Biomedical subjects

Katrin Marcus

Publications and source records attributed to Katrin Marcus.

At least 19 recordsLinked to original sources

PubMed2025

Proteomics of Duchenne Muscular Dystrophy Patient iPSC-Derived Skeletal Muscle Cells Reveal Differential Expression of Cytoskeletal and Extracellular Matrix Proteins.

Proteomics of dystrophic muscle samples is limited by the amount of protein that can be extracted from patient biopsies. Cells and tissues derived from patient-derived induced pluripotent stem cells (iPSCs) can be an expandable alternative source. We have patterned iPSCs from three Duchenne muscular dystrophy (DMD) patient lines into skeletal muscle cells using a two-dimensional as well as our three-dimensional organoid differentiation system. Probes with sufficient protein amounts could be extracted and prepared for mass spectrometry. In total, 3007 proteins in 2D and 2709 proteins in 3D were detected in DMD patient probes. A total of 83 proteins in 2D and 338 proteins in 3D can be described as differentially expressed between DMD and control patient probes in a post hoc test. We have identified and we propose Myosin-9, Collagen 18A, Tropomyosin 1, BASP1, RUVBL1, and NCAM1 as proteins specifically altered in their expression in DMD for further investigation. Proteomics of skeletal muscle organoids resulted in greater consistency of results between cell lines in comparison to the two-dimensional myogenic differentiation protocol.

Humans↗

PubMed2024

Genetic and functional analyses of CTBP2 in anorexia nervosa and body weight regulation.

The C-terminal binding protein 2 (CTBP2) gene (translational isoforms: CTBP2-L/S, RIBEYE) had been identified by a cross-trait analysis of genome-wide association studies for anorexia nervosa (AN) and body mass index (BMI). Here, we did a mutation analysis in CTBP2 by performing polymerase chain reactions with subsequent Sanger-sequencing to identify variants relevant for AN and body weight regulation and ensued functional studies. Analysis of the coding regions of CTBP2 in 462 female patients with AN (acute or recovered), 490 children and adolescents with severe obesity, 445 healthy-lean adult individuals and 168 healthy adult individuals with normal body weight detected 24 variants located in the specific exon of RIBEYE. In the initial analysis, three of these were rare non-synonymous variants (NSVs) detected heterozygously in patients with AN (p.Arg72Trp - rs146900874; p.Val289Met -rs375685611 and p.Gly362Arg - rs202010294). Four NSVs and one heterozygous frameshift variant were exclusively detected in children and adolescents with severe obesity (p.Pro53Ser - rs150867595; p.Gln175ArgfsTer45 - rs141864737; p.Leu310Val - rs769811964; p.Pro397Ala - rs76134089 and p.Pro402Ser - rs113477585). Ribeye mRNA was detected in mouse hypothalamus. No effect of fasting or overfeeding on murine hypothalamic Ribeye expression was determined. Yet, increased Ribeye expression was detected in hypothalami of leptin-treated Lepob/ob mice. This increase was not related to reduced food intake and leptin-induced weight loss. We detected rare and frequent variants in the RIBEYE specific exon in both patients with AN and in children and adolescents with severe obesity. Our data suggest RIBEYE as a relevant gene for weight regulation.

Humans↗

PubMed2007

Multidimensional chromatography: a powerful tool for the analysis of membrane proteins in mouse brain.

Understanding the function of membrane proteins is of fundamental importance due to their crucial roles in many cellular processes and their direct association with human disorders. However, their analysis poses a special challenge, largely due to their highly amphipathic nature. Until recently, analyses of proteomic samples mainly were performed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), due to the unprecedented separation power of the technique. However, in conventional 2D-PAGE membrane proteins are generally underrepresented due to their tendency to precipitate during isoelectric focusing and their inefficient transfer from the first to the second dimension. As a consequence, several other separation techniques, primarily based on liquid chromatography (LC), have been employed for analysis of this group of proteins. In the present study, different LC-based methods were compared for the analysis of crude protein extracts. One- and two-dimensional high-performance liquid chromatographic (1D- and 2D-HPLC) separations of brain protein tryptic digests with a predicted concentration range of up to 5 orders of magnitude were found to be insufficient, thus making a preceding fractionation step necessary. An additional protein separation step was introduced and a 3D-PAGE-HPLC analysis was performed. The results of these experiments are compared with results of 2D-PAGE/matrix-assisted laser desorption ionization mass spectrometric (MALDI MS) analyses of the same samples. Features, challenges, advantages, and disadvantages of the respective systems are discussed. The brain (mouse and human) was chosen as the analyzed tissue as it is of high interest in medical and pharmaceutical research into neurological diseases such as multiple sclerosis, stroke, Alzheimer's disease, and Parkinson's disease. The study is part of our ongoing research aimed at identifying new biomarkers for neurodegenerative diseases.

Animals↗

PubMed2006

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.

Aging↗

PubMed2006

4(th) HUPO Brain Proteome Project Workshop in Munich, Germany.

More than 70 interested colleagues attended the 4(th) Workshop of HUPO's Brain Proteome Project. The project was presented within nine talks mainly focusing on two running pilot studies as well as on data re-processing. A bioinformatics jamboree in Hinxton, UK, and the 5th Workshop taking place in Dublin next February were announced.

Brain↗

PubMed2006

The HUPO Brain Proteome Project jamboree: centralised summary of the pilot studies.

The Bioinformatics Committee of the HUPO Brain Proteome Project (HUPO BPP) meets regularly to execute the post-lab analyses of the data produced in the HUPO BPP pilot studies. On January 9-11, 2006 the members as well as invited analysts came together at the European Bioinformatics Institute in Hinxton, UK for the pilot studies jamboree. The results of the reprocessing were presented and tasks forces were initiated to compile, to interpret and to summarise the data obtained.

Animals↗

PubMed2006

Pilot study of the Human Proteome Organisation Brain Proteome Project: applying different 2-DE techniques to monitor proteomic changes during murine brain development.

The Human Proteome Organisation Brain Proteome Project aims at coordinating neuroproteomic activities with respect to analysis of development, aging, and evolution in human and mice and at analysing normal aging processes as well as neurodegenerative diseases. Our group participated in the mouse pilot study of this project using two different 2-DE systems, to find out the optimal conditions for comprehensive gel-based differential proteome analysis. Besides the assessment of the best methodical conditions the question of "How many biological replicate analyses have to be performed to get reliable statistically validated results?" was addressed. In total 420 differences were detected in all analyses. Both 2-DE methods were found to be suitable for comprehensive differential proteome analysis. Nevertheless, each of the methods showed substantial advantages and disadvantages resulting in the fact that modification of both systems is essential. From our results we can draw the conclusions that for the future optimal quantitative differential gel-based brain proteome analyses the sample preparation has to be slightly changed, the resolution of the first as well as the second dimension has to be advanced, the number of experiments has to be increased and that the 2D-DIGE system should be applied.

Aging↗

PubMed2006

Examination of 2-DE in the Human Proteome Organisation Brain Proteome Project pilot studies with the new RAIN gel matching technique.

The Human Proteome Organisation (HUPO) Brain Proteome Project (BPP) pilot studies have generated over 200 2-D gels from eight participating laboratories. This data includes 67 single-channel and 60 DIGE gels comparing 30 whole frozen C57/BL6 female mouse brains, ten each at embryonic day 16, postnatal day 7 (juvenile) and postnatal day 54-56 (adult); and ten single-channel and three DIGE gels comparing human epilepsy surgery of the temporal front lobe with a corresponding post-mortem specimen. The samples were generated centrally and distributed to the participating laboratories, but otherwise no restrictions were placed on sample preparation, running and staining protocols, nor on the 2-D gel analysis packages used. Spots were characterised by MS and the annotated gel images published on a ProteinScape web server. In order to examine the resultant differential expression and protein identifications, we have reprocessed a large subset of the gels using the newly developed RAIN (Robust Automated Image Normalisation) 2-D gel matching algorithm. Traditional approaches use symbolic representation of spots at the very early stages of the analysis, which introduces persistent errors due to inaccuracies in spot modelling and matching. With RAIN, image intensity distributions, rather than selected features, are used, where smooth geometric deformation and expression bias are modelled using multi-resolution image registration and bias-field correction. The method includes a new approach of volume-invariant warping which ensures the volume of protein expression under transformation is preserved. An image-based statistical expression analysis phase is then proposed, where small insignificant expression changes over one gel pair can be revealed when reinforced by the same consistent changes in others. Results of the proposed method as applied to the HUPO BPP data show significant intra-laboratory improvements in matching accuracy over a previous state-of-the-art technique, Multi-resolution Image Registration (MIR), and the commercial Progenesis PG240 package.

Algorithms↗

PubMed2006

2-D differential membrane proteome analysis of scarce protein samples.

Proteome studies with small sample amounts are difficult to perform, especially when membrane proteins are the focus of interest. In our study a new method for the analysis of scarce membrane protein samples combining large gel 2-D-CTAB/SDS-PAGE with fluorescence dye saturation labelling (satDIGE) was developed, allowing a highly sensitive differential analysis of different cell states. After Triton X-114 phase partitioning, enriched membrane protein samples of T cells were labelled at cysteine residues using fluorescence dyes and separated by large gel 2D-CTAB/SDS-PAGE. For a differential analysis 3 mug protein was found to be sufficient to detect proteins in a widespread well-separated diagonal spot pattern.

Amino Acid Sequence↗

PubMed2006

Bridging Proteomics and Medical Science - the 5th HUPO Brain Proteome Workshop in Dublin, Ireland.

More than 70 interested colleagues attended the 5th Workshop of the HUPO Brain Proteome Project (HUPO BPP) at the UCD Conway Institute, Dublin, Ireland. An overview of the outcome of the pilot study was presented and the new subprojects "Clinical Neuroproteomics of Human Body Fluids" as well as "Cerebellum 2D-mapping" were announced. In addition the election of the HUPO BPP committees and the future directions of this project were discussed and decided. The meeting was enhanced by several talks highlighting the application of proteomics in biomedical research.

Animals↗

PubMed2006

Gaining knowledge from previously unexplained spectra-application of the PTM-Explorer software to detect PTM in HUPO BPP MS/MS data.

A novel software tool named PTM-Explorer has been applied to LC-MS/MS datasets acquired within the Human Proteome Organisation (HUPO) Brain Proteome Project (BPP). PTM-Explorer enables automatic identification of peptide MS/MS spectra that were not explained in typical sequence database searches. The main focus was detection of PTMs, but PTM-Explorer detects also unspecific peptide cleavage, mass measurement errors, experimental modifications, amino acid substitutions, transpeptidation products and unknown mass shifts. To avoid a combinatorial problem the search is restricted to a set of selected protein sequences, which stem from previous protein identifications using a common sequence database search. Prior to application to the HUPO BPP data, PTM-Explorer was evaluated on excellently manually characterized and evaluated LC-MS/MS data sets from Alpha-A-Crystallin gel spots obtained from mouse eye lens. Besides various PTMs including phosphorylation, a wealth of experimental modifications and unspecific cleavage products were successfully detected, completing the primary structure information of the measured proteins. Our results indicate that a large amount of MS/MS spectra that currently remain unidentified in standard database searches contain valuable information that can only be elucidated using suitable software tools.

Amino Acid Sequence↗

PubMed2006

Functional annotation of proteins identified in human brain during the HUPO Brain Proteome Project pilot study.

The HUPO Brain Proteome Project is an initiative coordinating proteomics studies to characterise human and mouse brain proteomes. Proteins identified in human brain samples during the project's pilot phase were put into biological context through integration with various annotation sources followed by a bioinformatics analysis. The data set was related to the genome sequence via the genes encoding identified proteins including an assessment of splice variant identification as well as an analysis of tissue specificity of the respective transcripts. Proteins were furthermore categorised according to subcellular localisation, molecular function and biological process, grouped into protein families and mapped to biological pathways they are known to act in. Involvement in pathological conditions was examined based on association with entries in the online version of Mendelian Inheritance in Man and an interaction network was derived from curated protein-proteininteraction data. Overall a non-redundant set of 1804 proteins was identified in human brain samples. In the majority of cases splice variants could be unambiguously identified by unique peptides, including matches to several hypothetical transcripts of known as well as predicted genes.

Alternative Splicing↗

PubMed2006

Automated reprocessing pipeline for searching heterogeneous mass spectrometric data of the HUPO Brain Proteome Project pilot phase.

The newly available techniques for sensitive proteome analysis and the resulting amount of data require a new bioinformatics focus on automatic methods for spectrum reprocessing and peptide/protein validation. Manual validation of results in such studies is not feasible and objective enough for quality relevant interpretation. The necessity for tools enabling an automatic quality control is, therefore, important to produce reliable and comparable data in such big consortia as the Human Proteome Organization Brain Proteome Project. Standards and well-defined processing pipelines are important for these consortia. We show a way for choosing the right database model, through collecting data, processing these with a decoy database and end up with a quality controlled protein list merged from several search engines, including a known false-positive rate.

Algorithms↗

PubMed2006

HUPO Brain Proteome Project: summary of the pilot phase and introduction of a comprehensive data reprocessing strategy.

The Human Proteome Organisation (HUPO) initiated several projects focusing on the proteome analysis of distinct human organs. The Brain Proteome Project (BPP) is the initiative dedicated to the brain, its development and correlated diseases. Two pilot studies have been performed aiming at the comparison of techniques, laboratories and approaches. With the help of the results gained, objective data submission, storage and reprocessing workflow have been established. The biological relevance of the data will be drawn from the inter-laboratory comparisons as well as from the re-calculation of all data sets submitted by the different groups. In the following, results of the single groups as well as the centralised reprocessing effort will be summarised and compared, showing the added value of this concerted work.

Animals↗

PubMed2006

The power of cooperative investigation: summary and comparison of the HUPO Brain Proteome Project pilot study results.

Within the pilot phase of the HUPO Brain Proteome Project, nine participating laboratories analysed human (epilepsy and/or post mortem material) and mouse brain samples (embryonic, juvenile and adult), respectively, using a variety of different state of the art techniques. Thirty-seven different analytical approaches were accomplished. Of these analyses, 17 were done differentially, i.e. the protein expression patterns of the different samples (human or mouse) were compared. A catalogue of all proteins present in the respective sample was built in 20 analyses (mapping). All data were collected in the Data Collection Center in Bochum, Germany, and were reprocessed according to thoroughly defined parameters. In this report, a summary of all results and inter-laboratory comparisons with respect to the number of identified proteins, the analysed organism, and the used techniques is presented.

Animals↗

PubMed2005

Inactivation of the mitochondrial heat shock protein zim17 leads to aggregation of matrix hsp70s followed by pleiotropic effects on morphology and protein biogenesis.

The biogenesis of mitochondrial matrix proteins involves the translocase of the outer membrane, the presequence translocase of the inner membrane and the presequence translocase-associated motor. The mitochondrial heat shock protein 70 (mtHsp70) forms the central core of the motor. Recent studies led to the identification of Zim17, a mitochondrial zinc finger motif protein that interacts with mtHsp70. Different views have been reported on the localization of Zim17 in the mitochondrial inner membrane or matrix. Depletion of Zim17 impairs several critical mitochondrial processes, leading to inhibition of protein import, defects of Fe/S protein biogenesis and aggregation of Hsp70s in the matrix. Additionally, we found that inactivation of Zim17 altered the morphology of mitochondria. These pleiotropic effects raise the question of the specific function of Zim17 in mitochondria. Here, we report that Zim17 is a heat shock protein of the mitochondrial matrix that is loosely associated with the inner membrane. To address the function of Zim17 in organello, we generated a temperature-sensitive mutant allele of the ZIM17 gene in yeast. Upon a short-term shift of the yeast mutant cells to a non-permissive temperature, matrix Hsp70s aggregated while protein import, Fe/S protein activity and mitochondrial morphology were not, or only mildly, affected. Only after a long-term shift to non-permissive temperature, were strong defects in protein import, Fe/S protein activity and mitochondrial morphology observed. These findings suggest that the heat shock protein Zim17 plays a specific role in preventing protein aggregation in the mitochondrial matrix, and that aggregation of Hsp70s causes pleiotropic effects on protein biogenesis and mitochondrial morphology.

Dimerization↗

PubMed2005

"Subcellular proteomics" of neuromelanin granules isolated from the human brain.

"Subcellular proteomics" is currently the most effective approach to characterize subcellular compartments. Based on the powerful combination of subcellular fractionation and protein identification by LC-MS/MS we were able for the first time to 1) isolate intact neuromelanin granules from the human brain and 2) establish the first protein profile of these granules. This compartment containing neuromelanin (NM) is primarily located in the primate's substantia nigra, one of the main brain regions that severely degenerates in Parkinson disease. We used mechanic tissue disaggregation, discontinuous sucrose gradient centrifugation, cell disruption, and organelle separation to isolate NM granules from human substantia nigra. Using transmission electron microscopy we demonstrated that the morphological characteristics of the isolated NM granules are similar to those described in human brain tissue. Fundamentally we found numerous proteins definitely demonstrating a close relationship of NM-containing granules with lysosomes or lysosome-related organelles originating from the endosome-lysosome lineage. Intriguingly we further revealed the presence of endoplasmic reticulum-derived chaperones, especially the transmembrane protein calnexin, which recently has been located in lysosome-related melanosomes and has been suggested to be a melanogenic chaperone.

Calnexin↗

PubMed2005

Tryptic transpeptidation products observed in proteome analysis by liquid chromatography-tandem mass spectrometry.

Commonly, prior to mass spectrometry based analysis of proteins or protein mixtures, the proteins are subjected to specific enzymatic proteolysis. For this purpose trypsin is most frequently used. However, the process of proteolysis is not unflawed. For example, some side activities of trypsin are known and have already been described in the literature (e.g., chymotryptic activity). Here, we describe the occurrence of transpeptidated peptides during standard proteome analysis using two-dimensional polyacrylamide gel electrophoresis followed by mass spectrometric protein identification. Different types of transpeptidated peptides have been detected. The most frequently observed transpeptidation reaction is N-terminal addition of arginine or lysine to peptides. Furthermore, addition of two amino acids to the N-terminus of a peptide has also been detected. Another transpeptidation that we observed, is combination of two peptides, which were originally located in different regions of the analyzed protein. Currently, the full amount of peptides generated by transpeptidation is not clear. However, it should be recognized that protein information is presently lost as these effects are not detectable with available database search software.

Animals↗