Jan Moebius

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.

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.

Cytoskeleton assembly at endothelial cell–cell contacts is regulated by αII-spectrin–VASP complexes

Directed cortical actin assembly is the driving force for intercellular adhesion. Regulated by phosphorylation, vasodilator-stimulated phosphoprotein (VASP) participates in actin fiber formation. We screened for endothelial proteins, which bind to VASP, dependent on its phosphorylation status. Differential proteomics identified αII-spectrin as such a VASP-interacting protein. αII-Spectrin binds to the VASP triple GP5-motif via its SH3 domain. cAMP-dependent protein kinase–mediated VASP phosphorylation at Ser157 inhibits αII-spectrin–VASP binding. VASP is dephosphorylated upon formation of cell–cell contacts and in confluent, but not in sparse cells, αII-spectrin colocalizes with nonphosphorylated VASP at cell–cell junctions. Ectopic expression of the αII-spectrin SH3 domain at cell–cell contacts translocates VASP, initiates cortical actin cytoskeleton formation, stabilizes cell–cell contacts, and decreases endothelial permeability. Conversely, the permeability of VASP-deficient endothelial cells (ECs) and microvessels of VASP-null mice increases. Reconstitution of VASP-deficient ECs rescues barrier function, whereas αII-spectrin binding-deficient VASP mutants fail to restore elevated permeability. We propose that αII-spectrin–VASP complexes regulate cortical actin cytoskeleton assembly with implications for vascular permeability.

Enhanced N-Glycosylation Site Analysis of Sialoglycopeptides by Strong Cation Exchange Prefractionation Applied to Platelet Plasma Membranes

Elucidation of post-translational modifications to proteins, such as glycosylations or phosphorylations, is one of the major issues concerning ongoing proteomics studies. To reduce general sample complexity, a necessary prerequisite is specific enrichment of peptide subsets prior to mass spectrometric sequencing. Regarding analysis of overall N-glycosylation sites in the past, this has been achieved by several approaches proving to be more or less complicated and specific. Here we present a novel strategy to target N-glycosylation sites with application to platelet membrane proteins. Initial aqueous two-phase partitioning for membrane enrichment and single step strong cation exchange-based purification of glycopeptides resulted in identification of 148 glycosylation sites on 79 different protein species. Although 69% of these sites were not annotated in the Swiss-Prot database before, a high number of 75% plasma membrane-localized proteins were analyzed. Furthermore miniaturizations and relative quantification are comprised in the developed method suggesting further use in other proteome projects. Results on platelet glycosylation sites may imply an impact on research of bleeding disorders as well as potential new functions in inflammation and immunoactivity.

Two-Dimensional BAC/SDS-PAGE for Membrane Proteomics

Although often used in membrane proteome studies, conventional two-dimensional gel electrophoresis (2-DE) is not well suited for resolving hydrophobic proteins. Nevertheless, an alternative technique, two-dimensional BAC/SDS-PAGE (2-DB) using the cationic detergent benzyldimethyl-n-hexadecylammonium chloride (BAC) in the first and the anionic detergent SDS in the second dimension can be utilized as a powerful tool for the separation and analysis of membrane proteins. Systematic studies demonstrated the advantage of 2-DB over one-dimensional SDS-PAGE and 2-DE with regard to membrane proteomics. While in 2-DE gels, in particular proteins with more than one transmembrane domain (TMD) are underrepresented, one-dimensional SDS-PAGE lacks sufficient resolution for large scale analyses. In contrast, 2-DB enabled the identification of extremely hydrophobic proteins like cytochrome-c oxidase subunit I from S. cerevisiae with a total of 12 known TMD. Especially the application of tube gels in the first dimension as well as the recent introduction of improved buffer systems hold a great potential for future 2-DB-based membrane studies.

Ruthenium (II) tris‐bathophenanthroline disulfonate is well suitable for Tris‐Glycine PAGE but not for Bis‐Tris gels

Pre‐cast bis(2‐hydroxyethyl)iminotris(hydroxymethyl)methane (Bis‐Tris) gels have proven to be very suitable for pre‐fractionation for LC‐MS/MS analysis due to high reliability and long stability. To visualize proteins within gels fluorescence dyes proved to be a good tradeoff between sensitivity and MS‐compatibility. The custom‐made ruthenium dye represents a low‐cost alternative regarding fluorescence‐based protein visualization with high sensitivity. We demonstrate, that this dye is incompatible with Bis‐Tris gels, while using Tris‐Glycine gels a competitive sensitivity to commercially available stains can be achieved.

Platelet Proteomics: Essentials for Understanding and Application

Proteomics has become a promising technology for platelet research. The development of the last 10 years has provided a complex set of different separation tools and analytical techniques for proteins and their modifications. Several of these approaches have already been performed in platelet studies. Here, we present an overview of different methods used in the field of platelet proteomics, like, for instance, two-dimensional gel electrophoresis of proteins, proteolytic digestion of complete platelet lysates combined with subsequent high performance liquid chromatography (HPLC)-based peptide separation as well as strategies for the analysis of posttranslational modifications. We will focus on methodical aspects of the different techniques as well as their potential and limitations in order to provide an orientation guide for further projects.