Precursor ion scanning for detection and structural characterization of heterogeneous glycopeptide mixtures

AbstractThe structure of N-linked glycans is determined by a complex, anabolic, intracellular pathway but the exact role of individual glycans is not always clear. Characterization of carbohydrates attached to glycoproteins is essential to aid understanding of this complex area of biology. Specific mass spectral detection of glycopeptides from protein digests may be achieved by on-line HPLC-MS, with selected ion monitoring (SIM) for diagnostic product ions generated by cone voltage fragmentation, or by precursor ion scanning for terminal saccharide product ions, which can yield the same information more rapidly. When glycosylation is heterogeneous, however, these approaches can result in spectra that are complex and poorly resolved. We have developed methodology, based around precursor ion scanning for ions of high m/z, that allows site specific detection and structural characterization of glycans at high sensitivity and resolution. These methods have been developed using the standard glycoprotein, fetuin, and subsequently applied to the analysis of the N-linked glycans attached to the scrapie-associated prion protein, PrPSc. These glycans are highly heterogeneous and over 30 structures have been identified and characterized site specifically. Product ion spectra have been obtained on many glycopeptides confirming structure assignments. The glycans are highly fucosylated and carry Lewis X or sialyl Lewis X epitopes and the structures are in-line with previous results. [Abbreviations: Hex–Hexose, C6H12O6 carbohydrates, including mannnose and galactose; HexNAc—N-acetylhexosamine, C8H15NO6 carbohydrates, including N-acetylglucosamine and N-acetylgalactosamine; GlcNAc—N-acetylglucosamine; GalNAc—N-acetylgalactosamine; Fuc–Fucose; NeuAC—N-acetylneuraminic acid or sialic acid; TSE—Transmissible Spongiform Encephalopathy.

Elucidation of the anaerobic pathway for the corrin component of cobalamin (vitamin B12)

It has been known for the past 20 years that two pathways exist in nature for the de novo biosynthesis of the coenzyme form of vitamin B12, adenosylcobalamin, representing aerobic and anaerobic routes. In contrast to the aerobic pathway, the anaerobic route has remained enigmatic because many of its intermediates have proven technically challenging to isolate, because of their inherent instability. However, by studying the anaerobic cobalamin biosynthetic pathway in Bacillus megaterium and using homologously overproduced enzymes, it has been possible to isolate all of the intermediates between uroporphyrinogen III and cobyrinic acid. Consequently, it has been possible to detail the activities of purified cobinamide biosynthesis (Cbi) proteins CbiF, CbiG, CbiD, CbiJ, CbiET, and CbiC, as well as show the direct in vitro conversion of 5-aminolevulinic acid into cobyrinic acid using a mixture of 14 purified enzymes. This approach has resulted in the isolation of the long sought intermediates, cobalt-precorrin-6A and -6B and cobalt-precorrin-8. EPR, in particular, has proven an effective technique in following these transformations with the cobalt(II) paramagnetic electron in the dyz orbital, rather than the typical dz2. This result has allowed us to speculate that the metal ion plays an unexpected role in assisting the interconversion of pathway intermediates. By determining a function for all of the pathway enzymes, we complete the tool set for cobalamin biosynthesis and pave the way for not only enhancing cobalamin production, but also design of cobalamin derivatives through their combinatorial use and modification

High-Resolution Mass Spectrometry Based Proteomic Analysis of the Response to Vancomycin-Induced Cell Wall Stress in Streptomyces coelicolor A3(2).

Understanding how bacteria survive periods of cell wall stress is of fundamental interest and can help generate ideas for improved antibacterial treatments. In this study we use tandem mass tagging to characterize the proteomic response of vancomycin resistant Streptomyces coelicolor to the exposure to sublethal levels of the antibiotic. A common set of 804 proteins were identified in triplicate experiments. Contrasting changes in the abundance of proteins closely associated with the cytoplasmic membrane with those taking place in the cytosol identified aspects of protein spatial localization that are associated with the response to vancomycin. Enzymes for peptidoglycan precursor, mycothiol, ectoine and menaquinone biosynthesis together with a multisubunit nitrate reductase were recruited to the membrane following vancomycin treatment. Many proteins with regulatory functions (including sensor protein kinases) also exhibited significant changes in abundance exclusively in the membrane-associated protein fraction. Several enzymes predicted to be involved in extracellular peptidoglycan crossbridge formation became significantly depleted from the membrane. A comparison with data previously acquired on the changes in gene transcription following vancomycin treatment identified a common high-confidence set of changes in gene expression. Generalized changes in protein abundance indicate roles for proteolysis, the pentose phosphate pathway and a reorganization of amino acid biosynthesis in the stress response.HJH was supported by the Royal Society (516002.K5877/ROG) and by grant number GO700141 from the Medical Research Council.This is the final version of the article. It first appeared from ACS Publications via http://dx.doi.org/10.1021/acs.jproteome.5b0024

How to minimize repeat dental general anaesthetics

This article aims to provide general dental practitioners (GDPs) with the knowledge to improve their referrals primarily for children who they feel require a dental general anaesthetic. It discusses the impact of a general anaesthetic (GA) on a child and the financial impacts of dental general anaesthetics (DGAs). The risks of DGAs are well recognized and the ways in which the dental team in primary, secondary care and service commissioners can reduce the risk of repeat DGAs are discussed

Teaching about obesity: Caring, compassion, communication and courage in midwifery education

Teaching innovation can be used to promote the 6Cs in one of the most purportedly stigmatising areas of maternity care: obesity. As rates of maternal obesity continue to rise, getting this area of care right becomes more urgent. Although a great deal has been published about the unsatisfactory and stigmatising impact that staff attitudes can have on those with obesity, there is relatively little on how this problematic area of care should be taught to the next generation of midwives. This article presents a case study of learning about obesity management at the pre-registration, undergraduate level. The case study described is an effort to move away from what is currently largely an academic debate, towards a set of tangible practice recommendations

ATP-dependent substrate transport by the ABC transporter MsbA is proton-coupled.

ATP-binding cassette transporters mediate the transbilayer movement of a vast number of substrates in or out of cells in organisms ranging from bacteria to humans. Current alternating access models for ABC exporters including the multidrug and Lipid A transporter MsbA from Escherichia coli suggest a role for nucleotide as the fundamental source of free energy. These models involve cycling between conformations with inward- and outward-facing substrate-binding sites in response to engagement and hydrolysis of ATP at the nucleotide-binding domains. Here we report that MsbA also utilizes another major energy currency in the cell by coupling substrate transport to a transmembrane electrochemical proton gradient. The dependence of ATP-dependent transport on proton coupling, and the stimulation of MsbA-ATPase by the chemical proton gradient highlight the functional integration of both forms of metabolic energy. These findings introduce ion coupling as a new parameter in the mechanism of this homodimeric ABC transporter.Himansha Singh is supported by the Cambridge Commonwealth, European and International Trust. Saroj Velamakanni was a recipient of a Cambridge Nehru Scholarship. Shen L. Wei was funded by the Cambridge Overseas Trust. This research in the Van Veen group was supported by Biotechnology and Biological Sciences Research Council (BBSRC) grant BB/I002383/1 and BB/C004663/1, Medical Research Council (MRC) grant G0401165 and by further support from the Human Frontier Science Program (HFSP) and the British Society for Antimicrobial Chemotherapy (BSAC).This is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/ncomms1238

Professionalism and person-centredness: developing a practice based approach to leadership within NHS maternity services in the UK

This paper, based on data taken from in-depth interviews with senior midwives and obstetricians and conducted as part of a critical ethnographic study, argues for a greater appreciation of person-centred, value-led midwifery practice. The paper begins with a discussion of the way midwifery practice is shaped by encoded and embodied knowledge. The paper subsequently focuses on an emergent practice based leadership using an adapted Aristotelian conceptual framework derived from MacIntyre (2007). Professional dissonance is highlighted as a difficulty experienced by repositioned managers who are also expected to be leaders in their field. Using data gathered from in-depth interviews it is contended that establishing person-centred care might be better achieved through the development of practice based leadership (rather than solely by adherence to organisational requirements). This type of leadership could potentially nurture a professional environment that promotes qualities, such as agency, commitment and high levels of competence among midwives. Such leadership is central to UK government priorities and is applicable to a global practice development agenda