Biomedical subjects

Urs Lewandrowski

Publications and source records attributed to Urs Lewandrowski.

5 recordsLinked to original sources

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↗

PubMed2005

Laser-induced dissociation/high-energy collision-induced dissociation fragmentation using MALDI-TOF/TOF-MS instrumentation for the analysis of neutral and acidic oligosaccharides.

Producing detailed mass spectrometric fragmentation data of native oligosaccharides for the purpose of basic structure elucidation has become a readily accessible tool since the availability of enhanced technical equipment. In this report, high-energy collision-induced dissociation (heCID) in combination with MALDI-TOF/TOF technology for analysis of native neutral and acidic oligosaccharides is described. By providing complementary data, heCID-MALDI-TOF/TOF adds a variety of valuable cross-ring fragmentation information to the information of glycosidic fragmentation obtained preferably by laser-induced dissociation (LID). We examined parameters influencing fragmentation behavior of both-acidic and neutral-compounds. Results show a dependency of the fragmentation pattern for the employed matrix as well as the laser intensity provided for the ionization of the analytes and the complexity of the analytes. Due to instrument-specific settings, protonated glycosidic ion series within spectra of sodiated compounds could also be identified. Furthermore, acquired spectra could be readily used to identify compounds by comparison to existing glycan databases such as GlycoSuiteDB and GlycosciencesDB. The results show a better scoring of heCID data sets in comparison to LID-derived data. heCID-MALDI-TOF/TOF analysis in combination with database search algorithms is demonstrated to be suitable for an initial identification/classification of carbohydrates.

Carbohydrate Sequence↗

PubMed2004

Challenges in mass spectrometry-based proteomics.

During the last decade, protein analysis and proteomics have been established as new tools for understanding various biological problems. As the identification of proteins after classical separation techniques, such as two-dimensional gel electrophoresis, have become standard methods, new challenges arise in the field of proteomics. The development of "functional proteomics" combines functional characterization, like regulation, localization and modification, with the identification of proteins for deeper insight into cellular functions. Therefore, different mass spectrometric techniques for the analysis of post-translational modifications, such as phosphorylation and glycosylation, have been established as well as isolation and separation methods for the analysis of highly complex samples, e.g. protein complexes or cell organelles. Furthermore, quantification of protein levels within cells is becoming a focus of interest as mass spectrometric methods for relative or even absolute quantification have currently not been available. Protein or genome databases have been an essential part of protein identification up to now. Thus, de novo sequencing offers new possibilities in protein analytical studies of organisms not yet completely sequenced. The intention of this review is to provide a short overview about the current capabilities of protein analysis when addressing various biological problems.

Algorithms↗