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.

Quantitative Glycoproteomic Analysis Identifies Platelet-Induced Increase of Monocyte Adhesion via the Up-Regulation of Very Late Antigen 5.

Physiological stimuli, such as thrombin, or pathological stimuli, such as lysophosphatidic acid (LPA), activate platelets circulating in blood. Once activated, platelets bind to monocytes via P-selectin-PSGL-1 interactions but also release the stored contents of their granules. These platelet releasates, in addition to direct platelet binding, activate monocytes and facilitate their recruitment to atherosclerotic sites. Consequently, understanding the changes platelet releasates induce in monocyte membrane proteins is critical. We studied the glyco-proteome changes of THP-1 monocytic cells affected by LPA- or thrombin-induced platelet releasates. We employed lectin affinity chromatography combined with filter aided sample preparation to achieve high glyco- and membrane protein and protein sequence coverage. Using stable isotope labeling by amino acids in cell culture, we quantified 1715 proteins, including 852 membrane and 500 glycoproteins, identifying the up-regulation of multiple proteins involved in monocyte extracellular matrix binding and transendothelial migration. Flow cytometry indicated expression changes of integrin α5, integrin ß1, PECAM-1, and PSGL-1. The observed increase in monocyte adhesion to fibronectin was determined to be mediated by the up-regulation of very late antigen 5 via a P-selectin-PSGL-1 independent mechanism. This novel aspect could be validated on CD14+ human primary monocytes, highlighting the benefits of the improved enrichment method regarding high membrane protein coverage and reliable quantification.

Formaldehyde cross-linking and structural proteomics: Bridging the gap.

Proteins are dynamic entities constantly moving and altering their structures based on their functions and interactions inside and outside the cell. Formaldehyde cross-linking combined with mass spectrometry can accurately capture interactions of these rapidly changing biomolecules while maintaining their physiological surroundings. Even with its numerous established uses in biology and compatibility with mass spectrometry, formaldehyde has not yet been applied in structural proteomics. However, formaldehyde cross-linking is moving toward analyzing tertiary structure, which conventional cross-linkers have already accomplished. The purpose of this review is to describe the potential of formaldehyde cross-linking in structural proteomics by highlighting its applications, characteristics and current status in the field.

Identification of total reversible cysteine oxidation in an atherosclerosis model using a modified biotin switch assay.

Oxidative stress due to the imbalance of reactive oxygen species (ROS) and the resulting reversible cysteine oxidation (CysOX) are involved in the early proatherogenic aspect of atherosclerosis. Given that the corresponding redox signaling pathways are still unclear, a modified biotin switch assay was developed to quantify the reversible CysOX in an atherosclerosis model established by using a monocytic cell line treated with platelet releasate. The accumulation of ROS was observed in the model system and validated in human primary monocytes. Through the application of the modified biotin switch assay, we obtained the first reversible CysOX proteome for this model. A total of 75 peptides, corresponding to 53 proteins, were quantified with oxidative modification. The bioinformatics analysis of these CysOX-containing proteins highlighted biological processes including glycolysis, cytoskeleton arrangement, and redox regulation. Moreover, the reversible oxidation of three glycolysis enzymes was observed using this method, and the regulation influence was verified by an enzyme activity assay. NADPH oxidase (NOX) inhibition treatment, in conjunction with the modified biotin switch method, was used to evaluate the global CysOX status. In conclusion, this versatile modified biotin switch assay provides an approach for the quantification of all reversible CysOX and for the study of redox signaling in atherosclerosis as well as in diseases in other biological systems.

Development and application of a quantitative multiplexed small GTPase activity assay using targeted proteomics.

Small GTPases are a family of key signaling molecules that are ubiquitously expressed in various types of cells. Their activity is often analyzed by western blot, which is limited by its multiplexing capability, the quality of isoform-specific antibodies, and the accuracy of quantification. To overcome these issues, a quantitative multiplexed small GTPase activity assay has been developed. Using four different binding domains, this assay allows the binding of up to 12 active small GTPase isoforms simultaneously in a single experiment. To accurately quantify the closely related small GTPase isoforms, a targeted proteomic approach, i.e., selected/multiple reaction monitoring, was developed, and its functionality and reproducibility were validated. This assay was successfully applied to human platelets and revealed time-resolved coactivation of multiple small GTPase isoforms in response to agonists and differential activation of these isoforms in response to inhibitor treatment. This widely applicable approach can be used for signaling pathway studies and inhibitor screening in many cellular systems.

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.

Forced protein unfolding leads to highly elastic and tough protein hydrogels.

Protein-based hydrogels usually do not exhibit high stretchability or toughness, significantly limiting the scope of their potential biomedical applications. Here we report the engineering of a chemically cross-linked, highly elastic and tough protein hydrogel using a mechanically extremely labile, de novo-designed protein that assumes the classical ferredoxin-like fold structure. Due to the low mechanical stability of the ferredoxin-like fold structure, swelling of hydrogels causes a significant fraction of the folded domains to unfold. Subsequent collapse and aggregation of unfolded ferredoxin-like domains leads to intertwining of physically and chemically cross-linked networks, entailing hydrogels with unusual physical and mechanical properties: a negative swelling ratio, high stretchability and toughness. These hydrogels can withstand an average strain of 450% before breaking and show massive energy dissipation. Upon relaxation, refolding of the ferredoxin-like domains enables the hydrogel to recover its massive hysteresis. This novel biomaterial may expand the scope of hydrogel applications in tissue engineering.

Chemical proteomics and its impact on the drug discovery process.

Despite the rapid growth of postgenomic data and fast-paced technology advancement, drug discovery is still a lengthy and difficult process. More effective drug design requires a better understanding of the interaction between drug candidates and their targets/off-targets in various situations. The ability of chemical proteomics to integrate a multiplicity of disciplines enables the direct analysis of protein activities on a proteome-wide scale, which has enormous potential to facilitate drug target elucidation and lead drug verification. Over recent years, chemical proteomics has experienced rapid growth and provided a valuable method for drug target identification and inhibitor discovery. This review introduces basic concepts and technologies of different popular chemical proteomic approaches. It also covers the essential features and recent advances of each approach while underscoring their potentials in drug discovery and development.

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.

Liquid chromatography-atmospheric pressure electron capture dissociation mass spectrometry for the structural analysis of peptides and proteins.

Atmospheric pressure electron capture dissociation (AP-ECD) is an emerging technique with the potential to be a more accessible alternative to conventional ECD/electron transfer dissociation (ETD) methods because it can be implemented using a stand-alone ion source device suitable for use with any existing or future electrospray ionization mass spectrometer. With AP-ECD, no modification of the main instrument is required, so it may easily be retrofitted to instruments not originally equipped with ECD/ETD capabilities. Here, we present our first purpose-built AP-ECD source and demonstrate its use in conjunction with capillary LC for the analysis of substance P, a tryptic digest of bovine serum albumin, and a phosphopeptide mixture. Quality ECD spectra were obtained for all the samples at the low femtomole level, proving that LC-AP-ECD-MS is suitable for the structural analysis of peptides and protein digests, in this case using an unmodified quadrupole time-of-flight mass spectrometer built ca. 2002.

Deconstruction of the SS18-SSX fusion oncoprotein complex: insights into disease etiology and therapeutics.

Synovial sarcoma is a translocation-associated sarcoma where the underlying chromosomal event generates SS18-SSX fusion transcripts. In vitro and in vivo studies have shown that the SS18-SSX fusion oncoprotein is both necessary and sufficient to support tumorigenesis; however, its mechanism of action remains poorly defined. We have purified a core SS18-SSX complex and discovered that SS18-SSX serves as a bridge between activating transcription factor 2 (ATF2) and transducin-like enhancer of split 1 (TLE1), resulting in repression of ATF2 target genes. Disruption of these components by siRNA knockdown or treatment with HDAC inhibitors rescues target gene expression, leading to growth suppression and apoptosis. Together, these studies define a fundamental role for aberrant ATF2 transcriptional dysregulation in the etiology of synovial sarcoma.

A new ion source and procedures for atmospheric pressure-electron capture dissociation of peptides.

We introduce a new atmospheric pressure-electron capture dissociation (AP-ECD) source in which conventional nanospray emitters are coupled with the source block and photoionization lamp of a PhotoSpray APPI source. We also introduce procedures for data collection and processing, aimed at maximizing the signal-to-background ratio of ECD products. Representative data from Substance P are presented to demonstrate the performance of the technique. Further, we demonstrate the effects of two important experimental variables, source temperature and vacuum-interface declustering potential (DP), on the method. Last, we show that even when a high source temperature is used to maximize efficiency, AP-ECD fragments of a model phosphorylated peptide retain the modification.

A quick reality check for microRNA target prediction.

The regulation of protein abundance by microRNA (miRNA)-mediated repression of mRNA translation is a rapidly growing area of interest in biochemical research. In animal cells, the miRNA seed sequence does not perfectly match that of the mRNA it targets, resulting in a large number of possible miRNA targets and varied extents of repression. Several software tools are available for the prediction of miRNA targets, yet the overlap between them is limited. Jovanovic et al. have developed and applied a targeted, quantitative approach to validate predicted miRNA target proteins. Using a proteome database, they have set up and tested selected reaction monitoring assays for approximately 20% of more than 800 predicted let-7 targets, as well as control genes in Caenorhabditis elegans. Their results demonstrate that such assays can be developed quickly and with relative ease, and applied in a high-throughput setup to verify known and identify novel miRNA targets. They also show, however, that the choice of the biological system and material has a noticeable influence on the frequency, extent and direction of the observed changes. Nonetheless, selected reaction monitoring assays, such as those developed by Jovanovic et al., represent an attractive new tool in the study of miRNA function at the organism level.

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.

Accessibility governs the relative reactivity of basic residues in formaldehyde-induced protein modifications.

Cross-linking of proteins in a complex requires the chemical modification of the proteins in order to form a covalent link. This can be achieved in vivo using formaldehyde as it is small and rapidly permeates the cell membrane. Previous model studies of the speed and specificity of the first step of this reaction on peptides have suggested that residue accessibility and sequence micro-environment play a significant role in the production of the reactive intermediate necessary for cross-linking. This dependency was therefore further investigated on model proteins, which contain a more complex tertiary structure. Under mild reaction conditions, similar to those used for in vivo protein cross-linking, it was found that the vast majority of modification occurred on lysines, tertiary structure and solvent accessible surface area played a major role in regulating the extent of formaldehyde-induced modifications, and that the modifications on a folded protein did not significantly affect its tertiary structural stability.

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.

Applications of current proteomics techniques in modern drug design.

Proteins are currently the major drug targets and thus play a critical role in the process of modern drug design. This typically involves construction of drug compounds based on the structure of a drug target, validation for therapeutic efficacy of the drug compounds, evaluation of drug toxicity, and finally, clinical trial. Proteomics, defined as the comprehensive analysis of the proteins that are expressed in cells or tissues, can be employed at different stages of this process. Comparative proteomics can distinguish subtle changes in protein abundance at a depth of several thousand proteins at different conditions i.e. normal vs disease, to facilitate drug target identification. Also, chemical proteomics can be used to determine drug-target interactions and systematically analyze drug specificity and selectivity. Moreover, phosphoproteomics can be employed to monitor changes in phosphorylation events to characterize drug actions on cell signaling pathways. Similarly, functional proteomics can be utilized to investigate protein-protein and protein-ligand interactions for the clarification of the mechanism of drug action, identification of disease-related sub-networks and novel drug targets. Furthermore, quantitative proteomics can be used to characterize long-term drug effects on protein expression. In addition, computational approaches have emerged to convert complex proteomic data into sophisticated computer models of cellular protein networks. In this review, we will provide an overview of these state-of-the-art proteomics techniques, describe their underlying experimental concepts and compare them to each other, and discuss existing and future applications in the art of drug design and development.

A signaling pathway contributing to platelet storage lesion development: targeting PI3-kinase-dependent Rap1 activation slows storage-induced platelet deterioration.

BACKGROUND: The term platelet storage lesion (PSL) describes the structural and biochemical changes in platelets (PLTs) during storage. These are typified by alterations of morphologic features and PLT metabolism leading to reduced functionality and hence reduced viability for transfusion. While the manifestations of the storage lesion are well characterized, the biochemical pathways involved in the initiation of this process are unknown. STUDY DESIGN AND METHODS: A complementary proteomic approach has recently been applied to analyze changes in the PLT proteome during storage. By employing stringent proteomic criteria, 12 proteins were identified as significantly and consistently changing in relative concentration over a 7-day storage period. Microscopy, Western blot analysis, flow cytometry, and PLT functionality analyses were used to unravel the involvement of a subset of these 12 proteins, which are connected through integrin signaling in one potential signaling pathway underlying storage lesion development. RESULTS: Microscopic analysis revealed changes in localization of glycoprotein IIIa, Rap1, and talin during storage. Rap1 activation was observed to correlate with expression of the PLT activation marker CD62P. PLTs incubated for 7 days with the PI3-kinase inhibitor LY294002 showed diminished Rap1 activation as well as a moderate reduction in integrin alphaIIbbeta3 activation and release of alpha-granules. Furthermore, this inhibitor seemed to improve PLT integrity and quality during storage as several in vitro probes showed a deceleration of PLT activation. CONCLUSION: These results provide the first evidence for a signaling pathway mediating PSL in which PI3-kinase-dependent Rap1 activation leads to integrin alphaIIbbeta3 activation and PLT degranulation.