Characteristics of blood plasma proteome changes associated with the hemorrhagic purpura of cosmonauts on the first day after long-term space missions.

The interest in the role of the gravitational factor during landing after long-term space flights (SF) leads to the search for various innovative approaches to assessing the compliance of external changes observed by clinicians. The results of special research methods such as Omics technologies that may reflect physiological responses to the conditions created during landing are of great interest. Our purpose is to compare the blood plasma proteome changes associated with the trauma and endothelial dysfunction processes prior to launch and on the day of landing, as well as the groups of cosmonauts with and without the secondary hemorrhagic purpura. In our study, the concentrations of 125 plasma proteins in 18 Russian cosmonauts, measured using targeted proteomic analysis based on liquid chromatography and tandem mass spectrometry were analyzed. The results reveal the trends of 12 proteins participating in the processes that trigger hemorrhagic purpura under the effect of re-entry g-forces. Exposure to intense g-forces and return to the gravity are the key factors for external manifestations of changes in the body systems induced by a long-term stay in space microgravity. Our results may be useful for further research to experts in gravitational physiology, aviation and space medicine.

How iMALDI can improve clinical diagnostics.

Protein mass spectrometry (MS) is an indispensable tool to detect molecular signatures that can be associated with cellular dysregulation and disease. Despite its huge success in the life sciences, where it has led to novel insights into disease mechanisms and the identification of potential protein biomarkers, protein MS is rarely used for clinical protein assays. While conventional matrix-assisted laser desorption/ionization (MALDI) MS is not compatible with complex samples, liquid chromatography-MS (LC-MS)-based assays may be too complex and may lack the robustness and ease of automation required for routine use in the clinic. Therefore, clinical protein assays are dominated by immunohistochemistry and immunoassays which, however, often lack standardization and fully depend on antibody specificity. Immuno-MALDI (iMALDI) MS may overcome these hurdles by utilizing anti-peptide antibodies for the specific enrichment of targeted analytes and on-target detection of the captured analytes, thus combining the unique properties of MS for the unambiguous detection and quantitation of analytes with a workflow that can be fully automated. Here we discuss the requirements for clinical protein assays, the pitfalls of existing methods, how iMALDI has been successfully used to quantify endogenous peptides and proteins from clinical samples, as well as its potential as a powerful tool for companion diagnostics in the light of precision medicine.

Differential Proteomics for Distinguishing Ischemic Stroke from Controls: a Pilot Study of the SpecTRA Project.

A diagnostic blood test for stroke is desirable but will likely require multiple proteins rather than a single "troponin." Validating large protein panels requires large patient numbers. Mass spectrometry (MS) is a cost-effective tool for this task. We compared differences in the abundance of 147 protein markers to distinguish 20 acute cerebrovascular syndrome (ACVS) patients who presented to the Emergency Department of one urban hospital within < 24 h from onset) and from 20 control patients who were enrolled via an outpatient neurology clinic. We targeted proteins from the stroke literature plus cardiovascular markers previously studied in our lab. One hundred forty-one proteins were quantified using MS, 8 were quantified using antibody protein enrichment with MS, and 32 were measured using ELISA, with some proteins measured by multiple techniques. Thirty proteins (4 by ELISA and 26 by the MS techniques) were differentially abundant between mimic and stroke after adjusting for age in robust regression analyses (FDR < 0.20). A logistic regression model using the first two principal components of the proteins significantly improved discrimination between strokes and controls compared to a model based on age alone (p < 0.001, cross-validated AUC 0.93 vs. 0.78). Significant proteins included markers of inflammation (47%), coagulation (40%), atrial fibrillation (7%), neurovascular unit injury (3%), and other (3%). These results suggest the potential value of plasma proteins as biomarkers for ACVS diagnosis and the role of plasma-based MS in this area.

Current trends in quantitative proteomics - an update.

Proteins can provide insights into biological processes at the functional level, so they are very promising biomarker candidates. The quantification of proteins in biological samples has been routinely used for the diagnosis of diseases and monitoring the treatment. Although large-scale protein quantification in complex samples is still a challenging task, a great amount of effort has been made to advance the technologies that enable quantitative proteomics. Seven years ago, in 2009, we wrote an article about the current trends in quantitative proteomics. In writing this current paper, we realized that, today, we have an even wider selection of potential tools for quantitative proteomics. These tools include new derivatization reagents, novel sampling formats, new types of analyzers and scanning techniques, and recently developed software to assist in assay development and data analysis. In this review article, we will discuss these innovative methods, and their current and potential applications in proteomics. Copyright © 2017 John Wiley & Sons, Ltd.

Architecture of the RNA polymerase II-Mediator core initiation complex.

The conserved co-activator complex Mediator enables regulated transcription initiation by RNA polymerase (Pol) II. Here we reconstitute an active 15-subunit core Mediator (cMed) comprising all essential Mediator subunits from Saccharomyces cerevisiae. The cryo-electron microscopic structure of cMed bound to a core initiation complex was determined at 9.7 Å resolution. cMed binds Pol II around the Rpb4-Rpb7 stalk near the carboxy-terminal domain (CTD). The Mediator head module binds the Pol II dock and the TFIIB ribbon and stabilizes the initiation complex. The Mediator middle module extends to the Pol II foot with a 'plank' that may influence polymerase conformation. The Mediator subunit Med14 forms a 'beam' between the head and middle modules and connects to the tail module that is predicted to bind transcription activators located on upstream DNA. The Mediator 'arm' and 'hook' domains contribute to a 'cradle' that may position the CTD and TFIIH kinase to stimulate Pol II phosphorylation.

The Human Proteome Organization Chromosome 6 Consortium: integrating chromosome-centric and biology/disease driven strategies.

The Human Proteome Project (HPP) is designed to generate a comprehensive map of the protein-based molecular architecture of the human body, to provide a resource to help elucidate biological and molecular function, and to advance diagnosis and treatment of diseases. Within this framework, the chromosome-based HPP (C-HPP) has allocated responsibility for mapping individual chromosomes by country or region, while the biology/disease HPP (B/D-HPP) coordinates these teams in cross-functional disease-based groups. Chromosome 6 (Ch6) provides an excellent model for integration of these two tasks. This metacentric chromosome has a complement of 1002-1034 genes that code for known, novel or putative proteins. Ch6 is functionally associated with more than 120 major human diseases, many with high population prevalence, devastating clinical impact and profound societal consequences. The unique combination of genomic, proteomic, metabolomic, phenomic and health services data being drawn together within the Ch6 program has enormous potential to advance personalized medicine by promoting robust biomarkers, subunit vaccines and new drug targets. The strong liaison between the clinical and laboratory teams, and the structured framework for technology transfer and health policy decisions within Canada will increase the speed and efficacy of this transition, and the value of this translational research. BIOLOGICAL SIGNIFICANCE: Canada has been selected to play a leading role in the international Human Proteome Project, the global counterpart of the Human Genome Project designed to understand the structure and function of the human proteome in health and disease. Canada will lead an international team focusing on chromosome 6, which is functionally associated with more than 120 major human diseases, including immune and inflammatory disorders affecting the brain, skeletal system, heart and blood vessels, lungs, kidney, liver, gastrointestinal tract and endocrine system. Many of these chronic and persistent diseases have a high population prevalence, devastating clinical impact and profound societal consequences. As a result, they impose a multi-billion dollar economic burden on Canada and on all advanced societies through direct costs of patient care, the loss of health and productivity, and extensive caregiver burden. There is no definitive treatment at the present time for any of these disorders. The manuscript outlines the research which will involve a systematic assessment of all chromosome 6 genes, development of a knowledge base, and development of assays and reagents for all chromosome 6 proteins. We feel that the informatic infrastructure and MRM assays developed will place the chromosome 6 consortium in an excellent position to be a leading player in this major international research initiative. This article is part of a Special Issue: Can Proteomics Fill the Gap Between Genomics and Phenotypes?

Chemical fingerprinting of naphthenic acids and oil sands process waters-A review of analytical methods for environmental samples.

This article provides a review of the routine methods currently utilized for total naphthenic acid analyses. There is a growing need to develop chemical methods that can selectively distinguish compounds found within industrially derived oil sands process affected waters (OSPW) from those derived from the natural weathering of oil sands deposits. Attention is thus given to the characterization of other OSPW components such as oil sands polar organic compounds, PAHs, and heavy metals along with characterization of chemical additives such as polyacrylamide polymers and trace levels of boron species. Environmental samples discussed cover the following matrices: OSPW containments, on-lease interceptor well systems, on- and off-lease groundwater, and river and lake surface waters. There are diverse ranges of methods available for analyses of total naphthenic acids. However, there is a need for inter-laboratory studies to compare their accuracy and precision for routine analyses. Recent advances in high- and medium-resolution mass spectrometry, concomitant with comprehensive mass spectrometry techniques following multi-dimensional chromatography or ion-mobility separations, have allowed for the speciation of monocarboxylic naphthenic acids along with a wide range of other species including humics. The distributions of oil sands polar organic compounds, particularly the sulphur containing species (i.e., OxS and OxS2) may allow for distinguishing sources of OSPW. The ratios of oxygen- (i.e., Ox) and nitrogen-containing species (i.e., NOx, and N2Ox) are useful for differentiating organic components derived from OSPW from natural components found within receiving waters. Synchronous fluorescence spectroscopy also provides a powerful screening technique capable of quickly detecting the presence of aromatic organic acids contained within oil sands naphthenic acid mixtures. Synchronous fluorescence spectroscopy provides diagnostic profiles for OSPW and potentially impacted groundwater that can be compared against reference groundwater and surface water samples. Novel applications of X-ray absorption near edge spectroscopy (XANES) are emerging for speciation of sulphur-containing species (both organic and inorganic components) as well as industrially derived boron-containing species. There is strong potential for an environmental forensics application of XANES for chemical fingerprinting of weathered sulphur-containing species and industrial additives in OSPW.

Structure and control of a cell-cell adhesion complex associated with spermiation in rat seminiferous epithelium.

Spermiation, the release of late spermatids from the Sertoli cell, is disrupted by a number of toxicants. Control of the spermiation process, and the proteins that interact to adhere mature spermatids to Sertoli cells, is poorly understood. In these studies we used immunohistochemistry, coimmunoprecipitation/Western blotting, and mass spectrometry to refine an earlier model of sperm adhesion proposed by our laboratory. We have identified specific proteins linked together as part of a multiprotein complex, as well as several additional proteins (cortactin, ERK1/2, and 14-3-3 zeta) that may be functioning in both structural and signal transduction roles. The current and prior data suggest that protein phosphorylation is central to the control of spermiation. We also present and characterize an in vitro tubule culture system that allowed functional testing of the spermiation model by pharmacologic manipulation, and yielded data consistent with the importance of protein phosphorylation in spermiation.

Site-specific phosphorylation of human p53 protein determined by mass spectrometry.

Human recombinant p53 (r-p53) protein was studied by mass spectrometry (MS) to determine site-specific posttranslational differences between basal and hyperphosphorylated r-p53. Wild-type p53 was basally expressed after baculovirus infection while a parallel preparation was treated with the phosphatase inhibitor okadaic acid during the terminal stages of expression to create a hyperphosphorylated form of p53 known for its higher DNA binding and transcriptional activation. After immunoaffinity and HPLC purification, MALDI/MS measured a higher molecular mass for r-p53 from okadaic acid treatment relative to control, suggesting a higher phosphorylation state. This was supported by an acidic shift of r-p53 isoforms separated by gel isoelectric focusing. Employing a variety of mass spectrometric analyses combined with separation and affinity techniques, six specific phosphorylation sites of p53 were identified. The MS data indicated that hyperphosphorylated p53 showed a higher degree of phosphorylation than basal p53 at specific amino- and carboxy-terminal sites. In particular, ESI-MS demonstrated that Ser(315) was entirely phosphorylated after okadaic acid treatment, as confirmed biochemically by CDK2 kinase assay and by isoelectric focusing. In summary, MS analysis uniquely revealed increased, site-specific phosphorylations on p53 after phosphatase inhibition, particularly at Ser(315), which may be critical molecular events in defining p53 activity.

The dimerization motif of cytosolic sulfotransferases.

Cytosolic sulfotransferases sulfate steroids such as estrogens and hydroxysteroids. The enzymes, including human estrogen sulfotransferase (hEST) and hydroxysteroid sulfotransferase (hHST), are generally homodimers in solution with mouse estrogen sulfotransferase (mEST) being one of few exceptions. To identify the amino acid residues responsible for the dimerization, eight residues on the surface of hEST were mutated to their counterparts in mEST and mutated hESTs were then analyzed by gel filtration chromatography. A single mutation of Val(269) to Glu was sufficient to convert hEST to a monomer and the corresponding mutation of Val(260) also altered hHST to a monomer. The hHST crystal structure revealed a short stretch of peptide with the side-chains from two hHST monomers forming a hydrophobic zipper-like structure enforced by ion pairs at both ends. This peptide consisted of 10 residues near the C-terminus that, including the critical Val residue, is conserved as KXXXTVXXXE in nearly all cytosolic sulfotransferases. When mEST underwent the double mutations Pro269Thr/Glu270Val dimerization resulted. Thus, the KXXXTVXXXE sequence appears to be the common protein-protein interaction motif that mediates the homo- as well as heterodimerization of cytosolic sulfotransferases.