Application of label-free mass spectrometry-based proteomics to biomarker discovery

Mass spectrometry is an analytical technique which is used extensively in the fields of chemistry and physics. Developments in the field over the last two decades have permitted the analysis of a wide variety of biological molecules from a range of sources. The term proteomics relates to the study of the protein complement of a cell or organism with particular interest in the identification and quantification of these analytes. A biomarker is a characteristic that can be measured and evaluated to give an indication of normal, biological processes, or pharmacological responses to a therapeutic intervention. Bodily fluids are a rich source of potential biomarkers as they can be obtained in reasonable quantity and their extraction is generally minimally invasive. The plasma proteome, which contains many thousands of analytes spanning a dynamic range greater than 10 orders of magnitude, reflects the status of the many tissues and organs in the body serving as an ideal medium for potential biomarker discovery. The analytical challenges posed by the plasma proteome are significant. Depletion of the highly abundant proteins is usually a prerequisite of any biomarker study and no technique has the dynamic range to study all of the proteins present. Comprehensive characterisation of the plasma proteome requires significant experimental effort and cost. Use of pooled samples in biomarker studies is widespread and the majority of biomarkers, which have been identified in the discovery phase, have not passed clinical validation. A data independent, label-free quantitative approach has been evaluated for the study of depleted maternal plasma proteomes taken in the first trimester. Plasma was characterised from individual and groups of patients from three obstetric conditions using single and multi-dimensional chromatography. Potential biomarkers from each source were identified and evaluated. Multi-dimensional chromatography was used to simplify the complexity of the analytes introduced to the mass spectrometer and the benefits and limitations of the approach in terms of biomarker discovery have been demonstrated

Standardised method for reporting exercise programmes : protocol for a modified Delphi study

Introduction Exercise is integral to health across the lifespan and important for people with chronic health conditions. A systematic review of exercise trials for chronic conditions reported suboptimal descriptions of the evaluated interventions and concluded that this hinders interpretation and replication. The aim of this project is to develop a standardised method for reporting essential exercise programme details being evaluated in clinical trials. Methods and analysis A modified Delphi technique will be used to gain consensus among international exercise experts. We will use three sequential rounds of anonymous online questionnaires to refine a standardised checklist. A draft checklist of potentially relevant items was developed based on the results of a systematic review of exercise systematic reviews. An international panel of experts was identified by exercise systematic review authorship, established international profile in exercise research and practice and by peer referral. In round 1, the international panel of experts will be asked to rate the importance of each draft item and provide additional suggestions for revisions or new items. Consensus will be considered reached if at least 70% of the panel strongly agree/disagree that an item should be included or excluded. Where agreement is not reached or there are suggestions for altered or new items, these will be taken to round 2 together with an aggregated summary of round 1 responses. Following the second round, a ranking of item importance will be made to rationalise the number of items. The final template will be distributed to panel members for approval. Ethics and dissemination Ethics approval was received from The Cabrini Institute Ethics Committee, Melbourne, Australia (HREC 02-07-04-14). We plan to use a stepwise process to develop and refine a standardised and internationally agreed template for explicit reporting of exercise programmes. The template will be generalisable across all types of exercise interventions. The findings will be disseminated through peer-reviewed publications and conference presentations

Exercise and progressive supranuclear palsy : the need for explicit exercise reporting

Background Progressive Supranuclear Palsy (PSP) is the most frequent form of atypical Parkinsonism. Although there is preliminary evidence for the benefits of gait rehabilitation, balance training and oculomotor exercises in PSP, the quality of reporting of exercise therapies appears mixed. The current investigation aims to evaluate the comprehensiveness of reporting of exercise and physical activity interventions in the PSP literature. Methods Two independent reviewers used the Consensus on Exercise Reporting Template (CERT) to extract all exercise intervention data from 11 studies included in a systematic review. CERT items covered: ‘what’ (materials), ‘who’ (instructor qualifications), ‘how’ (delivery), ‘where’ (location), ‘when’, ‘how much’ (dosage), ‘tailoring’ (what, how), and ‘how well’ (fidelity) exercise delivery complied with the protocol. Each exercise item was scored ‘1’ (adequately reported) or ‘0’ (not adequately reported or unclear). The CERT score was calculated, as well as the percentage of studies that reported each CERT item. Results The CERT scores ranged from 3 to 12 out of 19. No PSP studies adequately described exercise elements that would allow exact replication of the interventions. Well-described items included exercise equipment, exercise settings, exercise therapy scheduling, frequency and duration. Poorly described items included decision rules for exercise progression, instructor qualifications, exercise adherence, motivation strategies, safety and adverse events associated with exercise therapies. Discussion The results revealed variability in the reporting of physical therapies for people living with PSP. Future exercise trials need to more comprehensively describe equipment, instructor qualifications, exercise and physical activity type, dosage, setting, individual tailoring of exercises, supervision, adherence, motivation strategies, progression decisions, safety and adverse events. Conclusion Although beneficial for people living with PSP, exercise and physical therapy interventions have been inadequately reported. It is recommended that evidence-based reporting templates be utilised to comprehensively document therapeutic exercise design, delivery and evaluation

Site specific genetic incorporation of azidophenylalanine in Schizosaccharomyces pombe

The diversity of protein functions is impacted in significant part by the chemical properties of the twenty amino acids, which are used as building blocks for nearly all proteins. The ability to incorporate unnatural amino acids (UAA) into proteins in a site specific manner can vastly expand the repertoire of protein functions and also allows detailed analysis of protein function. In recent years UAAs have been incorporated in a site-specific manner into proteins in a number of organisms. In nearly all cases, the amber codon is used as a sense codon, and an orthogonal tRNA/aminoacyl-tRNA synthetase (RS) pair is used to generate amber suppressing tRNAs charged with the UAA. In this work, we have developed tools to incorporate the cross-linking amino acid azido-phenylalanine (AzF) through the use of bacterial tRNATyr and a modified version of TyrRS, AzFRS, in Schizosaccharomyces pombe, which is an attractive model organism for the study of cell behavior and function. We have incorporated AzF into three different proteins. We show that the majority of AzF is modified to amino-phenyl alanine, but protein cross-linking was still observed. These studies set the stage for exploitation of this new technology for the analysis of S. pombe proteins

A combined approach for comparative exoproteome analysis of Corynebacterium pseudotuberculosis

Background: Bacterial exported proteins represent key components of the host-pathogen interplay. Hence, we sought to implement a combined approach for characterizing the entire exoproteome of the pathogenic bacterium Corynebacterium pseudotuberculosis, the etiological agent of caseous lymphadenitis (CLA) in sheep and goats. Results: An optimized protocol of three-phase partitioning (TPP) was used to obtain the C. pseudotuberculosis exoproteins, and a newly introduced method of data-independent MS acquisition (LC-MSE) was employed for protein identification and label-free quantification. Additionally, the recently developed tool SurfG+ was used for in silico prediction of sub-cellular localization of the identified proteins. In total, 93 different extracellular proteins of C. pseudotuberculosis were identified with high confidence by this strategy; 44 proteins were commonly identified in two different strains, isolated from distinct hosts, then composing a core C. pseudotuberculosis exoproteome. Analysis with the SurfG+ tool showed that more than 75% (70/93) of the identified proteins could be predicted as containing signals for active exportation. Moreover, evidence could be found for probable non-classical export of most of the remaining proteins. Conclusions: Comparative analyses of the exoproteomes of two C. pseudotuberculosis strains, in addition to comparison with other experimentally determined corynebacterial exoproteomes, were helpful to gain novel insights into the contribution of the exported proteins in the virulence of this bacterium. The results presented here compose the most comprehensive coverage of the exoproteome of a corynebacterial species so far

Identificaction and quantificationof proteins from Methylophaga Thiooxidans and Methylocella Silvestris using label-free LC/MS

Comunicaciones a congreso

Post-traumatic stress disorder following childbirth: an update of current issues and recommendations for future research

Objective: This paper aimed to report the current status of research in the field of post-traumatic stress disorder following childbirth (PTSD FC), and to update the findings of an earlier 2008 paper. Background: A group of international researchers, clinicians and service users met in 2006 to establish the state of clinical and academic knowledge relating to PTSD FC. A paper identified four key areas of research knowledge at that time. Methods: Fourteen clinicians and researchers met in Oxford, UK to update the previously published paper relating to PTSD FC. The first part of the meeting focused on updating the four key areas identified previously, and the second part on discussing new and emerging areas of research within the field. Results: A number of advances have been made in research within the area of PTSD FC. Prevalence is well established within mothers, several intervention studies have been published, and there is growing interest in new areas: staff and pathways; prevention and early intervention; impact on families and children; special populations; and post-traumatic growth. Conclusion: Despite progress, significant gaps remain within the PTSD FC knowledge base. Further research continues to be needed across all areas identified in 2006, and five areas were identified which can be seen as ‘new and emerging’. All of these new areas require further extensive research. Relatively little is still known about PTSD FC

Ancient role of vasopressin/oxytocin-type neuropeptides as regulators of feeding revealed in an echinoderm.

BACKGROUND: Vasopressin/oxytocin (VP/OT)-type neuropeptides are well known for their roles as regulators of diuresis, reproductive physiology and social behaviour. However, our knowledge of their functions is largely based on findings from studies on vertebrates and selected protostomian invertebrates. Little is known about the roles of VP/OT-type neuropeptides in deuterostomian invertebrates, which are more closely related to vertebrates than protostomes. RESULTS: Here, we have identified and functionally characterised a VP/OT-type signalling system comprising the neuropeptide asterotocin and its cognate G-protein coupled receptor in the starfish (sea star) Asterias rubens, a deuterostomian invertebrate belonging to the phylum Echinodermata. Analysis of the distribution of asterotocin and the asterotocin receptor in A. rubens using mRNA in situ hybridisation and immunohistochemistry revealed expression in the central nervous system (radial nerve cords and circumoral nerve ring), the digestive system (including the cardiac stomach) and the body wall and associated appendages. Informed by the anatomy of asterotocin signalling, in vitro pharmacological experiments revealed that asterotocin acts as a muscle relaxant in starfish, contrasting with the myotropic actions of VP/OT-type neuropeptides in vertebrates. Furthermore, in vivo injection of asterotocin had a striking effect on starfish behaviour-triggering fictive feeding where eversion of the cardiac stomach and changes in body posture resemble the unusual extra-oral feeding behaviour of starfish. CONCLUSIONS: We provide a comprehensive characterisation of VP/OT-type signalling in an echinoderm, including a detailed anatomical analysis of the expression of both the VP/OT-type neuropeptide asterotocin and its cognate receptor. Our discovery that asterotocin triggers fictive feeding in starfish provides important new evidence of an evolutionarily ancient role of VP/OT-type neuropeptides as regulators of feeding in animals

Substrate and Stereochemical Control of Peptidoglycan Cross-Linking by Transpeptidation by Escherichia coli PBP1B

Penicillin binding proteins (PBPs) catalyzing transpeptidation reactions that stabilize the peptidoglycan component of the bacterial cell wall are the targets of β-lactams, the most clinically successful antibiotics to date. However, PBP-transpeptidation enzymology has evaded detailed analysis, because of the historical unavailability of kinetically competent assays with physiologically relevant substrates and the previously unappreciated contribution of protein cofactors to PBP activity. By re-engineering peptidoglycan synthesis, we have constructed a continuous spectrophotometric assay for transpeptidation of native or near native peptidoglycan precursors and fragments by Escherichia coli PBP1B, allowing us to (a) identify recognition elements of transpeptidase substrates, (b) reveal a novel mechanism of stereochemical editing within peptidoglycan transpeptidation, (c) assess the impact of peptidoglycan substrates on β-lactam targeting of transpeptidation, and (d) demonstrate that both substrates have to be bound before transpeptidation occurs. The results allow characterization of high molecular weight PBPs as enzymes and not merely the targets of β-lactam acylation