Cytoskeleton assembly at endothelial cell-cell contacts is regulated by alphaII-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.

Phosphoproteome of resting human platelets.

Beside their main physiological function in hemostasis, platelets are also highly involved in pathological processes, such as atherothrombosis and inflammation. During hemostasis, binding of adhesive substrates to tyrosine-kinase-linked adhesion receptors and/or soluble agonists to G-protein coupled receptors leads to a cascade of intracellular signaling processes based on substrate (de)phosphorylation. The same mechanisms are involved in platelet activation at sites of atherosclerotic plaque rupture, contributing to vessel occlusion and consequently to pathologic states, such as myocardial infarction, stroke, or peripheral artery disease.

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.

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.

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.

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.

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.

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.

Differential analysis of phosphorylated proteins in resting and thrombin-stimulated human platelets.

Blood platelets are important components of haemostasis. After their activation they cause healing of wounds by forming plugs and initiate repair processes. One important event in regulating this activation is the phosphorylation/dephosphorylation of multiple proteins on various tyrosine, serine and threonine residues.