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Albert Sickmann

Publications and source records attributed to Albert Sickmann.

At least 19 recordsLinked to original sources
PubMed2026

Cardiac mitochondrial proteome of lean, healthy Ossabaw minipigs with predisposition to metabolic syndrome versus that of Göttingen minipigs.

Ossabaw minipigs differ from other (mini)pig strains by their genetic predisposition to develop full metabolic syndrome and their nonresponsiveness to cardioprotective interventions, even before developing the diseased phenotype. Previous DNA sequencing data revealed differences in a cluster of mitochondrial protein-coding genes between Ossabaw and Göttingen minipigs-a large animal model without such a genetic predisposition and a responsiveness to cardioprotection. Alterations in mitochondrial protein composition affect mitochondrial function, and mitochondria play a crucial role in the development of metabolic syndrome and for cardioprotection. Therefore, we aimed to compare the cardiac mitochondrial proteome between lean Ossabaw minipigs with a healthy phenotype and Göttingen minipigs to gain initial insights into potential differences in mitochondrial protein composition and function. Cardiac mitochondria (left ventricular tissue) of both minipig strains (male/female pigs) were isolated, and the proteome was analyzed by liquid chromatography-tandem mass spectrometry. An unbiased, nonhypothesis-driven proteome analysis identified 97% overlap in the proteome. Among the 3% of differentially expressed proteins, 19 were related to mitochondrial metabolism, 8 to transcription and translation, 3 to small molecule transport, 2 to oxidative phosphorylation, and 1 to dynamics and surveillance. These small differences in protein composition were associated with an altered mitochondrial energy turnover-ATP production was reduced by 49% in Ossabaw compared with Göttingen minipig mitochondria. This proteome analysis provides a broader basis to understand how genetic alterations result in changes of the mitochondrial proteome and function, which might be relevant for the development and progression of metabolic syndrome and/or the primordial nonresponsiveness to cardioprotection in Ossabaw minipigs.NEW & NOTEWORTHY Our comprehensive cardiac mitochondrial proteome of Ossabaw and Göttingen minipigs is a valuable resource for cardiac biomedical research. Moreover, our proteome analysis provides a broader basis for understanding how genetic alterations result in changes of the mitochondrial proteome and support a mechanistic link between subtle, strain-specific mitochondrial proteomic signatures and altered mitochondrial energy turnover. These changes may be relevant for the development and progression of metabolic syndrome and/or primordial nonresponsiveness to cardioprotection in Ossabaw minipigs.

Animals↗
PubMed2024

Proteomic Characterization of 1000 Human and Murine Neutrophils Freshly Isolated From Blood and Sites of Sterile Inflammation.

Neutrophils are indispensable for defense against pathogens. Injured tissue-infiltrated neutrophils can establish a niche of chronic inflammation and promote degeneration. Studies investigated transcriptome of single-infiltrated neutrophils which could misinterpret molecular states of these post mitotic cells. However, neutrophil proteome characterization has been challenging due to low harvests from affected tissues. Here, we present a workflow to obtain proteome of 1000 murine and human tissue-infiltrated neutrophils. We generated spectral libraries containing ∼6200 mouse and ∼5300 human proteins from circulating neutrophils. 4800 mouse and 3400 human proteins were recovered from 1000 cells with 102-108 copies/cell. Neutrophils from stroke-affected mouse brains adapted to the glucose-deprived environment with increased mitochondrial activity and ROS-production, while cells invading inflamed human oral cavities increased phagocytosis and granule release. We provide an extensive protein repository for resting human and mouse neutrophils, identify proteins lost in low input samples, thus enabling the proteomic characterization of limited tissue-infiltrated neutrophils.

Proteomics↗
PubMed2006

A comprehensive interaction map of the human survival of motor neuron (SMN) complex.

Assembly of the Sm-class of U-rich small nuclear ribonucleoprotein particles (U snRNPs) is a process facilitated by the macromolecular survival of motor neuron (SMN) complex. This entity promotes the binding of a set of factors, termed LSm/Sm proteins, onto snRNA to form the core structure of these particles. Nine factors, including the SMN protein, the product of the spinal muscular atrophy (SMA) disease gene, Gemins 2-8 and unrip have been identified as the major components of the SMN complex. So far, however, only little is known about the architecture of this complex and the contribution of individual components to its function. Here, we present a comprehensive interaction map of all core components of the SMN complex based upon in vivo and in vitro methods. Our studies reveal a modular composition of the SMN complex with the three proteins SMN, Gemin8, and Gemin7 in its center. Onto this central building block the other components are bound via multiple interactions. Furthermore, by employing a novel assay, we were able to reconstitute the SMN complex from individual components and confirm the interaction map. Interestingly, SMN protein carrying an SMA-causing mutation was severely impaired in formation of the SMN complex. Finally, we show that the peripheral component Gemin5 contributes an essential activity to the SMN complex, most likely the transfer of Sm proteins onto the U snRNA. Collectively, the data presented here provide a basis for the detailed mechanistic and structural analysis of the assembly machinery of U snRNPs.

Carrier Proteins↗
PubMed2006

Phosphoproteomics of human platelets: A quest for novel activation pathways.

Besides their role in hemostasis, platelets are also highly involved in the pathogenesis and progression of cardiovascular diseases. Since important and initial steps of platelet activation and aggregation are regulated by phosphorylation events, a comprehensive study aimed at the characterization of phosphorylation-driven signaling cascades might lead to the identification of new target proteins for clinical research. However, it becomes increasingly evident that only a comprehensive phosphoproteomic approach may help to characterize functional protein networks and their dynamic alteration during physiological and pathophysiological processes in platelets. In this review, we discuss current methodologies in phosphoproteome research including their potentials as well as limitations, from sample preparation to classical approaches like radiolabeling and state-of-the-art mass spectrometry techniques.

Blood Platelets↗
PubMed2006

Identification and isolation of Dictyostelium microtubule-associated protein interactors by tandem affinity purification.

Tandem affinity purification (TAP) is a method originally established in yeast to isolate highly purified protein complexes in a very gentle and efficient way. In this work, we have modified TAP for Dictyostelium applications and have proved it as a useful method to specifically isolate and identify microtubule-associated protein (MAP) complexes. MAPs are known to interact with other proteins to fulfill their complex functions in balancing the dynamic instability of microtubules as well as anchoring microtubules at the cell cortex, controlling mitosis at the centrosome and guiding transport along them. DdEB1 and the Dictyostelium member of the XMAP215 protein family, DdCP224, are known to be part of complexes at the microtubule tips as well as at the centrosome. Employing TAP and mass spectrometry we were able to prove an interaction between EB1 and the DdCP224. Additionally, among other interactions that remain to be confirmed by other methods, an interaction between DdCP224 and a TACC-family protein could be shown for the first time in Dictyostelium and was confirmed by colocalization and co-immunoprecipitation analyses.

Animals↗
PubMed2006

Feedback of the kinesin-1 neck-linker position on the catalytic site.

Kinesin-1 motor proteins step along microtubules by a mechanism in which the heads cycle through microtubule-bound and unbound states in an interlaced fashion. An important contribution to head-head coordination arises from the action of the neck-linker that docks onto the core motor domain upon ATP binding. We show here that the docked neck-linker not only guides the microtubule-unbound head to the next microtubule binding site but also signals its position to the head to which it is attached. Cross-linking studies on mutated kinesin constructs reveal that residues at the interface motor core/docked neck-linker, among them most importantly a conserved tyrosine, are involved in this feedback. The primary effect of the docked neck-linker is a reduced microtubule binding affinity in the ADP state.

Adenosine Diphosphate↗
PubMed2006

Proteomic analysis of the yeast mitochondrial outer membrane reveals accumulation of a subclass of preproteins.

Mitochondria consist of four compartments-outer membrane, intermembrane space, inner membrane, and matrix--with crucial but distinct functions for numerous cellular processes. A comprehensive characterization of the proteome of an individual mitochondrial compartment has not been reported so far. We used a eukaryotic model organism, the yeast Saccharomyces cerevisiae, to determine the proteome of highly purified mitochondrial outer membranes. We obtained a coverage of approximately 85% based on the known outer membrane proteins. The proteome represents a rich source for the analysis of new functions of the outer membrane, including the yeast homologue (Hfd1/Ymr110c) of the human protein causing Sjögren-Larsson syndrome. Surprisingly, a subclass of proteins known to reside in internal mitochondrial compartments were found in the outer membrane proteome. These seemingly mislocalized proteins included most top scorers of a recent genome-wide analysis for mRNAs that were targeted to mitochondria and coded for proteins of prokaryotic origin. Together with the enrichment of the precursor form of a matrix protein in the outer membrane, we conclude that the mitochondrial outer membrane not only contains resident proteins but also accumulates a conserved subclass of preproteins destined for internal mitochondrial compartments.

Aldehyde Oxidoreductases↗
PubMed2006

Vegetative insecticidal protein (Vip1Ac) of Bacillus thuringiensis HD201: evidence for oligomer and channel formation.

The binding component (Vip1Ac) of the ADP-ribosylating vegetative insecticidal protein (Vip) of Bacillus thuringiensis HD201 was isolated from the supernatant of cell cultures. Vip1Ac protein solubilized at room temperature ran as oligomers on SDS-PAGE. These oligomers were not resistant to heating. Mass spectroscopic analysis of this high molecular mass band identified it as Vip1Ac. The protein formed in artificial lipid bilayer membranes channels with two conductance states of about 350 and 700 pS in 1 M KCl. The channel conductance showed a linear dependence on the bulk aqueous KCl concentration, which indicated that the channel properties were more general than specific. Zero-current membrane potential measurements showed that the Vip1Ac channel has a slightly higher permeability for chloride than for potassium ions. Asymmetric addition of Vip1Ac to lipid bilayer membranes resulted in an asymmetric voltage dependence, indicating its full orientation within the membrane. The functional role of Vip1Ac and its relationship to other ADP-ribosylating toxins are discussed.

Adenosine Diphosphate Ribose↗
PubMed2006

A comprehensive dictionary of protein accession codes for complete protein accession identifier alias resolving.

In mass spectrometry-based proteomics, protein identification results usually consist of peptide sequences and database-dependent accession identifiers of the matching proteins. Often certain annotations are only available in particular databases that in turn must be queried by a certain identifier. In order to simplify and unify the tracing of identified proteins back to their original annotation information, a system capable of set-oriented mapping the different accession identifiers of proteins derived from multiple sequence database sources has been developed. This allows unification of the access to protein information and tracing to other online resources providing additional information as well as resolving cross-references of protein identifications. The interface of seqDB is available via http://www.protein-ms.de following the link to seqDB.

Database Management Systems↗
PubMed2006

Identification of novel centrosomal proteins in Dictyostelium discoideum by comparative proteomic approaches.

The centrosome functions as the main microtubule-organization center of the cell and is of importance for all microtubule-dependent processes such as organelle transport and directionality of cell migration. One of the major model organisms in centrosome research is the slime mold Dictyostelium discoideum. Since only 10 centrosomal proteins are known so far in Dictyostelium discoideum, the elucidation of new centrosomal components may give a more comprehensive understanding of centrosomal function. To distinguish between centrosomal and contaminating proteins we established different separation and relative quantification strategies including techniques such as iTRAQ and DIGE. In this work, we present the identification of several known components as well as more than 70 new candidates--currently subject of further investigations--for the protein inventory of the Dictyostelium centrosome. Among these protein identifications, 44% represent hypothetical proteins of still unknown function associated with the centrosome.

Animals↗
PubMed2006

Toward the complete yeast mitochondrial proteome: multidimensional separation techniques for mitochondrial proteomics.

Proteomic analyses of different subcellular compartments, so-called organellar proteomics, facilitate the understanding of cellular functions on a molecular level. In this work, various orthogonal multidimensional separation techniques both on the protein and on the peptide level are compared with regard to the number of identified proteins as well as the classes of proteins accessible by the respective methodology. The most complete overview was achieved by a combination of such orthogonal techniques as shown by the analysis of the yeast mitochondrial proteome. A total of 851 different proteins (PROMITO dataset) were identified by use of multidimensional LC-MS/MS, 1D-SDS-PAGE combined with nano-LC-MS/MS and 2D-PAGE with subsequent MALDI-mass fingerprinting. Our PROMITO approach identified the 749 proteins, which were found in the largest previous study on the yeast mitochondrial proteome, and additionally 102 proteins including 42 open reading frames with unknown function, providing the basis for a more detailed elucidation of mitochondrial processes. Comparison of the different approaches emphasizes a bias of 2D-PAGE against proteins with very high isoelectric points as well as large and hydrophobic proteins, which can be accessed more appropriately by the other methods. While 2D-PAGE has advantages in the possible separation of protein isoforms and quantitative differential profiling, 1D-SDS-PAGE with nano-LC-MS/MS and multidimensional LC-MS/MS are better suited for efficient protein identification as they are less biased against distinct classes of proteins. Thus, comprehensive proteome analyses can only be realized by a combination of such orthogonal approaches, leading to the largest dataset available for the mitochondrial proteome of yeast.

Mitochondrial Proteins↗
PubMed2006

An improved method for Dictyostelium centrosome isolation.

The Dictyostelium dicoideum centrosome consists of a box-shaped, layered core structure surrounded by dense nodules embedded in amorphous material, which make up the so-called corona. Thus, it differs markedly from centriole-containing centrosomes in animal cells or the plaque structure of yeast spindle pole bodies. For a long time, purification of Dictyostelium centrosomes was hampered by its extraordinarily tight linkage to the nucleus, which resisted all attempts to dissociate centrosomes and nuclei without destruction of the centrosome itself. Fortunately, we were able to solve this problem, and have already published a centrosome isolation protocol that is based on treatment of nucleus/centrosome complexes with sodium pyrophosphate and shear forces, followed by centrosome isolation through sedimentation and filtration techniques. However, isolated centrosomes prepared according to this protocol still contained too many impurities to allow mass spectrometrical analyses. Here, we present an improved protocol for the isolation of Dictyostelium centrosomes that contain considerably less contaminations with cytosolic and nuclear proteins.

Animals↗
PubMed2005

Efficient analysis and extraction of MS/MS result data from Mascot result files.

BACKGROUND: Mascot is a commonly used protein identification program for MS as well as for tandem MS data. When analyzing huge shotgun proteomics datasets with Mascot's native tools, limits of computing resources are easily reached. Up to now no application has been available as open source that is capable of converting the full content of Mascot result files from the original MIME format into a database-compatible tabular format, allowing direct import into database management systems and efficient handling of huge datasets analyzed by Mascot. RESULTS: A program called mres2x is presented, which reads Mascot result files, analyzes them and extracts either selected or all information in order to store it in a single file or multiple files in formats which are easier to handle downstream of Mascot. It generates different output formats. The output of mres2x in tab format is especially designed for direct high-performance import into relational database management systems using native tools of these systems. Having the data available in database management systems allows complex queries and extensive analysis. In addition, the original peak lists can be extracted in DTA format suitable for protein identification using the Sequest program, and the Mascot files can be split, preserving the original data format. During conversion, several consistency checks are performed. mres2x is designed to provide high throughput processing combined with the possibility to be driven by other computer programs. The source code including supplement material and precompiled binaries is available via http://www.protein-ms.de and http://sourceforge.net/projects/protms/. CONCLUSION: The database upload allows regrouping of the MS/MS results using a database management system and complex analyzing queries using SQL without the need to run new Mascot searches when changing grouping parameters.

Databases, Factual↗
PubMed2005

Transformation and other factors of the peptide mass spectrometry pairwise peak-list comparison process.

BACKGROUND: Biological Mass Spectrometry is used to analyse peptides and proteins. A mass spectrum generates a list of measured mass to charge ratios and intensities of ionised peptides, which is called a peak-list. In order to classify the underlying amino acid sequence, the acquired spectra are usually compared with synthetic ones. Development of suitable methods of direct peak-list comparison may be advantageous for many applications. RESULTS: The pairwise peak-list comparison is a multistage process composed of matching of peaks embedded in two peak-lists, normalisation, scaling of peak intensities and dissimilarity measures. In our analysis, we focused on binary and intensity based measures. We have modified the measures in order to comprise the mass spectrometry specific properties of mass measurement accuracy and non-matching peaks. We compared the labelling of peak-list pairs, obtained using different factors of the pairwise peak-list comparison, as being the same or different to those determined by sequence database searches. In order to elucidate how these factors influence the peak-list comparison we adopted an analysis of variance type method with the partial area under the ROC curve as a dependent variable. CONCLUSION: The analysis of variance provides insight into the relevance of various factors influencing the outcome of the pairwise peak-list comparison. For large MS/MS and PMF data sets the outcome of ANOVA analysis was consistent, providing a strong indication that the results presented here might be valid for many various types of peptide mass measurements.

Algorithms↗
PubMed2005

Elucidation of N-glycosylation sites on human platelet proteins: a glycoproteomic approach.

Among known platelet proteins, a prominent and functionally important group is represented by glycoprotein isoforms. They account e.g. for secretory proteins and plasma membrane receptors including integrins and glycoprotein VI as well as intracellular components of cytosol and organelles including storage proteins (multimerin 1 etc.). Although many of those proteins have been studied for some time with regard to their function, little attention has been paid with respect to their glycosylation sites. Here we report the analysis of N-glycosylation sites of human platelet proteins. For the enrichment of glycopeptides, lectin affinity chromatography as well as chemical trapping of protein bound oligosaccharides was used. Therefore, concanavalin A was used for specific interaction with carbohydrate species along with periodic acid oxidation and hydrazide bead trapping of glycosylated proteins. Derivatization by peptide:N-glycosidase F yielded deglycosylated peptides, which provided the basis for the elucidation of proteins and their sites of modification. Using both methods in combination with nano-LC-ESI-MS/MS analysis 70 different glycosylation sites within 41 different proteins were identified. Comparison with the Swiss-Prot database established that the majority of these 70 sites have not been specifically determined by previous research projects. With this approach including hydrazide bead affinity trapping, the immunoglobulin receptor G6f, which is known to couple to the Ras-mitogen-activated protein kinase pathway in the immune system, was shown here for the first time to be present in human platelets.

Amino Acid Sequence↗
PubMed2005

Proteomic analysis of brain plasma membranes isolated by affinity two-phase partitioning.

A comprehensive analysis of plasma membrane proteins is essential to in-depth understanding of brain development, function, and diseases. Proteomics offers the potential to perform such a comprehensive analysis, yet it requires efficient protocols for the purification of the plasma membrane compartment. Here, we present a novel and efficient protocol for the separation and enrichment of brain plasma membrane proteins. It lasts only 4 h and is easy to perform. It highly enriches plasma membrane proteins and can be applied to small amounts of brain tissue, such as the cerebellum of a single rat, which was used in the present study. The protocol is based on affinity partitioning of microsomes in an aqueous two-phase system. Marker enzyme assays demonstrated a more than 12-fold enrichment of plasma membranes and a strong reduction of other compartments, such as mitochondria and the endoplasmic reticulum. 506 different proteins were identified when the enriched proteins underwent LC-MS/MS analysis subsequent to protein separation by SDS-PAGE. Using gene ontology, 146 proteins were assigned to a subcellular compartment. Ninety-three of those (64%) were membrane proteins, and 49 (34%) were plasma membrane proteins. A combined literature and database search for all 506 identified proteins revealed subcellular information on 472 proteins, of which 197 (42%) were plasma membrane proteins. These comprised numerous transporters, channels, and neurotransmitter receptors, e.g. the inward rectifying potassium channel Kir7.1 and the cerebellum-specific gamma-aminobutyric acid receptor GABRA6. Surface proteins involved in cell-cell contact and disease-related proteins were also identified. Six of the 146 assigned proteins were derived from mitochondrial membranes and 5 from membranes of the endoplasmic reticulum. Taken together, our protocol represents a simple, rapid, and reproducible tool for the proteomic characterization of brain plasma membranes. Because it conserves membrane structure and protein interactions, it is also suitable to enrich multimeric protein complexes from the plasma membrane for subsequent analysis.

Alkaline Phosphatase↗
PubMed2005

The human platelet membrane proteome reveals several new potential membrane proteins.

We present the first focused proteome study on human platelet membranes. Due to the removal of highly abundant cytoskeletal proteins a wide spectrum of known platelet membrane proteins and several new and hypothetical proteins were accessible. In contrast to other proteome studies we focused on prefractionation and purification of membranes from human platelets according to published protocols to reduce sample complexity and enrich interesting membrane proteins. Subsequently protein separation by common one-dimensional SDS-PAGE as well as the combined benzyldimethyl-n-hexadecylammonium chloride/SDS separation technique was performed prior to mass spectrometry analysis by nano-LC-ESI-MS/MS. We demonstrate that the application of both separation systems in parallel is required for maximization of protein tagging out of a complex sample. Furthermore the identification of several potential membrane proteins in human platelets yields new potential targets in functional platelet research.

Amino Acid Sequence↗