A Health Check of Avondale\u27s Distance Education Program: Where Have we Been? Where are we Going Next?

Avondale College of Higher Education has been offering tertiary courses for over 120 years. In the past two decades, this institution has extended its programs to include distance courses for students who opt to study online or are not able to attend on-campus courses at Avondale’s Lake Macquarie and Sydney campuses. While all of the institutions courses are evaluated on a regular basis, no formal evaluation had ever been undertaken of the distance education program as a whole. During 2017, a mixed methods research project was conducted to gather evaluative data from recent and current distance students using questionnaires and focus groups. The results of the study provide insight into the extent to which the distance education program at the College provides a space in which learning relationships can develop in online communities. Also, suggestions for future improvement and further research recommendations are provided. Findings of this study may be of interest to educators and administrators who incorporate online components in their curricula

Isotope Depletion Mass Spectrometry (ID-MS) for Accurate Mass Determination and Improved Top-Down Sequence Coverage of Intact Proteins

Top-down mass spectrometry (MS) is an increasingly important technique for protein characterization. However, in many biological MS experiments, the practicality of applying top-down methodologies is still limited at higher molecular mass. In large part, this is due to the detrimental effect resulting from the partitioning of the mass spectral signal into an increasing number of isotopic peaks as molecular mass increases. Reducing the isotopologue distribution of proteins via depletion of heavy stable isotopes was first reported over 20 years ago (Marshall, A. G.; Senko, M. W.; Li, W.; Li, M.; Dillon, S., Guan, S.; Logan, T. M.. Protein Molecular Mass to 1 Da by 13C, 15N Double-Depletion and FT-ICR Mass Spectrometry. J. Am. Chem. Soc. 1997, 119, 433−434.) and has been demonstrated for several small proteins. Here we extend this approach, introducing a new highly efficient method for the production of recombinant proteins depleted in 13C and 15N and demonstrating its advantages for top-down analysis of larger proteins (up to ∼50 kDa). FT-ICR MS of isotopically depleted proteins reveals dramatically reduced isotope distributions with monoisotopic signal observed up to 50 kDa. In top-down fragmentation experiments, the reduced spectral complexity alleviates fragment-ion signal overlap, the presence of monoisotopic signals allows assignment with higher mass accuracy, and the dramatic increase in signal-to-noise ratio (up to 7-fold) permits vastly reduced acquisition times. These compounding benefits allow the assignment of ∼3-fold more fragment ions than comparable analyses of proteins with natural isotopic abundances. Finally, we demonstrate greatly increased sequence coverage in time-limited top-down experiments—highlighting advantages for top-down LC–MS/MS workflows and top-down proteomics

Determining the location of the α-synuclein dimer Interface using native top-down fragmentation and isotope depletion-mass spectrometry

α-Synuclein (αSyn), a 140-residue intrinsically disordered protein, comprises the primary proteinaceous component of pathology-associated Lewy body inclusions in Parkinson’s disease (PD). Due to its association with PD, αSyn is studied extensively; however, the endogenous structure and physiological roles of this protein are yet to be fully understood. Here, ion mobility-mass spectrometry and native top-down electron capture dissociation fragmentation have been used to elucidate the structural properties associated with a stable, naturally occurring dimeric species of αSyn. This stable dimer appears in both wild-type (WT) αSyn and the PD-associated variant A53E. Furthermore, we integrated a novel method for generating isotopically depleted protein into our native top-down workflow. Isotope depletion increases signal-to-noise ratio and reduces the spectral complexity of fragmentation data, enabling the monoisotopic peak of low abundant fragment ions to be observed. This enables the accurate and confident assignment of fragments unique to the αSyn dimer to be assigned and structural information about this species to be inferred. Using this approach, we were able to identify fragments unique to the dimer, which demonstrates a C-terminal to C-terminal interaction between the monomer subunits. The approach in this study holds promise for further investigation into the structural properties of endogenous multimeric species of αSyn

Determination of protein thiol reduction potential by isotope labeling and intact mass measurement

Oxidation/reduction of thiol residues in proteins is an important type of post-translational modification that is implicated in regulating a range of biological processes. The nature of the modification makes it possible to define a quantifiable electrochemical potential, E⊕, for oxidation/reduction that allows cysteine-containing proteins to be ranked based on their propensity to be oxidized. Measuring oxidation of cysteine residues in proteins is difficult using standard electrochemical methods but recently top-down mass-spectrometry has been shown to enable the quantification of E⊕ for thiol oxidations. In this paper we demonstrate that mass spectrometry of intact proteins can be used in combination with an isotopic labeling strategy and an automated data analysis algorithm to measure E⊕ for the thiols in both E Coli Thioredoxin 1 and Human Thioredoxin 1. Our methodology relies on accurate mass measurement of proteins using LC-MS analyses and does not necessarily require top-down fragmentation. As well as analyzing homogeneous protein samples, we also demonstrate that our methodology can be used to determine thiol E⊕ measurements in samples which contain mixtures of proteins. Thus the combination of experiential methodology and data analysis regime have the potential to make such measurements in a high-throughput manner and in a manner more accessible to a broad community of protein scientists

The development of a field-based preservation method for total mercury in water samples using functionalised C18 solid-phase extraction

Mercury (Hg) is considered one of the most toxic elements to human health, due to its persistent and bioaccumulative properties, and is present in all spheres of the environment. Artisanal smallscale gold mining (ASGM) activities in countries, such as in Kenya, use Hg as a method to amalgamate gold from the geological matrix, with the potential to release Hg into the environment and subsequent public health exposure. In order to measure Hg in environmental samples improvements are required on existing recommended preservation methods for Hg in water samples that may not be fit for purpose e.g. acidification or use of glass bottles are potentially hazardous to operators in the field. Additionally, challenges are faced when samples are collected in remote locations far from laboratories with sufficient analytical sensitivity for Hg, requiring a preservation method that is safe to use during fieldwork, will preserve the analytical integrity of the sample and provide sufficient stability over a time period to allow for return to an appropriate laboratory. Therefore, a dithizone functionalised C18 solid phase extraction cartridge (SPE) was developed to preserve Hg in water samples, with the aim of presenting minimal risk to the operator when used in the field and to provide sufficient stability over a minimum of four weeks for subsequent elution and measurement in a laboratory environment – in this case, by ICP-MS. Performance characteristics were defined using a 0.8 µg L-1 Hg spike of a synthetic water matrix typical from an ASGM outflow – 30ml of this spike was passed through the functionalised cartridge and Hg eluted with 15ml of 2-mercaptoethanol (1% v/v with deionised water). The SPE cartridge retained 100% of Hg in the spike solution and provided stability for Hg preservation across a 57-day period, with recoveries of >75% Hg achieved following elution. Further work shows promising recovery rates of up to 90% with adjusted dithizone functionalisation of the SPE, without compromising retention of Hg on the SPE. Initial test data will be presented for ASGM sites in Kakamega County, Kenya

Anticipated effects of abiotic environmental change on intraspecific social interactions

Peer reviewedPublisher PD

Comprehensive glycosylation profiling of IgG and IgG-fusion proteins by top-down MS with multiple fragmentation techniques

We employed top- and middle-down analyses with multiple fragmentation techniques including electron transfer dissociation (ETD), electron capture dissociation (ECD), and matrix-assisted laser desorption ionization in-source decay (MALDI-ISD) for characterization of a reference monoclonal antibody (mAb) IgG1 and a fusion IgG protein. Fourier transform ion cyclotron resonance (FT-ICR) or high performance liquid chromatography electrospray ionization (HPLC-ESI) on an Orbitrap was employed. These experiments provided a comprehensive view on the protein species; especially for different glycosylation level in these two proteins, which showed good agreement with oligosaccharide profiling. Top- and middle-down MS provided additional information regarding glycosylation sites and different combinational protein species that were not available from oligosaccharide mapping or conventional bottom-up analysis. Finally, incorporating a limited enzymatic digestion by immunoglobulin G-degrading enzyme of Streptococcus pyogene (IdeS) with MALDI-ISD analysis enabled extended sequence coverage of the internal region of protein without pre-fractionation. Biological significance: Oligosaccharide profiling together with top- and middle-down methods enabled: 1) detection of heterogeneous glycosylated protein species and sites in intact IgG1 and fusion proteins with high mass accuracy, 2) estimation of relative abundance levels of protein species in the sample, 3) confirmation of the protein termini structural information, and 4) improved sequence coverage by MALDI-ISD analysis for the internal regions of the proteins without sample pre-fractionation