Publications by authors named "Rainer Deutzmann"

38 Publications

Chromatin regulation by Histone H4 acetylation at Lysine 16 during cell death and differentiation in the myeloid compartment.

Nucleic Acids Res 2019 06;47(10):5016-5037

Nanomaterials and Nanotechnology Research Center (CINN-CSIC), Universidad de Oviedo-Principado de Asturias, Spain.

Histone H4 acetylation at Lysine 16 (H4K16ac) is a key epigenetic mark involved in gene regulation, DNA repair and chromatin remodeling, and though it is known to be essential for embryonic development, its role during adult life is still poorly understood. Here we show that this lysine is massively hyperacetylated in peripheral neutrophils. Genome-wide mapping of H4K16ac in terminally differentiated blood cells, along with functional experiments, supported a role for this histone post-translational modification in the regulation of cell differentiation and apoptosis in the hematopoietic system. Furthermore, in neutrophils, H4K16ac was enriched at specific DNA repeats. These DNA regions presented an accessible chromatin conformation and were associated with the cleavage sites that generate the 50 kb DNA fragments during the first stages of programmed cell death. Our results thus suggest that H4K16ac plays a dual role in myeloid cells as it not only regulates differentiation and apoptosis, but it also exhibits a non-canonical structural role in poising chromatin for cleavage at an early stage of neutrophil cell death.
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http://dx.doi.org/10.1093/nar/gkz195DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6547425PMC
June 2019

DiSUMO-LIKE Interacts with RNA-Binding Proteins and Affects Cell-Cycle Progression during Maize Embryogenesis.

Curr Biol 2018 05 3;28(10):1548-1560.e5. Epub 2018 May 3.

Cell Biology and Plant Biochemistry, Biochemie-Zentrum Regensburg, University of Regensburg, 93053 Regensburg, Germany. Electronic address:

Embryogenesis in flowering plants is initiated by an asymmetric zygote division, generating two daughter cells that are the precursors of different cell lineages. Little is known about the molecular players regulating activation and progression of zygote development, establishment of asymmetry, and the plant-specific process of cell-plate formation. Here, we report the function of the ubiquitin-like modifier DiSUMO-LIKE (DSUL) for early embryo development in maize. Introducing a DSUL-RNAi construct by sperm cells affects cytokinesis generating non-separated zygotic daughter nuclei or multinucleate embryonic cells lacking cell plates. DSUL accumulates in the cytoplasm partly in granules, in the nucleus, as well as in the cell division zone. The enzymatic DSULyation cascade involves maturation and the same enzymatic machinery for activation and conjugation as was previously shown for SUMO1. Identification of DSUL targets suggests predominant roles of DSULylation in regulation of cytoplasmic RNA metabolism as well as in cell-cycle progression and cell-plate formation. A comparison of DSUL and SUMO1 localization during the cell cycle and of their substrates indicates strong functional diversification between these two SUMO family modifiers.
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http://dx.doi.org/10.1016/j.cub.2018.03.066DOI Listing
May 2018

CHD3 and CHD4 form distinct NuRD complexes with different yet overlapping functionality.

Nucleic Acids Res 2017 Oct;45(18):10534-10554

Institute of Biochemistry, Genetics and Microbiology, University of Regensburg, 93053 Regensburg, Germany.

CHD3 and CHD4 (Chromodomain Helicase DNA binding protein), two highly similar representatives of the Mi-2 subfamily of SF2 helicases, are coexpressed in many cell lines and tissues and have been reported to act as the motor subunit of the NuRD complex (nucleosome remodeling and deacetylase activities). Besides CHD proteins, NuRD contains several repressors like HDAC1/2, MTA2/3 and MBD2/3, arguing for a role as a transcriptional repressor. However, the subunit composition varies among cell- and tissue types and physiological conditions. In particular, it is unclear if CHD3 and CHD4 coexist in the same NuRD complex or whether they form distinct NuRD complexes with specific functions. We mapped the CHD composition of NuRD complexes in mammalian cells and discovered that they are isoform-specific, containing either the monomeric CHD3 or CHD4 ATPase. Both types of complexes exhibit similar intranuclear mobility, interact with HP1 and rapidly accumulate at UV-induced DNA repair sites. But, CHD3 and CHD4 exhibit distinct nuclear localization patterns in unperturbed cells, revealing a subset of specific target genes. Furthermore, CHD3 and CHD4 differ in their nucleosome remodeling and positioning behaviour in vitro. The proteins form distinct CHD3- and CHD4-NuRD complexes that do not only repress, but can just as well activate gene transcription of overlapping and specific target genes.
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http://dx.doi.org/10.1093/nar/gkx711DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5737555PMC
October 2017

Phosphorylation of Argonaute proteins affects mRNA binding and is essential for microRNA-guided gene silencing .

EMBO J 2017 07 23;36(14):2088-2106. Epub 2017 Jun 23.

Biochemistry Center Regensburg (BZR), Laboratory for RNA Biology, University of Regensburg, Regensburg, Germany

Argonaute proteins associate with microRNAs and are key components of gene silencing pathways. With such a pivotal role, these proteins represent ideal targets for regulatory post-translational modifications. Using quantitative mass spectrometry, we find that a C-terminal serine/threonine cluster is phosphorylated at five different residues in human and In human, hyper-phosphorylation does not affect microRNA binding, localization, or cleavage activity of Ago2. However, mRNA binding is strongly affected. Strikingly, on Ago2 mutants that cannot bind microRNAs or mRNAs, the cluster remains unphosphorylated indicating a role at late stages of gene silencing. In , the phosphorylation of the conserved cluster of ALG-1 is essential for microRNA function Furthermore, a single point mutation within the cluster is sufficient to phenocopy the loss of its complete phosphorylation. Interestingly, this mutant retains its capacity to produce and bind microRNAs and represses expression when artificially tethered to an mRNA Altogether, our data suggest that the phosphorylation state of the serine/threonine cluster is important for Argonaute-mRNA interactions.
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http://dx.doi.org/10.15252/embj.201696386DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5510005PMC
July 2017

Epimerisation of chiral hydroxylactones by short-chain dehydrogenases/reductases accounts for sex pheromone evolution in Nasonia.

Sci Rep 2016 10 5;6:34697. Epub 2016 Oct 5.

Institute of Biophysics and Physical Biochemistry, University of Regensburg, 93053 Regensburg, Germany.

Males of all species of the parasitic wasp genus Nasonia use (4R,5S)-5-hydroxy-4-decanolide (RS) as component of their sex pheromone while only N. vitripennis (Nv), employs additionally (4R,5R)-5-hydroxy-4-decanolide (RR). Three genes coding for the NAD-dependent short-chain dehydrogenases/reductases (SDRs) NV10127, NV10128, and NV10129 are linked to the ability of Nv to produce RR. Here we show by assaying recombinant enzymes that SDRs from both Nv and N. giraulti (Ng), the latter a species with only RS in the pheromone, epimerise RS into RR and vice versa with (4R)-5-oxo-4-decanolide as an intermediate. Nv-derived SDR orthologues generally had higher epimerisation rates, which were also influenced by NAD availability. Semiquantitative protein analyses of the pheromone glands by tandem mass spectrometry revealed that NV10127 as well as NV10128 and/or NV10129 were more abundant in Nv compared to Ng. We conclude that the interplay of differential expression patterns and SDR epimerisation rates on the ancestral pheromone component RS accounts for the evolution of a novel pheromone phenotype in Nv.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5050451PMC
http://dx.doi.org/10.1038/srep34697DOI Listing
October 2016

Argonaute Family Protein Expression in Normal Tissue and Cancer Entities.

PLoS One 2016 12;11(8):e0161165. Epub 2016 Aug 12.

Institute of Biochemistry, Emil-Fischer-Zentrum, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany.

The members of the Argonaute (AGO) protein family are key players in miRNA-guided gene silencing. They enable the interaction between small RNAs and their respective target mRNA(s) and support the catalytic destruction of the gene transcript or recruit additional proteins for downstream gene silencing. The human AGO family consists of four AGO proteins (AGO1-AGO4), but only AGO2 harbors nuclease activity. In this study, we characterized the expression of the four AGO proteins in cancer cell lines and normal tissues with a new mass spectrometry approach called AGO-APP (AGO Affinity Purification by Peptides). In all analyzed normal tissues, AGO1 and AGO2 were most prominent, but marked tissue-specific differences were identified. Furthermore, considerable changes during development were observed by comparing fetal and adult tissues. We also identified decreased overall AGO expression in melanoma derived cell lines compared to other tumor cell lines and normal tissues, with the largest differences in AGO2 expression. The experiments described in this study suggest that reduced amounts of AGO proteins, as key players in miRNA processing, have impact on several cellular processes. Deregulated miRNA expression has been attributed to chromosomal aberrations, promoter regulation and it is known to have a major impact on tumor development and progression. Our findings will further increase our basic understanding of the molecular basis of miRNA processing and its relevance for disease.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0161165PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4982624PMC
August 2017

Functional consequence of fibulin-4 missense mutations associated with vascular and skeletal abnormalities and cutis laxa.

Matrix Biol 2016 12 23;56:132-149. Epub 2016 Jun 23.

Department of Experimental Medicine I, Nikolaus-Fiebiger Center of Molecular Medicine, University of Erlangen-Nürnberg, 91054 Erlangen, Germany.

Fibulin-4 is a 60kDa calcium binding glycoprotein that has an important role in development and integrity of extracellular matrices. It interacts with elastin, fibrillin-1 and collagen IV as well as with lysyl oxidases and is involved in elastogenesis and cross-link formation. To date, several mutations in the fibulin-4 gene (FBLN4/EFEMP2) are known in patients whose major symptoms are vascular deformities, aneurysm, cutis laxa, joint laxity, or arachnodactyly. The pathogenetic mechanisms how these mutations translate into the clinical phenotype are, however, poorly understood. In order to elucidate these mechanisms, we expressed fibulin-4 mutants recombinantly in HEK293 cells, purified the proteins in native forms and analyzed alterations in protein synthesis, secretion, matrix assembly, and interaction with other proteins in relation to wild type fibulin-4. Our studies show that different mutations affect these properties in multiple ways, resulting in fibulin-4 deficiency and/or impaired ability to form elastic fibers. The substitutions E126K and C267Y impaired secretion of the protein, but not mRNA synthesis. Furthermore, the E126K mutant showed less resistance to proteases, reduced binding to collagen IV and fibrillin-1, as well as to LTBP1s and LTBP4s. The A397T mutation introduced an extra O-glycosylation site and deleted binding to LTBP1s. We show that fibulin-4 binds stronger than fibulin-3 and -5 to LTBP1s, 3, and 4s, and to the lysyl oxidases LOX and LOXL1; the binding of fibulin-4 to the LOX propeptide was strongly reduced by the mutation E57K. These findings show that different mutations in the fibulin-4 gene result in different molecular defects affecting secretion rates, protein stability, LOX-induced cross-linking, or binding to other ECM components and molecules of the TGF-β pathway, and thus illustrate the complex role of fibulin-4 in connective tissue assembly.
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http://dx.doi.org/10.1016/j.matbio.2016.06.003DOI Listing
December 2016

PSCD Domains of Pleuralin-1 from the Diatom Cylindrotheca fusiformis: NMR Structures and Interactions with Other Biosilica-Associated Proteins.

Structure 2016 07 16;24(7):1178-91. Epub 2016 Jun 16.

Institute of Biophysics und Physical Biochemistry, Centre of Magnetic Resonance in Chemistry and Biomedicine, University of Regensburg, 93040 Regensburg, Germany. Electronic address:

Diatoms are eukaryotic unicellular algae characterized by silica cell walls and associated with three unique protein families, the pleuralins, frustulins, and silaffins. The NMR structure of the PSCD4 domain of pleuralin-1 from Cylindrotheca fusiformis contains only three short helical elements and is stabilized by five unique disulfide bridges. PSCD4 contains two binding sites for Ca(2+) ions with millimolar affinity. NMR-based interaction studies show an interaction of the domain with native silaffin-1A as well as with α-frustulins. The interaction sites of the two proteins mapped on the PSCD4 structure are contiguous and show only a small overlap. A plausible functional role of pleuralin could be to bind simultaneously silaffin-1A located inside the cell wall and α-frustulin coating the cell wall, thus connecting the interfaces between hypotheca and epitheca at the girdle bands. Restrained molecular dynamics calculations suggest a bead-chain-like structure of the central part of pleuralin-1.
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http://dx.doi.org/10.1016/j.str.2016.04.021DOI Listing
July 2016

Loss of fibulin-4 results in abnormal collagen fibril assembly in bone, caused by impaired lysyl oxidase processing and collagen cross-linking.

Matrix Biol 2016 Mar 9;50:53-66. Epub 2015 Dec 9.

Department of Experimental Medicine I, Nikolaus-Fiebiger Center of Molecular Medicine, University of Erlangen-Nürnberg, 91054 Erlangen, Germany.

The extracellular matrix protein fibulin-4 has been shown to be indispensable for elastic fiber assembly, but there is also evidence from human mutations that it is involved in controlling skeletal development and bone stability. Fibulin-4 mutations were identified in patients suffering from vascular abnormality and/or cutis laxa, and some of these patients exhibited bone fragility, arachnodactyly and joint laxity. In order to elucidate the role of fibulin-4 in bone structure and skeletal development, we analyzed structural changes in skeletal tissues of Fbln4(-/-) mice. Immunostaining confirmed that fibulin-4 is highly expressed in cartilage, bone, ligaments and tendons. No morphological abnormalities were found in the skeleton of Fbln4(-/-) mice as compared to wild type littermates except forelimb contractures as well as unusually thick collagen fibrils. Furthermore, fibulin-4 deficiency caused enhanced susceptibility of bone collagen for acid extraction, consistent with significantly reduced lysylpyridinoline and hydroxylysylpyridinoline cross-links in bone. In accordance with that, the amount of lysyl oxidase in long bones and calvaria was strongly decreased and proteolytic activation of lysyl oxidase was reduced in fibulin-4 deficient osteoblasts, while addition of recombinant fibulin-4 rescued the activation. The finding suggested that fibulin-4 is important for the proteolytic activation of lysyl oxidase which has a pivotal role in cross-linking of collagen and elastin.
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http://dx.doi.org/10.1016/j.matbio.2015.12.002DOI Listing
March 2016

Biochemical isolation of Argonaute protein complexes by Ago-APP.

Proc Natl Acad Sci U S A 2015 Sep 8;112(38):11841-5. Epub 2015 Sep 8.

Biochemistry Center Regensburg, Laboratory for RNA Biology, University of Regensburg, 93053 Regensburg, Germany;

During microRNA (miRNA)-guided gene silencing, Argonaute (Ago) proteins interact with a member of the TNRC6/GW protein family. Here we used a short GW protein-derived peptide fused to GST and demonstrate that it binds to Ago proteins with high affinity. This allows for the simultaneous isolation of all Ago protein complexes expressed in diverse species to identify associated proteins, small RNAs, or target mRNAs. We refer to our method as "Ago protein Affinity Purification by Peptides" (Ago-APP). Furthermore, expression of this peptide competes for endogenous TNRC6 proteins, leading to global inhibition of miRNA function in mammalian cells.
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http://dx.doi.org/10.1073/pnas.1506116112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4586862PMC
September 2015

Binding of the termination factor Nsi1 to its cognate DNA site is sufficient to terminate RNA polymerase I transcription in vitro and to induce termination in vivo.

Mol Cell Biol 2014 Oct 4;34(20):3817-27. Epub 2014 Aug 4.

Universität Regensburg, Biochemie Zentrum Regensburg, Lehrstuhl Biochemie III, Regensburg, Germany

Different models have been proposed explaining how eukaryotic gene transcription is terminated. Recently, Nsi1, a factor involved in silencing of ribosomal DNA (rDNA), was shown to be required for efficient termination of rDNA transcription by RNA polymerase I (Pol I) in the yeast Saccharomyces cerevisiae. Nsi1 contains Myb-like DNA binding domains and associates in vivo near the 3' end of rRNA genes to rDNA, but information about which and how DNA sequences might influence Nsi1-dependent termination is lacking. Here, we show that binding of Nsi1 to a stretch of 11 nucleotides in the correct orientation was sufficient to pause elongating Pol I shortly upstream of the Nsi1 binding site and to release the transcripts in vitro. The same minimal DNA element triggered Nsi1-dependent termination of pre-rRNA synthesis using an in vivo reporter assay. Termination efficiency in the in vivo system could be enhanced by inclusion of specific DNA sequences downstream of the Nsi1 binding site. These data and the finding that Nsi1 blocks efficiently only Pol I-dependent RNA synthesis in an in vitro transcription system improve our understanding of a unique mechanism of transcription termination.
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http://dx.doi.org/10.1128/MCB.00395-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4187712PMC
October 2014

Interatrial differences of basal molecular set-up and changes in tachycardia-induced heart failure-a proteomic profiling study.

Eur J Heart Fail 2014 Aug 12;16(8):835-45. Epub 2014 Jul 12.

Department of Internal Medicine II, University Hospital Regensburg; Department of Genetic Epidemiology, University of Regensburg.

Aims: Left and right atria show compelling differences regarding organogenesis and specific clinical diseases. In congestive heart failure (CHF), remodelling of the atria occurs leading to increased arrhythmogenic susceptibility and deterioration of clinical symptoms. We aimed to assess the basal left and right atrial molecular set-up and different chamber-specific atrial changes in heart failure.

Methods And Results: We combined an animal model of rapid ventricular pacing induced heart failure in the rabbit and a gel-based proteomic screening of left and right atrial specimen. A gene ontology over-representation analysis was performed for biological function. Ultrastructural adaptations were evaluated using transmission electron microscopy. Comparing left and right atria of healthy control animals (CTRL), 39 proteins displayed significant expression differences involving various biological functions. Upon further statistical analyses, four pathways of energy metabolism were confirmed to be significantly over-represented beneath the other biological processes. Rapid ventricular pacing induced severe left ventricular systolic dysfunction, symptomatic heart failure and a macroscopic atrial remodelling. In CHF versus CTRL, metabolic and antioxidative enzymes were differentially expressed and showed chamber-specific bidirectional alterations. Transmission electron microscopy visualized a remarkable and again chamber-specific ultrastructural disturbance of mitochondrial morphology.

Conclusions: Our data indicate a diverging basal left and right atrial molecular set-up in the adult healthy heart. In addition, metabolic and antioxidative enzymes are profoundly and chamber-specifically altered during atrial remodelling in progressive heart failure.
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http://dx.doi.org/10.1002/ejhf.122DOI Listing
August 2014

Experimental heart failure induces alterations of the lung proteome--insight into molecular mechanisms.

Cell Physiol Biochem 2014 7;33(3):692-704. Epub 2014 Mar 7.

Department of Internal Medicine II, University Hospital Regensburg, Regensburg, Germany.

Background: Heart failure (CHF) is characterized by dyspnea and pulmonary changes. The underlying molecular adaptations are unclear, but might provide targets for therapeutic interventions. We therefore conceived a study to determine molecular changes of early pulmonary stress failure in a model of tachycardia-induced heart failure.

Methods: CHF was induced in rabbits by progessive right ventricular pacing (n=6). Invasive blood pressure measurements and echocardiography were repeatedly performed. Untreated animals served as controls (n=6). Pulmonary tissue specimens were subjected to two-dimensional gel electrophoresis, and differentially expressed proteins were identified by mass spectrometry. Selected proteins were validated by Western Blot analysis and localized by immunohistochemical staining.

Results: CHF animals were characterized by significantly altered functional, morphological, and hemodynamic parameters. Upon proteomic profiling, a total of 33 proteins was found to be differentially expressed in pulmonary tissue of CHF animals (18 up-regulated, and 15 down-regulated) belonging to 4 functional groups: 1. proteins involved in maintaining cytoarchitectural integrity, 2. plasma proteins indicating impaired alveolar-capillary permeability, 3. proteins with antioxidative properties, and 4. proteins participating in the metabolism of selenium compounds

Conclusion: Experimental heart failure profoundly alters the pulmonary proteome. Our results supplement the current knowledge of pulmonary stress failure by specifying its molecular fundament.
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http://dx.doi.org/10.1159/000358645DOI Listing
August 2016

The transcript elongation factor SPT4/SPT5 is involved in auxin-related gene expression in Arabidopsis.

Nucleic Acids Res 2014 Apr 4;42(7):4332-47. Epub 2014 Feb 4.

Department of Cell Biology & Plant Biochemistry, Biochemie-Zentrum Regensburg (BZR), University of Regensburg, Universitätsstrasse 31, D-93053 Regensburg, Germany, Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University, Sohngaardsholmsvej 49, DK-9000 Aalborg, Denmark, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstrasse 3, 06466 Gatersleben, Germany and Institute for Biochemistry I, Biochemie-Zentrum Regensburg (BZR), University of Regensburg, Universitätsstrasse 31, D-93053 Regensburg, Germany.

The heterodimeric complex SPT4/SPT5 is a transcript elongation factor (TEF) that directly interacts with RNA polymerase II (RNAPII) to regulate messenger RNA synthesis in the chromatin context. We provide biochemical evidence that in Arabidopsis, SPT4 occurs in a complex with SPT5, demonstrating that the SPT4/SPT5 complex is conserved in plants. Each subunit is encoded by two genes SPT4-1/2 and SPT5-1/2. A mutant affected in the tissue-specifically expressed SPT5-1 is viable, whereas inactivation of the generally expressed SPT5-2 is homozygous lethal. RNAi-mediated downregulation of SPT4 decreases cell proliferation and causes growth reduction and developmental defects. These plants display especially auxin signalling phenotypes. Consistently, auxin-related genes, most strikingly AUX/IAA genes, are downregulated in SPT4-RNAi plants that exhibit an enhanced auxin response. In Arabidopsis nuclei, SPT5 clearly localizes to the transcriptionally active euchromatin, and essentially co-localizes with transcribing RNAPII. Typical for TEFs, SPT5 is found over the entire transcription unit of RNAPII-transcribed genes. In SPT4-RNAi plants, elevated levels of RNAPII and SPT5 are detected within transcribed regions (including those of downregulated genes), indicating transcript elongation defects in these plants. Therefore, SPT4/SPT5 acts as a TEF in Arabidopsis, regulating transcription during the elongation stage with particular impact on the expression of certain auxin-related genes.
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http://dx.doi.org/10.1093/nar/gku096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3985667PMC
April 2014

Analysis of histone posttranslational modifications from nucleolus-associated chromatin by mass spectrometry.

Methods Mol Biol 2014 ;1094:277-93

Biochemistry Center Regensburg, University of Regensburg, Regensburg, Germany.

Chromatin is unevenly distributed within the eukaryote nucleus and it contributes to the formation of morphologically and functionally distinct substructures, called chromatin domains and nuclear bodies. Here we describe an approach to assess specific chromatin features, the histone posttranslational modifications (PTMs), of the largest nuclear sub-compartment, the nucleolus. In this chapter, methods for the isolation of nucleolus-associated chromatin from native or formaldehyde-fixed cells and the effect of experimental procedures on the outcome of mass spectrometry analysis of histone PTMs are compared.
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http://dx.doi.org/10.1007/978-1-62703-706-8_22DOI Listing
May 2014

Compositional and structural analysis of selected chromosomal domains from Saccharomyces cerevisiae.

Nucleic Acids Res 2014 Jan 7;42(1):e2. Epub 2013 Oct 7.

Universität Regensburg, Biochemie-Zentrum Regensburg (BZR), Lehrstuhl für Biochemie III, 93053 Regensburg, Germany and Department of Molecular, Cell, and Developmental Biology, University of California Santa Cruz, Santa Cruz, CA 95064, USA.

Chromatin is the template for replication and transcription in the eukaryotic nucleus, which needs to be defined in composition and structure before these processes can be fully understood. We report an isolation protocol for the targeted purification of specific genomic regions in their native chromatin context from Saccharomyces cerevisiae. Subdomains of the multicopy ribosomal DNA locus containing transcription units of RNA polymerases I, II or III or an autonomous replication sequence were independently purified in sufficient amounts and purity to analyze protein composition and histone modifications by mass spectrometry. We present and discuss the proteomic data sets obtained for chromatin in different functional states. The native chromatin was further amenable to electron microscopy analysis yielding information about nucleosome occupancy and positioning at the single-molecule level. We also provide evidence that chromatin from virtually every single copy genomic locus of interest can be purified and analyzed by this technique.
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http://dx.doi.org/10.1093/nar/gkt891DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3874202PMC
January 2014

Horizontal gene transfer contributed to the evolution of extracellular surface structures: the freshwater polyp Hydra is covered by a complex fibrous cuticle containing glycosaminoglycans and proteins of the PPOD and SWT (sweet tooth) families.

PLoS One 2012 27;7(12):e52278. Epub 2012 Dec 27.

Department Biologie II, Ludwig-Maximilians-University, Munich, Germany.

The single-cell layered ectoderm of the fresh water polyp Hydra fulfills the function of an epidermis by protecting the animals from the surrounding medium. Its outer surface is covered by a fibrous structure termed the cuticle layer, with similarity to the extracellular surface coats of mammalian epithelia. In this paper we have identified molecular components of the cuticle. We show that its outermost layer contains glycoproteins and glycosaminoglycans and we have identified chondroitin and chondroitin-6-sulfate chains. In a search for proteins that could be involved in organising this structure we found PPOD proteins and several members of a protein family containing only SWT (sweet tooth) domains. Structural analyses indicate that PPODs consist of two tandem β-trefoil domains with similarity to carbohydrate-binding sites found in lectins. Experimental evidence confirmed that PPODs can bind sulfated glycans and are secreted into the cuticle layer from granules localized under the apical surface of the ectodermal epithelial cells. PPODs are taxon-specific proteins which appear to have entered the Hydra genome by horizontal gene transfer from bacteria. Their acquisition at the time Hydra evolved from a marine ancestor may have been critical for the transition to the freshwater environment.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0052278PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3531485PMC
July 2013

Rrp5p, Noc1p and Noc2p form a protein module which is part of early large ribosomal subunit precursors in S. cerevisiae.

Nucleic Acids Res 2013 Jan 2;41(2):1191-210. Epub 2012 Dec 2.

Universität Regensburg, Biochemie-Zentrum Regensburg (BZR), Lehrstuhl Biochemie III, 93053 Regensburg, Germany.

Eukaryotic ribosome biogenesis requires more than 150 auxiliary proteins, which transiently interact with pre-ribosomal particles. Previous studies suggest that several of these biogenesis factors function together as modules. Using a heterologous expression system, we show that the large ribosomal subunit (LSU) biogenesis factor Noc1p of Saccharomyces cerevisiae can simultaneously interact with the LSU biogenesis factor Noc2p and Rrp5p, a factor required for biogenesis of the large and the small ribosomal subunit. Proteome analysis of RNA polymerase-I-associated chromatin and chromatin immunopurification experiments indicated that all members of this protein module and a specific set of LSU biogenesis factors are co-transcriptionally recruited to nascent ribosomal RNA (rRNA) precursors in yeast cells. Further ex vivo analyses showed that all module members predominantly interact with early pre-LSU particles after the initial pre-rRNA processing events have occurred. In yeast strains depleted of Noc1p, Noc2p or Rrp5p, levels of the major LSU pre-rRNAs decreased and the respective other module members were associated with accumulating aberrant rRNA fragments. Therefore, we conclude that the module exhibits several binding interfaces with pre-ribosomes. Taken together, our results suggest a co- and post-transcriptional role of the yeast Rrp5p-Noc1p-Noc2p module in the structural organization of early LSU precursors protecting them from non-productive RNase activity.
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http://dx.doi.org/10.1093/nar/gks1056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3553968PMC
January 2013

Proteomic profiling implies mitochondrial dysfunction in tachycardia-induced heart failure.

J Card Fail 2012 Aug;18(8):660-73

Department of Internal Medicine II, University Regensburg, Regensburg, Germany.

Background/objectives: Molecular mechanisms of congestive heart failure as reflected by alterations of protein expression patterns are still incompletely analyzed. We therefore investigated intraventricular (ie, left ventricular congestive heart failure [LV-CHF] vs. LV-control [CTRL], and right ventricular [RV]-CHF vs. RV-CTRL) and interventricular (ie, LV-CHF vs. RV-CHF, and LV-CTRL vs. RV-CTRL) protein expression differences in an animal model.

Methods: The model of rapid ventricular pacing in rabbits was combined with a proteomic approach using 2-dimensional gel electrophoresis. Identification of proteins was done by matrix-assisted laser desorption/ionization-tandem mass spectrometry (MALDI-MS/MS).

Results: Rapid ventricular pacing-induced heart failure was characterized by LV dilatation, dysfunction, and hypotension as well as by increased BNP gene expression. By comparing LV-CHF vs. LV-CTRL, proteins were found to be underexpressed at 3 crucial points of cellular energy metabolism. In RV-CHF vs. RV-CTRL, proteins belonging to respiratory chain complexes were underexpressed, but additionally a disturbance in the nitric oxide-generating enzymatic apparatus was seen. Regarding the interventricular analyses, a stronger expression of energetic pathways was accompanied by an underexpression of contractile and stress response proteins in failing left vs. right ventricles. Finally, significant protein expression differences were found in LV-CTRL vs. RV-CTRL reflecting a higher expression of contractile, stress response, and respiratory chain proteins in LV tissue.

Conclusions: In tachycardia-induced heart failure, significant inter- and intraventricular protein expression patterns were found with a predominance of proteins, which are involved in cellular energy metabolism.
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http://dx.doi.org/10.1016/j.cardfail.2012.06.418DOI Listing
August 2012

Interrelationships between yeast ribosomal protein assembly events and transient ribosome biogenesis factors interactions in early pre-ribosomes.

PLoS One 2012 14;7(3):e32552. Epub 2012 Mar 14.

Lehrstuhl für Biochemie III, Universität Regensburg, Regensburg, Germany.

Early steps of eukaryotic ribosome biogenesis require a large set of ribosome biogenesis factors which transiently interact with nascent rRNA precursors (pre-rRNA). Most likely, concomitant with that initial contacts between ribosomal proteins (r-proteins) and ribosome precursors (pre-ribosomes) are established which are converted into robust interactions between pre-rRNA and r-proteins during the course of ribosome maturation. Here we analysed the interrelationship between r-protein assembly events and the transient interactions of ribosome biogenesis factors with early pre-ribosomal intermediates termed 90S pre-ribosomes or small ribosomal subunit (SSU) processome in yeast cells. We observed that components of the SSU processome UTP-A and UTP-B sub-modules were recruited to early pre-ribosomes independently of all tested r-proteins. On the other hand, groups of SSU processome components were identified whose association with early pre-ribosomes was affected by specific r-protein assembly events in the head-platform interface of the SSU. One of these components, Noc4p, appeared to be itself required for robust incorporation of r-proteins into the SSU head domain. Altogether, the data reveal an emerging network of specific interrelationships between local r-protein assembly events and the functional interactions of SSU processome components with early pre-ribosomes. They point towards some of these components being transient primary pre-rRNA in vivo binders and towards a role for others in coordinating the assembly of major SSU domains.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0032552PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3303783PMC
July 2012

Mineralization of the metre-long biosilica structures of glass sponges is templated on hydroxylated collagen.

Nat Chem 2010 Dec 23;2(12):1084-8. Epub 2010 Nov 23.

Institute of Bioanalytical Chemistry, Dresden University of Technology, D-01062 Dresden, Germany.

The minerals involved in the formation of metazoan skeletons principally comprise glassy silica, calcium phosphate or carbonate. Because of their ancient heritage, glass sponges (Hexactinellida) may shed light on fundamental questions such as molecular evolution, the unique chemistry and formation of the first skeletal silica-based structures, and the origin of multicellular animals. We have studied anchoring spicules from the metre-long stalk of the glass rope sponge (Hyalonema sieboldi; Porifera, Class Hexactinellida), which are remarkable for their size, durability, flexibility and optical properties. Using slow-alkali etching of biosilica, we isolated the organic fraction, which was revealed to be dominated by a hydroxylated fibrillar collagen that contains an unusual [Gly-3Hyp-4Hyp] motif. We speculate that this motif is predisposed for silica precipitation, and provides a novel template for biosilicification in nature.
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http://dx.doi.org/10.1038/nchem.899DOI Listing
December 2010

Laminin-121--recombinant expression and interactions with integrins.

Matrix Biol 2010 Jul 23;29(6):484-93. Epub 2010 May 23.

Max-Planck-Institut für Biochemie, Martinsried, Germany.

Laminin-121, previously referred as to laminin-3, was expressed recombinantly in human embryonic kidney (HEK) 293 cells by triple transfection of full-length cDNAs encoding mouse laminin α1, β2 and γ1 chains. The recombinant laminin-121 was purified using Heparin-Sepharose followed by molecular sieve chromatography and shown to be correctly folded by electron microscopy and circular dichroism (CD). The CD spectra of recombinant laminin-121 were very similar to those of laminin-111 isolated from Engelbreth-Holm-Swarm tumor (EHS-laminin) but its T(m) value was smaller than EHS-laminin and recombinant lamnin-111 suggesting that the replacement of the β chain reduced the stability of the coiled-coil structure of laminin-121. Its binding to integrins was compared with EHS-laminin, laminin-3A32 purified from murine epidermal cell line and recombinantly expressed laminins-111, -211 and -221. Laminin-121 showed the highest affinity to α6β1 and α7β1 integrins and furthermore, laminin-121 most effectively supported neurite outgrowth. Together, this suggests that the β2 laminins have higher affinity for integrins than the β1 laminins.
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http://dx.doi.org/10.1016/j.matbio.2010.05.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2939186PMC
July 2010

A local role for the small ribosomal subunit primary binder rpS5 in final 18S rRNA processing in yeast.

PLoS One 2010 Apr 19;5(4):e10194. Epub 2010 Apr 19.

Institut für Biochemie, Genetik und Mikrobiologie, University of Regensburg, Regensburg, Germany.

In vivo depletion of the yeast small ribosomal subunit (SSU) protein S5 (rpS5) leads to nuclear degradation of nascent SSUs and to a perturbed global assembly state of the SSU head domain. Here, we report that rpS5 plays an additional local role at the head/platform interface in efficient SSU maturation. We find that yeast small ribosomal subunits which incorporated an rpS5 variant lacking the seven C-terminal amino acids have a largely assembled head domain and are exported to the cytoplasm. On the other hand, 3' processing of 18S rRNA precursors is inhibited in these ribosomal particles, although they associate with the putative endonuclease Nob1p and other late acting 40S biogenesis factors. We suggest that the SSU head component rpS5 and platform components as rpS14 are crucial constituents of a highly defined spatial arrangement in the head-platform interface of nascent SSUs, which is required for efficient processing of the therein predicted SSU rRNA 3' end. Positioning of rpS5 in nascent SSUs, including its relative orientation towards platform components in the head-platform cleft, will depend on the general assembly and folding state of the head domain. Therefore, the suggested model can explain 18S precursor rRNA 3' processing phenotypes observed in many eukaryotic SSU head assembly mutants.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0010194PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2856670PMC
April 2010

Analysis of ribosome biogenesis factor-modules in yeast cells depleted from pre-ribosomes.

Nucleic Acids Res 2010 May 25;38(9):3068-80. Epub 2010 Jan 25.

Institut für Biochemie, Genetik und Mikrobiologie, University of Regensburg, Universitätsstr. 31, 93053 Regensburg, Germany.

Formation of eukaryotic ribosomes requires more than 150 biogenesis factors which transiently interact with the nascent ribosomal subunits. Previously, many pre-ribosomal intermediates could be distinguished by their protein composition and rRNA precursor (pre-rRNA) content. We purified complexes of ribosome biogenesis factors from yeast cells in which de novo synthesis of rRNA precursors was down-regulated by genetic means. We compared the protein composition of these largely pre-rRNA free assemblies with the one of analogous pre-ribosomal preparations by semi-quantitative mass spectrometry. The experimental setup minimizes the possibility that the analysed pre-rRNA free protein modules were derived from (partially) disrupted pre-ribosomal particles and provides thereby strong evidence for their pre-ribosome independent existence. In support of the validity of this approach (i) the predicted composition of the analysed protein modules was in agreement with previously described rRNA-free complexes and (ii) in most of the cases we could identify new candidate members of reported protein modules. An unexpected outcome of these analyses was that free large ribosomal subunits are associated with a specific set of ribosome biogenesis factors in cells where neo-production of nascent ribosomes was blocked. The data presented strengthen the idea that assembly of eukaryotic pre-ribosomal particles can result from transient association of distinct building blocks.
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http://dx.doi.org/10.1093/nar/gkp1244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2875017PMC
May 2010

Insight into the proteome of the hyperthermophilic Crenarchaeon Ignicoccus hospitalis: the major cytosolic and membrane proteins.

Arch Microbiol 2008 Sep 27;190(3):379-94. Epub 2008 Jun 27.

Centre for Electron Microscopy, Faculty for Biology and Preclinical Medicine, University of Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany.

Ignicoccus hospitalis, a hyperthermophilic, chemolithoautotrophic Crenarchaeon, is the host of Nanoarchaeum equitans. Together, they form an intimate association, the first among Archaea. Membranes are of fundamental importance for the interaction of I. hospitalis and N. equitans, as they harbour the proteins necessary for the transport of macromolecules like lipids, amino acids, and cofactors between these organisms. Here, we investigated the protein inventory of I. hospitalis cells, and were able to identify 20 proteins in total. Experimental evidence and predictions let us conclude that 11 are soluble cytosolic proteins, eight membrane or membrane-associated proteins, and a single one extracellular. The quantitatively dominating proteins in the cytoplasm (peroxiredoxin; thermosome) antagonize oxidative and temperature stress which I. hospitalis cells are exposed to at optimal growth conditions. Three abundant membrane protein complexes are found: the major protein of the outer membrane, which might protect the cell against the hostile environment, forms oligomeric complexes with pores of unknown selectivity; two other complexes of the cytoplasmic membrane, the hydrogenase and the ATP synthase, play a key role in energy production and conversion.
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http://dx.doi.org/10.1007/s00203-008-0399-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2755778PMC
September 2008

Ucma, a novel secreted cartilage-specific protein with implications in osteogenesis.

J Biol Chem 2008 Mar 21;283(11):7082-93. Epub 2007 Dec 21.

Department of Experimental Medicine I, Nikolaus-Fiebiger Center of Molecular Medicine, University of Erlangen-Nuremberg, 91054 Erlangen, Germany.

Here we report on the structure, expression, and function of a novel cartilage-specific gene coding for a 17-kDa small, highly charged, and secreted protein that we termed Ucma (unique cartilage matrix-associated protein). The protein is processed by a furin-like protease into an N-terminal peptide of 37 amino acids and a C-terminal fragment (Ucma-C) of 74 amino acids. Ucma is highly conserved between mouse, rat, human, dog, clawed frog, and zebrafish, but has no homology to other known proteins. Remarkable are 1-2 tyrosine sulfate residues/molecule and dense clusters of acidic and basic residues in the C-terminal part. In the developing mouse skeleton Ucma mRNA is expressed in resting chondrocytes in the distal and peripheral zones of epiphyseal and vertebral cartilage. Ucma is secreted into the extracellular matrix as an uncleaved precursor and shows the same restricted distribution pattern in cartilage as Ucma mRNA. In contrast, antibodies prepared against the processed C-terminal fragment located Ucma-C in the entire cartilage matrix, indicating that it either diffuses or is retained until chondrocytes reach hypertrophy. During differentiation of an MC615 chondrocyte subclone in vitro, Ucma expression parallels largely the expression of collagen II and decreases with maturation toward hypertrophic cells. Recombinant Ucma-C does not affect expression of chondrocyte-specific genes or proliferation of chondrocytes, but interferes with osteogenic differentiation of primary osteoblasts, mesenchymal stem cells, and MC3T3-E1 pre-osteoblasts. These findings suggest that Ucma may be involved in the negative control of osteogenic differentiation of osteochondrogenic precursor cells in peripheral zones of fetal cartilage and at the cartilage-bone interface.
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http://dx.doi.org/10.1074/jbc.M702792200DOI Listing
March 2008

Pir proteins of Saccharomyces cerevisiae are attached to beta-1,3-glucan by a new protein-carbohydrate linkage.

J Biol Chem 2006 Apr 22;281(17):11523-9. Epub 2006 Feb 22.

Lehrstuhl für Zellbiologie und Pflanzenphysiologie, Universitaet Regensburg, Universitaetsstrasse 31, 93053 Regensburg, Germany.

A family of covalently linked cell wall proteins of Saccharomyces cerevisiae, called Pir proteins, are characterized by up to 10 conserved repeating units. Ccw5/Pir4p contains only one complete repeating sequence and its deletion caused a release of the protein into the medium. The exchange of each of three glutamines (Gln69, Gln74, Gln76) as well as one aspartic acid (Asp72) within the repeating unit leads to a loss of the protein from the cell wall. Amino acid sequencing revealed that only Gln74 is modified. Release of the protein with mild alkali, changed Gln74 to to glutamic acid, suggesting that Gln74 is involved in the linkage. Analysis by mass spectrometry showed that 5 hexoses are attached to Gln/Glu74. Sugar analysis revealed glucose as the only constituent. It is suggested that Pir proteins form novel, alkali labile ester linkages between the gamma-carboxyl group of glutamic acids, arising from specific glutamines, with hydroxyl groups of glucoses of beta-1,3-glucan chains. This transglutaminase-type reaction could take place extracellularly and would energetically proceed on the account of amido group elimination.
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http://dx.doi.org/10.1074/jbc.M600314200DOI Listing
April 2006

A simplified laminin nomenclature.

Matrix Biol 2005 Aug;24(5):326-32

Center for Biochemistry, Medical Faculty, University of Cologne, Cologne, Germany.

A simplification of the laminin nomenclature is presented. Laminins are multidomain heterotrimers composed of alpha, beta and gamma chains. Previously, laminin trimers were numbered with Arabic numerals in the order discovered, that is laminins-1 to -5. We introduce a new identification system for a trimer using three Arabic numerals, based on the alpha, beta and gamma chain numbers. For example, the laminin with the chain composition alpha5beta1gamma1 is termed laminin-511, and not laminin-10. The current practice is also to mix two overlapping domain and module nomenclatures. Instead of the older Roman numeral nomenclature and mixed nomenclature, all modules are now called domains. Some domains are renamed or renumbered. Laminin epidermal growth factor-like (LE) domains are renumbered starting at the N-termini, to be consistent with general protein nomenclature. Domain IVb of alpha chains is named laminin 4a (L4a), domain IVa of alpha chains is named L4b, domain IV of gamma chains is named L4, and domain IV of beta chains is named laminin four (LF). The two coiled-coil domains I and II are now considered one laminin coiled-coil domain (LCC). The interruption in the coiled-coil of beta chains is named laminin beta-knob (Lbeta) domain. The chain origin of a domain is specified by the chain nomenclature, such as alpha1L4a. The abbreviation LM is suggested for laminin. Otherwise, the nomenclature remains unaltered.
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http://dx.doi.org/10.1016/j.matbio.2005.05.006DOI Listing
August 2005

Quaternary ammonium groups in silica-associated proteins.

Angew Chem Int Ed Engl 2004 Nov;43(44):5933-6

Lehrstuhl Biochemie I, Universität Regensburg, 93040 Regensburg, Germany.

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http://dx.doi.org/10.1002/anie.200461236DOI Listing
November 2004

Structural characterization of proteins and peptides.

Authors:
Rainer Deutzmann

Methods Mol Med 2004 ;94:269-97

Institute for Biochemistry, University of Regensburg, Germany.

The primary structure of proteins is nowadays determined by DNA sequencing, and a variety of genomes are already known. Nevertheless, protein sequencing/identification is still indispensable to analyze the proteins expressed in a cell, to identify specific proteins, and to determine posttranslational modifications. Proteins of interest are typically available in low microgram amounts or even less. The separation method of choice is gel electrophoresis, followed by blotting to PVDF membrane for N-terminal sequencing or by in-gel digestion to generate peptides that can be separated by HPLC. Structural analysis can be done by Edman degradation or mass spectrometry (MS). Edman degradation is the older method based on successive removal of N-terminal amino acids by chemical methods. Sequencing of a peptide requires many hours, the sensitivity is in the range of 2-5 pmol of a purified peptide. Nevertheless, Edman degradation is still the workhorse in the lab for routine work such as identification of blotted proteins. It is also the method of choice for sequencing unknown proteins/ peptides and modified peptides. MS has routinely been used with peptides in the range of 100 fmol or even less. In contrast to Edman degradation, complex mixtures such as tryptic digests can be analyzed, making HPLC separation of peptides unnecessary. MS is a very fast method that can be automated. It is the method of choice for sensitive analysis and large-scale applications (proteomics). Two different ionization methods are commonly used to generate peptide/protein ions for MS analysis. These are MALDI (matrix assisted laser desorption and ionization) and ESI (electrospray ionization). They can be combined with a variety of mass analyzers (TOF, quadrupole, ion trap). Proteins are either identified by searching databases with the masses of proteolytic peptides (peptide mass fingerprinting) or using fragmentation data (raw MS/MS spectra or sequence tags). This approach requires that the protein is known and listed in the database. De novo sequencing by MS of peptides is possible, but very time consuming and not a routine application, in contrast to Edman degradation. The aim of this chapter is to introduce to basic theory, practical applications and limitations of the various methods, to enable the non-expert scientist to decide which method is best suited for his project and which kind of sample preparation is necessary.
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http://dx.doi.org/10.1385/1-59259-679-7:269DOI Listing
April 2004