Joel Steele (BMedSc Hons1) is a current PhD candidate and casual academic at UTS, having held affiliate positions at the University of Sydney and the Defence Science Technology Group. Joel has several years of experience in proteomics beginning with exploration of Staphylococcus aureus for novel protein’s existing outside the predicted proteome. (N-Terminomics)
His second project was a collaboration with the Department of Primary Industries where-by he performed discovery-based proteomics on the hand dissected reproductive material of the Queensland fruit fly in pursuit of an antibody-based breeding assay for the spread of sterilised fruit flies.
Joel’s current field of research is on how non-coded amino acids play a role in Neurodegenerative diseases, this project includes the growth of neuronal cells for the modeling of complex biomolecular pathways and numerous molecular analysis methods.
Joel has expertise in human molecular biology with the application of mass spectrometry (LC-MS/MS, MALDI, analytical-QQQ LC-MS/MS) the application of various fractionation methods for top-down and bottom-up proteomics (Gel-based, Chromatography, Centrifugation, Affinity, Solubility).
Joel has further expertise in Neurological immunohistochemistry, tissue homoginization/handling (Brain), culturing of pathogens, microscopy (Light, Confocal) and the bioinformatic analysis of molecular networks especially disease states (Cytoscape, R, Reactome, String, KEGG).
Violi, JP, Bishop, DP, Padula, MP, Steele, JR & Rodgers, KJ 2020, 'Considerations for amino acid analysis by liquid chromatography-tandem mass spectrometry: a tutorial review', TrAC Trends in Analytical Chemistry, pp. 116018-116018.View/Download from: Publisher's site
O'Rourke, MB, Town, SEL, Dalla, PV, Bicknell, F, Koh Belic, N, Violi, JP, Steele, JR & Padula, MP 2019, 'What is Normalization? The Strategies Employed in Top-Down and Bottom-Up Proteome Analysis Workflows.', Proteomes, vol. 7, no. 3.View/Download from: Publisher's site
The accurate quantification of changes in the abundance of proteins is one of the main applications of proteomics. The maintenance of accuracy can be affected by bias and error that can occur at many points in the experimental process, and normalization strategies are crucial to attempt to overcome this bias and return the sample to its regular biological condition, or normal state. Much work has been published on performing normalization on data post-acquisition with many algorithms and statistical processes available. However, there are many other sources of bias that can occur during experimental design and sample handling that are currently unaddressed. This article aims to cast light on the potential sources of bias and where normalization could be applied to return the sample to its normal state. Throughout we suggest solutions where possible but, in some cases, solutions are not available. Thus, we see this article as a starting point for discussion of the definition of and the issues surrounding the concept of normalization as it applies to the proteomic analysis of biological samples. Specifically, we discuss a wide range of different normalization techniques that can occur at each stage of the sample preparation and analysis process.
Facey, JA, Steele, JR, Violi, JP, Mitrovic, SM & Cranfield, C 2019, 'An examination of microcystin-LR accumulation and toxicity using tethered bilayer lipid membranes (tBLMs).', Toxicon, vol. 158, pp. 51-56.View/Download from: Publisher's site
Microcystin-LR (MC-LR) is a potent cyanobacterial toxin responsible for animal and human poisonings worldwide. MC-LR is found in organisms throughout the foodweb, however there is conjecture regarding whether it biomagnifies. Few studies have investigated how MC-LR interacts with lipid membranes, a determinant of biomagnification potential. We tested whether 1 μM MC-LR irreversibly associates with lipid bilayers or causes the creation of pore defects upon short and long-term exposure. Using tethered bilayer lipid membranes (tBLMs), we observed an increase in membrane conduction in tBLMs, representing an interaction of microcystin-LR with the lipid bilayer and a change in membrane packing properties. However, there were minimal changes in membrane capacitance upon short and long-term exposure, and MC-LR exhibited a rapid off-rate. Upon 24 h exposure to the toxin, no lipophilic multimeric complexes were detected capable of altering the toxin's off-rate. There was no evidence of the creation of new pores. This study demonstrates that MC-LR does not irreversibly imbed itself into lipids membranes after short or long-term exposure and suggests MC-LR does not biomagnify through the food web via lipid storage.
Jarocki, VM, Steele, JR, Widjaja, M, Tacchi, JL, Padula, MP & Djordjevic, SP 2019, 'Formylated N-terminal methionine is absent from the Mycoplasma hyopneumoniae proteome: Implications for translation initiation.', International journal of medical microbiology : IJMM, vol. 309, no. 5, pp. 288-298.View/Download from: Publisher's site
N-terminal methionine excision (NME) is a proteolytic pathway that cleaves the N-termini of proteins, a process that influences where proteins localise in the cell and their turnover rates. In bacteria, protein biosynthesis is initiated by formylated methionine start tRNA (fMet-tRNAfMet). The formyl group is attached by formyltransferase (FMT) and is subsequently removed by peptide deformylase (PDF) in most but not all proteins. Methionine aminopeptidase then cleaves deformylated methionine to complete the process. Components of NME, particularly PDF, are promising therapeutic targets for bacterial pathogens. In Mycoplasma hyopneumoniae, a genome-reduced, major respiratory pathogen of swine, pdf and fmt are absent from its genome. Our bioinformatic analysis uncovered additional enzymes involved in formylated N-terminal methionine (fnMet) processing missing in fourteen mycoplasma species, including M. hyopneumoniae but not in Mycoplasma pneumoniae, a major respiratory pathogen of humans. Consistent with our bioinformatic studies, an analysis of in-house tryptic peptide libraries confirmed the absence of fnMet in M. hyopneumoniae proteins but, as expected fnMet peptides were detected in the proteome of M. pneumoniae. Additionally, computational molecular modelling of M. hyopneumoniae translation initiation factors reveal structural and sequence differences in areas known to interact with fMet-tRNAfMet. Our data suggests that some mycoplasmas have evolved a translation process that does not require fnMet.
Widjaja, M, Harvey, KL, Hagemann, L, Berry, IJ, Jarocki, V, Raymond, BBA, Tacchi, JL, Gründel, A, Steele, JR, Padula, MP, Charles, IG, Dumke, R & Djordjevic, SP 2017, 'Elongation factor Tu is a multifunctional and processed moonlighting protein.', Scientific Reports, vol. 7, no. 1, pp. 1-17.View/Download from: Publisher's site
Many bacterial moonlighting proteins were originally described in medically, agriculturally, and commercially important members of the low G + C Firmicutes. We show Elongation factor Tu (Ef-Tu) moonlights on the surface of the human pathogens Staphylococcus aureus (SaEf-Tu) and Mycoplasma pneumoniae (MpnEf-Tu), and the porcine pathogen Mycoplasma hyopneumoniae (MhpEf-Tu). Ef-Tu is also a target of multiple processing events on the cell surface and these were characterised using an N-terminomics pipeline. Recombinant MpnEf-Tu bound strongly to a diverse range of host molecules, and when bound to plasminogen, was able to convert plasminogen to plasmin in the presence of plasminogen activators. Fragments of Ef-Tu retain binding capabilities to host proteins. Bioinformatics and structural modelling studies indicate that the accumulation of positively charged amino acids in short linear motifs (SLiMs), and protein processing promote multifunctional behaviour. Codon bias engendered by an A + T rich genome may influence how positively-charged residues accumulate in SLiMs.
Berry, IJ, Steele, JR, Padula, MP & Djordjevic, SP 2016, 'The Application of terminomics for the identification of protein start sites and proteoforms in bacteria.', Proteomics, vol. 16, no. 2, pp. 257-272.View/Download from: Publisher's site
Protein terminomics, or the study of amino acids sequences at the protein amino or carboxyl
terminus has rapidly evolved as a proteomic discipline due to significant methodological improvements
in the labelling and recovery of terminal peptides as well as the increased speed
and sensitivity of current mass spectrometry instrumentation. The most significant benefi-
ciaries of these developments include an increased awareness and understanding of complex
proteolytic cascades that regulate key biological processes and in genome annotation. Most terminomics
research to date has focused on gaining insight into important biological processes
such as inflammation, wound healing and cancer. The application of terminomics to the study
of important biological questions in prokaryotes is gaining traction. Here we review current
applications and progress of terminomics in prokaryotes, discuss the significance of protease
research in bacterial pathogenesis and protein maturation, and suggest novel applications of
terminomics in the study of infectious disease.