disease, proteoforms that may be differently altered by pathology. In consequence, it is illogical to expect that the whole protein family produces a reliably usable biomarker. I...

Amyotrophic Lateral Sclerosis and Frontotemporal Degeneration, 2016

Neurofilaments are leading neurochemical biomarkers for amyotrophic lateral sclerosis (ALS). Here... more Neurofilaments are leading neurochemical biomarkers for amyotrophic lateral sclerosis (ALS). Here, we investigated the effect of preanalytical factors on neurofilament concentrations in cerebrospinal fluid (CSF) in a "reverse" round-robin with 15 centers across Europe/U.S. Samples from ALS and control patients (5/5 each center, n = 150) were analyzed for phosphorylated neurofilament heavy chain (pNfH) and neurofilament light chain (NfL) at two laboratories. CSF pNfH was increased (p < 0.05) in ALS in 10 out of 15 centers and NfL in 5 out of 12 centers. The coefficient of variation (CV%) of pNfH measurements between laboratories was 18.7 ± 19.1%. We calculated a diagnostic cut-off of >568.5 pg/mL for pNfH (sensitivity 78.7%, specificity 93.3%) and >1,431pg/mL for NfL (sensitivity 79.0%, specificity 86.4%). Values in ALS patients are already comparable between most centers, supporting eventual implementation into clinical routine. However, continuous quality control programs will be necessary for inclusion in the diagnostic work-up.

GBM Fall meeting Hamburg 2007, 2007

BMC veterinary research, Jan 25, 2015

Cumulating reports suggest that acute phase proteins (APPs) do not only play a role as systemic i... more Cumulating reports suggest that acute phase proteins (APPs) do not only play a role as systemic inflammatory mediators, but are also expressed in different tissues as local reaction to inflammatory stimuli. The present study aimed to evaluate presence and changes in luminal lung concentrations of the APPs haptoglobin (Hp), lipopolysaccharide binding protein (LBP), C-reactive protein (CRP), and lactoferrin (Lf) in calves with an acute respiratory disease experimentally induced by Chlamydia (C.) psittaci. Intra-bronchial inoculation of the pathogen resulted in a consistent respiratory illness. In venous blood of the infected calves (n = 13), concentrations of plasma proteins and serum LBP were assessed (i) before exposure and (ii) 8 times within 14 days after inoculation (dpi). Increasing clinical illness correlated significantly with increasing LBP-and decreasing albumin concentrations in blood, both verifying a systemic acute phase response. Broncho-alveolar lavage fluid (BALF) was ...

GBM Annual Fall meeting M�nster 2004, 2004

Analytical and bioanalytical chemistry, 2015

Triton X-100 has been widely used in many analytical and preparative protocols for a long time. N... more Triton X-100 has been widely used in many analytical and preparative protocols for a long time. Nevertheless, mass spectrometry, chromatographic separation, and spectrophotometric readout may be considerably hampered by this detergent due to signal suppression, complex formation, and high blank values, respectively. Additionally, Triton X-100 is not safe to remove prior to analytics. Here, microdialysis is introduced as a parallelizable, high-throughput method to clean samples from Triton X-100 with high efficacy and precision. To achieve this, we exploit the potential to considerably increase the critical micellar concentration of Triton X-100 by alteration of matrix properties. To that end, addition of several chaotropic compounds and organic solvents has been shown to increase the critical micellar concentration as well as the removal rate of the detergent. For application, matrix additives can be selected for analyte stability requirements out of a variety of compounds. Convenie...

PROTEOMICS - Clinical Applications, 2010

Aim of the study was to identify long-term differences of middle and high-molecular-weight serum ... more Aim of the study was to identify long-term differences of middle and high-molecular-weight serum constituents under high- and low-flux hemodialysis treatments. Thus, the entire predialytic serum proteomes had to be analyzed using identical hemodialysis membrane material but with different cut-off values. A cross-over study and a global native chromatographic proteomic approach were used to analyze serum compositions of 16 patients suffering from end-stage renal disease. No significant or reproducible differences were found between predialytic serum samples from high- and low-flux dialysis treatments using UV-absorbance and fluorescence spectrometry, PMF, or sequence tags. In contrast, there are characteristic differences in the predialytic serum composition of the patients considered and two control sets, which include samples obtained post-dialytically from patients and samples from healthy controls. Only a fraction of β(2)-microglobulin, an example of so-called middle molecules, exhibits the expected molecular weight. A small fraction was found with high molecular weight unaffected by any dialysis treatment. Moreover, immunoreactivity of fragments of β(2)-microglobulin, surprisingly, was also not affected by the cut-off of dialysis membranes. Thus, simply increasing the pore size of a hemodialysis membrane may not have any long-term effect on serum composition.