Quantitative Protein Sulfenic Acid Analysis Identifies Platelet Releasate-Induced Activation of Integrin ß2 on Monocytes via NADPH Oxidase.

Physiological stimuli such as thrombin, or pathological stimuli such as lysophosphatidic acid (LPA), activate platelets. The activated platelets bind to monocytes through P-selectin-PSGL-1 interactions but also release the contents of their granules, commonly called "platelet releasate". It is known that monocytes in contact with platelet releasate produce reactive oxygen species (ROS). Reversible cysteine oxidation by ROS is considered to be a potential regulator of protein function. In a previous study, we used THP-1 monocytic cells exposed to LPA- or thrombin-induced platelet releasate and a modified biotin switch assay to unravel the biological processes that are influenced by reversible cysteine oxidation. To gain a better understanding of the redox regulation of monocytes in atherosclerosis, we have now altered the modified biotin switch to selectively quantify protein sulfenic acid, a subpopulation of reversible cysteine oxidation. Using arsenite as reducing agent in the modified biotin switch assay, we were able to quantify 1161 proteins, in which more than 100 sulfenic acid sites were identified. Bioinformatics analysis of the quantified sulfenic acid sites highlighted the relevant, previously missed biological process of monocyte transendothelial migration, which included integrin ß2. Flow cytometry validated the activation of LFA-1 (αLß2) and Mac-1 (αMß2), two subfamilies of integrin ß2 complexes, on human primary monocytes following platelet releasate treatment. The activation of LFA-1 was mediated by ROS from NADPH oxidase (NOX) activation. Production of ROS and activation of LFA-1 in human primary monocytes were independent of P-selectin-PSGL-1 interaction. Our results proved the modified biotin switch assay to be a powerful tool with the ability to reveal new regulatory mechanisms and identify new therapeutic targets.

Investigating Deformylase and Deacylase Activity of Mammalian and Bacterial Sirtuins.

Lysine acylation constitutes a major group of post-translational modifications of proteins, and is found in the proteomes of organisms from all kingdoms of life. Sirtuins are considered the main erasers of these modification marks, and thus contribute to acylation-dependent regulation of enzyme activity, and potentially of protein quality control. We have established a substrate scaffold to enable the analysis of sirtuin activity with a broad range of acyl-lysine modifications, including hydrophobic fatty acids. Characterization of the deacylase activity of the bacterial SrtN, which is encoded by the yhdZ gene of Bacillus subtilis, showed that this enzyme is capable of removing a broad range of acyl groups. These investigations further showed that SrtN and human SIRT1 are efficient lysine-deformylases, thereby providing a first clue as to how this nonenzymatic modification might be removed from affected proteins.

Global proteome analysis identifies active immunoproteasome subunits in human platelets.

The discovery of new functions for platelets, particularly in inflammation and immunity, has expanded the role of these anucleate cell fragments beyond their primary hemostatic function. Here, four in-depth human platelet proteomic data sets were generated to explore potential new functions for platelets based on their protein content and this led to the identification of 2559 high confidence proteins. During a more detailed analysis, consistently high expression of the proteasome was discovered, and the composition and function of this complex, whose role in platelets has not been thoroughly investigated, was examined. Data set mining resulted in identification of nearly all members of the 26S proteasome in one or more data sets, except the ß5 subunit. However, ß5i, a component of the immunoproteasome, was identified. Biochemical analyses confirmed the presence of all catalytically active subunits of the standard 20S proteasome and immunoproteasome in human platelets, including ß5, which was predominantly found in its precursor form. It was demonstrated that these components were assembled into the proteasome complex and that standard proteasome as well as immunoproteasome subunits were constitutively active in platelets. These findings suggest potential new roles for platelets in the immune system. For example, the immunoproteasome may be involved in major histocompatibility complex I (MHC I) peptide generation, as the MHC I machinery was also identified in our data sets.

Advancing formaldehyde cross-linking towards quantitative proteomic applications.

Formaldehyde is a key fixation reagent. This review explores its application in combination with qualitative and quantitative mass spectrometry (MS). Formalin-fixed and paraffin-embedded (FFPE) tissues form a large reservoir of biologically valuable samples and their investigation by MS has only recently started. Furthermore, formaldehyde can be used to stabilise protein-protein interactions in living cells. Because formaldehyde is able to modify proteins, performing MS analysis on these samples can pose a challenge. Here we discuss the chemistry of formaldehyde cross-linking, describe the problems of and progress in these two applications and their common aspects, and evaluate the potential of these methods for the future.

In silico protein interaction analysis using the global proteome machine database.

Experiments to probe for protein-protein interactions are the focus of functional proteomic studies, thus proteomic data repositories are increasingly likely to contain a large cross-section of such information. Here, we use the Global Proteome Machine database (GPMDB), which is the largest curated and publicly available proteomic data repository derived from tandem mass spectrometry, to develop an in silico protein interaction analysis tool. Using a human histone protein for method development, we positively identified an interaction partner from each histone protein family that forms the histone octameric complex. Moreover, this method, applied to the α subunits of the human proteasome, identified all of the subunits in the 20S core particle. Furthermore, we applied this approach to human integrin αIIb and integrin ß3, a major receptor involved in the activation of platelets. We identified 28 proteins, including a protein network for integrin and platelet activation. In addition, proteins interacting with integrin ß1 obtained using this method were validated by comparing them to those identified in a formaldehyde-supported coimmunoprecipitation experiment, protein-protein interaction databases and the literature. Our results demonstrate that in silico protein interaction analysis is a novel tool for identifying known/candidate protein-protein interactions and proteins with shared functions in a protein network.

Mass spectrometry-based proteomics in biomedical research: emerging technologies and future strategies.

In recent years, the technology and methods widely available for mass spectrometry (MS)-based proteomics have increased in power and potential, allowing the study of protein-level processes occurring in biological systems. Although these methods remain an active area of research, established techniques are already helping answer biological questions. Here, this recent evolution of MS-based proteomics and its applications are reviewed, including standard methods for protein and peptide separation, biochemical fractionation, quantitation, targeted MS approaches such as selected reaction monitoring, data analysis and bioinformatics. Recent research in many of these areas reveals that proteomics has moved beyond simply cataloguing proteins in biological systems and is finally living up to its initial potential - as an essential tool to aid related disciplines, notably health research. From here, there is great potential for MS-based proteomics to move beyond basic research, into clinical research and diagnostics.

Optimization of formaldehyde cross-linking for protein interaction analysis of non-tagged integrin beta1.

Formaldehyde cross-linking of protein complexes combined with immunoprecipitation and mass spectrometry analysis is a promising technique for analysing protein-protein interactions, including those of transient nature. Here we used integrin beta1 as a model to describe the application of formaldehyde cross-linking in detail, particularly focusing on the optimal parameters for cross-linking, the detection of formaldehyde cross-linked complexes, the utility of antibodies, and the identification of binding partners. Integrin beta1 was found in a high molecular weight complex after formaldehyde cross-linking. Eight different anti-integrin beta1 antibodies were used for pull-down experiments and no loss in precipitation efficiency after cross-linking was observed. However, two of the antibodies could not precipitate the complex, probably due to hidden epitopes. Formaldehyde cross-linked complexes, precipitated from Jurkat cells or human platelets and analyzed by mass spectrometry, were found to be composed of integrin beta1, alpha4 and alpha6 or beta1, alpha6, alpha2, and alpha5, respectively.

Inefficient processing of mRNA for the membrane form of IgE is a genetic mechanism to limit recruitment of IgE-secreting cells.

Immunoglobulin E (IgE) is the key effector element in allergic diseases ranging from innocuous hay fever to life-threatening anaphylactic shock. Compared to other Ig classes, IgE serum levels are very low. In its membrane-bound form (mIgE), IgE behaves as a classical antigen receptor on B lymphocytes. Expression of mIgE is essential for subsequent recruitment of IgE-secreting cells. We show that in activated, mIgE-bearing B cells, mRNA for the membrane forms of both murine and human epsilon (epsilon) heavy chains (HC) are poorly expressed compared to mRNA for the secreted forms. In contrast, in mIgG-bearing B cells, mRNA for the membrane forms of murine gamma-1 (gamma1) and the corresponding human gamma4 HC are expressed at a much higher level than mRNA for the respective secreted forms. We show that these findings correlate with the presence of deviant polyadenylation signal hexamers in the 3' untranslated region (UTR) of both murine and human epsilon genes, causing inefficient processing of primary transcripts and thus poor expression of the proteins and poor recruitment of IgE-producing cells in the immune response. Thus, we have identified a genetic steering mechanism in the regulation of IgE synthesis that represents a further means to restrain potentially dangerous, high serum IgE levels.