Dr Matt Padula, while being a Senior Lecturer in the School of Life Sciences, also operates the Proteomics Core Facility, the main purpose of which is to provide high level technical support and the instrumentation required for researchers to perform their research projects without having to spend long periods of time learning the complicated techniques and instruments needed to answer the scientific question they want answered. The Core is equipped with the knowledge, expertise and instrumentation to perform almost all proteomic, protein chemistry, lipidomic and metabolomic experiments that could be of use to the faculty’s researchers. In addition, Core staff have built relationships with other institutions and research groups to gain access to instrumentation and expertise not available within UTS.
I have 21 years experience in protein chemistry, liquid chromatography, mass spectrometry analysis and characterization of peptides and proteins by liquid chromatography/mass spectrometry (LC/MS), electrophoresis and sample preparation by native and denaturing methodologies. In this time I have performed protein extraction, fractionation and analysis on organisms such as Mycoplasma, bacteria, yeast, mammalian tissue and cells, plant tissue, parasites including Fasciola, Toxoplasma, Eimeria, Haemonchus and Plasmodium, paralysis ticks, coral, snake venom and the pathogenic fungus Cryptococcus. I have expertise in nanoelectrospray mass spectrometric analysis of peptides and proteins, the quantitation of peptides and proteins using mass spectrometry and the determination of de novo peptide sequence from tandem mass spectrometry data.
In addition, he has expertise in the fractionation of peptides and proteins by analytical and preparative liquid chromatography and analytical and preparative electrophoresis techniques such as 1D and 2D-PAGE, liquid phase isoelectric focusing and continuous elution electrophoresis.
Recently, the focus of my interests has expanded to include lipids and metabolites, specifically developing methods and techniques to extract and fractionate these molecules, subject them to mass spectrometry for identification and quantification, and perform bioinformatic analysis to determine whether changes in abundance in these molecules is significant.
There are many opportunities for undergraduate students to gain experience and perform internships within the Core Facility by engaging in small parts of larger projects, all of which are aimed at developing methodologies and techniques that can be further applied to biological problems. A partial list of these projects appears below and a more extensive list can be provided and discussed by contacting me by email. Not all of these projects would be laboratory based but focused on bioinformatic, statistical and interpretative work on previously generated data.
Australian Proteomics Society - Member.
Can supervise: YES
Development of fractionation and analysis techniques for the analysis of proteins, protein expression, post-translational modifications of proteins, and protein complexes in their native and denatured states. These techniques use chromatographic and electrophoretic approaches combined with classical and targeted mass spectrometry techniques. The model wall-less bacteria Mycoplasma, a product of reductive evolution, is used extensively to develop these tecnniques.
Internship and other projects.
- Mapping the ENTIRE E.coli proteome by 2D-PAGE and LC/MS/MS.
- Software pipelines for proteomics, metabolomics and lipidomics data analysis and visualisation.
- Improving protein extraction methodologies.
- Using multiple solvent systems to fractionate and analyse the bacterial lipidome and metabolome.
- And many more!
Program Director - Masters of Medical Biotechnology
Subject co-ordinator for Proteomics (91536; post graduate coursework subject) and Biotechnology Research Internship Projects (60112, 60113, 60114, 60115).
Lecturer in Analytical Biochemistry (Mass spectrometry, Electrophoresis and Proteomics) and Molecular Biology 2 (Proteomics and Systems Biology).
Aili, SR, Touchard, A, Hayward, R, Robinson, SD, Pineda, SS, Lalagüe, H, Mrinalini, Vetter, I, Undheim, EAB, Kini, RM, Escoubas, P, Padula, MP, Myers, GSA & Nicholson, GM 2020, 'An Integrated Proteomic and Transcriptomic Analysis Reveals the Venom Complexity of the Bullet Ant Paraponera clavata.', Toxins, vol. 12, no. 5.View/Download from: Publisher's site
A critical hurdle in ant venom proteomic investigations is the lack of databases to comprehensively and specifically identify the sequence and function of venom proteins and peptides. To resolve this, we used venom gland transcriptomics to generate a sequence database that was used to assign the tandem mass spectrometry (MS) fragmentation spectra of venom peptides and proteins to specific transcripts. This was performed alongside a shotgun liquid chromatography-mass spectrometry (LC-MS/MS) analysis of the venom to confirm that these assigned transcripts were expressed as proteins. Through the combined transcriptomic and proteomic investigation of Paraponera clavata venom, we identified four times the number of proteins previously identified using 2D-PAGE alone. In addition to this, by mining the transcriptomic data, we identified several novel peptide sequences for future pharmacological investigations, some of which conform with inhibitor cysteine knot motifs. These types of peptides have the potential to be developed into pharmaceutical or bioinsecticide peptides.
Caulfield, MP & Padula, MP 2020, 'HPLC MS-MS Analysis Shows Measurement of Corticosterone in Egg Albumen Is Not a Valid Indicator of Chicken Welfare.', Animals : an open access journal from MDPI, vol. 10, no. 5.View/Download from: Publisher's site
Assessment of animal welfare can include analysis of physiological parameters, as well as behavior and health. Levels of adrenocortical hormones such as cortisol (and corticosterone in chickens) have been relied on as indicators of stress. Elevations in those hormones have been said to be correlated with poor welfare, while levels in the normal range have been interpreted to mean that animals are in a good state of welfare. Procuring blood samples from animals for hormone measures can in itself be stressful and cause increases in the target hormones. To overcome this problem, indirect measures of cortisol and corticosterone have been developed. In chickens, corticosterone levels in egg albumen are said to be a useful indirect measure, and have been used in several recent studies as indicators of chicken welfare. All of the measures of chicken egg albumen corticosterone in welfare studies have used immunoassays, and have reported values ranging from about 0.5 to over 20 ng/g. Using these measures, egg albumen from chickens housed in conventional cages or free ranging has been said to have indistinguishable corticosterone levels. This has been used to support the conclusion that chickens kept in conventional cages are not experiencing stress and are in a good state of welfare. In this study, we have used high-pressure liquid chromatography (HPLC) coupled with mass spectrometry (MS) to measure corticosterone in egg albumen. We found levels of corticosterone (median level about 50 pg/g) in egg albumen which were just above the limit of detection. By contrast, we found significant levels of progesterone and cortisol, hormones which would be expected to cross react with anti-corticosterone antibodies, and which therefore might explain the high reported levels of corticosterone using immunoassay. We conclude that because corticosterone levels in egg albumen are negligible, they cannot be used as an indicator of chicken welfare.
Coorssen, JR & Padula, MP 2020, 'Special issue "Top-down proteomics: In memory of dr alfred yergey". alfred linwood yergey, iii, 17 september 1941-27 may 2018', Proteomes, vol. 8, no. 1.View/Download from: Publisher's site
© 2020 Elsevier Inc. Lipids and bioactive lipid mediators are essential for platelet function. The lipid profile of platelets is highly dynamic due to free exchange of lipids with the plasma, release of extracellular vesicles, and both enzymatic and nonenzymatic lipid conversion. The lipidome of platelets changes in response to activation to accommodate the functional requirements of platelets, particularly for maintenance of hemostasis. Furthermore, when stored at room temperature as a component for transfusion, the lipid profile of platelets is altered. Although there is a growing interest in alternate storage conditions, such as refrigeration and cryopreservation, few contemporary studies have examined the impact of these storage modes on the lipid profile. However, evidence exists that bioactive lipid mediators produced over the storage of blood products may have functional implications once these products are transfused. As such, there is a need to determine the changes occurring to the lipid profile of these products over storage. This review outlines the role of lipids in platelets and discusses the current state of lipidomics for studying platelet components for transfusion in an effort to highlight the necessity for additional transfusion-focused investigations.
Jolly, C, Lee, Q, Padula, M, Pinello, N, Williams, S, O’Rourke, M, Fumagalli, MJ, Orkin, J, Shaban, B, Brenner, O, Weninger, W, de Souza, WM, Melin, A, Wong, JJ-L, Crim, M, Monette, S & Roediger, B 2020, 'MKPV (aka MuCPV) and related chapparvoviruses are nephro-tropic and encode novel accessory proteins p15 and NS2'.View/Download from: Publisher's site
Abstract Mouse kidney parvovirus (MKPV) is a member of the provisional Chapparvovirus genus that causes renal disease in immune-compromised mice, with a disease course reminiscent of polyomavirus-associated nephropathy in immune-suppressed kidney transplant patients. Here we map four MKPV transcripts, created by alternative splicing, to a common transcription initiation region, and use mass spectrometry to identify “p10” and “p15” as novel chapparvovirus accessory proteins produced in MKPV-infected kidneys. p15 and a splicing-dependent putative accessory protein NS2 are conserved in all near-complete tetrapod chapparvovirus genomes currently available (from mammals, birds and a reptile). In contrast, p10 may be encoded only by viruses with >60% amino acid identity to MKPV. We show that MKPV is kidney-tropic and that the bat chapparvovirus DrPV-1 and a non-human primate chapparvovirus, CKPV, are also found in the kidneys of their hosts. We propose, therefore, that chapparvoviruses with >60% VP1 amino acid identity to MKPV be classified into a genus dubbed Nephroparvovirus , which is consistent with nomenclature for the genus Erythroparvovirus .
Lee, Q, Padula, MP, Pinello, N, Williams, SH, O'Rourke, MB, Fumagalli, MJ, Orkin, JD, Song, R, Shaban, B, Brenner, O, Pimanda, JE, Weninger, W, de Souza, WM, Melin, AD, Wong, JJ-L, Crim, MJ, Monette, S, Roediger, B & Jolly, CJ 2020, 'Murine and related chapparvoviruses are nephro-tropic and produce novel accessory proteins in infected kidneys', PLOS PATHOGENS, vol. 16, no. 1.View/Download from: Publisher's site
Waters, L, Padula, MP, Marks, DC & Johnson, L 2020, 'Calcium chelation: a novel approach to reduce cryopreservation-induced damage to frozen platelets', TRANSFUSION.View/Download from: Publisher's site
Widjaja, M, Berry, IJ, Jarocki, VM, Padula, MP, Dumke, R & Djordjevic, SP 2020, 'Cell surface processing of the P1 adhesin of Mycoplasma pneumoniae identifies novel domains that bind host molecules', Scientific Reports, vol. 10, no. 1.View/Download from: Publisher's site
© 2020, The Author(s). Mycoplasma pneumoniae is a genome reduced pathogen and causative agent of community acquired pneumonia. The major cellular adhesin, P1, localises to the tip of the attachment organelle forming a complex with P40 and P90, two cleavage fragments derived by processing Mpn142, and other molecules with adhesive and mobility functions. LC-MS/MS analysis of M. pneumoniae M129 proteins derived from whole cell lysates and eluents from affinity matrices coupled with chemically diverse host molecules identified 22 proteoforms of P1. Terminomics was used to characterise 17 cleavage events many of which were independently verified by the identification of semi-tryptic peptides in our proteome studies and by immunoblotting. One cleavage event released 1597TSAAKPGAPRPPVPPKPGAPKPPVQPPKKPA1627 from the C-terminus of P1 and this peptide was shown to bind to a range of host molecules. A smaller synthetic peptide comprising the C-terminal 15 amino acids, 1613PGAPKPPVQPPKKPA1627, selectively bound cytoskeletal intermediate filament proteins cytokeratin 7, cytokeratin 8, cytokeratin 18, and vimentin from a native A549 cell lysate. Collectively, our data suggests that ectodomain shedding occurs on the surface of M. pneumoniae where it may alter the functional diversity of P1, Mpn142 and other surface proteins such as elongation factor Tu via a mechanism similar to that described in Mycoplasma hyopneumoniae.
Barolo, L, Abbriano, RM, Commault, AS, George, J, Kahlke, T, Fabris, M, Padula, MP, Lopez, A, Ralph, PJ & Pernice, M 2020, 'Perspectives for Glyco-Engineering of Recombinant Biopharmaceuticals from Microalgae', CELLS, vol. 9, no. 3.View/Download from: Publisher's site
Harrison, JA, Kelso, C, Padula, MP, Nicholson, GM & Beck, JL 2019, 'Structural characterization of protein toxins from Australian snake venoms using native mass spectrometry', Toxicon, vol. 158, pp. S43-S43.View/Download from: Publisher's site
Jarocki, VM, Raymond, BBA, Tacchi, JL, Padula, MP & Djordjevic, SP 2019, 'Mycoplasma hyopneumoniae surface-associated proteases cleave bradykinin, substance P, neurokinin A and neuropeptide Y.', Scientific reports, vol. 9, no. 1.View/Download from: Publisher's site
Mycoplasma hyopneumoniae is an economically-devastating and geographically-widespread pathogen that colonises ciliated epithelium, and destroys mucociliary function. M. hyopneumoniae devotes ~5% of its reduced genome to encode members of the P97 and P102 adhesin families that are critical for colonising epithelial cilia, but mechanisms to impair mucociliary clearance and manipulate host immune response to induce a chronic infectious state have remained elusive. Here we identified two surface exposed M. hyopneumoniae proteases, a putative Xaa-Pro aminopeptidase (MHJ_0659; PepP) and a putative oligoendopeptidase F (MHJ_0522; PepF), using immunofluorescence microscopy and two orthogonal proteomic methodologies. MHJ_0659 and MHJ_0522 were purified as polyhistidine fusion proteins and shown, using a novel MALDI-TOF MS assay, to degrade four pro-inflammatory peptides that regulate lung homeostasis; bradykinin (BK), substance P (SP), neurokinin A (NKA) and neuropeptide Y (NPY). These findings provide insight into the mechanisms used by M. hyopneumoniae to influence ciliary beat frequency, impair mucociliary clearance, and initiate a chronic infectious disease state in swine, features that are a hallmark of disease caused by this pathogen.
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.
O'Rourke, MB & Padula, MP 2019, 'An Inexpensive, simple calibration method for MALDI TOF/TOF systems.', Journal of mass spectrometry : JMS, vol. 54, no. 12, pp. 1003-1007.View/Download from: Publisher's site
The array of analytes that can be measured by MADLI MS has created an equally vast range of calibration mixtures. The inherent problem with this is that acquiring all of them at commercial rates can be prohibitively expensive. With this in mind, we have created a low-cost alternative to the most commonly used peptide calibrants. We were able to achieve an overall 78 ppm mass accuracy across a mass range of 900 to 2500 Da which was comparable to the mass accuracy achievable with commercial peptide mixes and hence has become a viable alternative.
O'Rourke, MB, Smith, CC, De La Monte, SM, Sutherland, GT & Padula, MP 2019, 'Higher Mass Accuracy MALDI-TOF/TOF Lipid Imaging of Human Brain Tissue in Alzheimer's Disease', Current Protocols in Molecular Biology, vol. 126, no. 1.View/Download from: Publisher's site
© 2019 John Wiley & Sons, Inc. Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) is a well-established technique for elucidating the location and relative abundance of a range of biomolecules. More recently, research into this technique has shifted from simple discovery and demonstration of utility to application in biomedical research. Here, we describe a protocol utilizing MALDI-IMS for the spatial mapping of lipids in brain tissue from normal human brains and brains from patients with Alzheimer's disease, in the context of Alzheimer's disease. Improved accuracy calibration of the instrument from the tissue surface is emphasized, as this allows for significantly improved mass determination in time of flight (TOF)-based instruments enabling more confident preliminary lipid identification. This improved initial result allows MALDI-IMS data to be complemented with additional instrumentation, such as liquid chromatography mass spectrometry workflows or specialized non-TOF systems such as Fourier transform cyclotron resonance instruments. This method is not limited to human tissue and can be applied to virtually any lipid-rich formalin-fixed tissue. © 2019 by John Wiley & Sons, Inc.
O'Rourke, MB, Smith, CC, Tse, BCY, Sutherland, GT, Crossett, B & Padula, MP 2019, ''What did i do wrong?' An empirical evaluation of sample preparation methodologies in matrix-assisted laser desorption/ionization-mass spectrometry imaging', Future Science OA, vol. 5, no. 4.View/Download from: Publisher's site
© 2019 MO Rourke. Aim: This guide aims to broaden the uptake of MALDI-MSI biomedical research by removing the initial 'lag phase' associated with empirical determination in sample preparation and data analysis. Methods: Samples from several tissue types were prepared for lipid, protein and peptide MSI analysis. Broadly, samples were cryo sectioned, mounted onto conductive MALDI slides and sublimed with an analyte specific matrix, recrystallised and analyzed in a Bruker UltrafleXtreme MALDI TOF/TOF. Results/conclusion: Here we present a general guide that serves as the first comprehensive, explanatory index for curation and verification of both sample preparation and data generation during the MALDI-MSI process. Lay abstract The field of mass spectrometry tissue imaging is a complex field that is designed to provide a map of the molecules on the surface of tissue sections. It often requires a significant investment of time and resources before useful data can be generated; therefore, this paper provides a visual troubleshooting guide that will act as a reference point for a range of sample preparation mistakes and explanations for unusual or suboptimal data. Elimination of the lag phase associated with the development of new techniques will help to expedite the growth and application of this technology.
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.
Santos, J, Milthorpe, BK & Padula, MP 2019, 'Correction: Santos, J., et al. Proteomic Analysis of Cyclic Ketamine Compounds Ability to Induce Neural Differentiation in Human Adult Mesenchymal Stem Cells. International Journal of Molecular Sciences 2019, 20, 523.', International journal of molecular sciences, vol. 20, no. 14.View/Download from: Publisher's site
The authors wish to make the following corrections to this paper [...].
Santos, J, Milthorpe, BK & Padula, MP 2019, 'Proteomic Analysis of Cyclic Ketamine Compounds Ability to Induce Neural Differentiation in Human Adult Mesenchymal Stem Cells.', International journal of molecular sciences, vol. 20, no. 3.View/Download from: Publisher's site
Neural regeneration is of great interest due to its potential to treat traumatic brain injuries and diseases that impact quality of life. Growth factor mediated differentiation can take up to several weeks to months to produce the cell of interest whereas chemical stimulation may be as minimal as a few hours. The smaller time scale is of great clinical relevance. Adipose derived stem cells (ADSCs) were treated for up to 24 h with a novel differentiation media containing the cyclic ketamine compounds to direct neurogenic induction. The extent of differentiation was investigated by proteome changes occurring during the process. The treatments indicated the ADSCs responded favorably to the neurogenic induction media by presenting a number of morphological cues of neuronal phenotype previously seen and a higher cell population post induction compared to previous studies. Furthermore, approximately 3500 proteins were analyzed and identified by mass spectrometric iTRAQ analyses. The bioinformatics analyses revealed hundreds of proteins whose expression level changes were statistically significant and biologically relevant to neurogenesis and annotated as being involved in neurogenic development. Complementing this, the Bioplex cytokine assay profiles present evidence of decreased panel of stress response cytokines and a relative increase in those involved in neurogenesis.
Li, G, Chan, YL, Sukjamnong, S, Anwer, AG, Vindin, H, Padula, M, Zakarya, R, George, J, Oliver, BG, Saad, S & Chen, H 2019, 'A Mitochondrial Specific Antioxidant Reverses Metabolic Dysfunction and Fatty Liver Induced by Maternal Cigarette Smoke in Mice.', Nutrients, vol. 11, no. 7.View/Download from: Publisher's site
Maternal smoking leads to glucose and lipid metabolic disorders and hepatic damage in the offspring, potentially due to mitochondrial oxidative stress. Mitoquinone mesylate (MitoQ) is a mitochondrial targeted antioxidant with high bioavailability. This study aimed to examine the impact of maternal cigarette smoke exposure (SE) on offspring's metabolic profile and hepatic damage, and whether maternal MitoQ supplementation during gestation can affect these changes. Female Balb/c mice (eight weeks) were either exposed to air or SE for six weeks prior to mating and throughout gestation and lactation. A subset of the SE dams were supplied with MitoQ in the drinking water (500 µmol/L) during gestation and lactation. Intraperitoneal glucose tolerance test was performed in the male offspring at 12 weeks and the livers and plasma were collected at 13 weeks. Maternal SE induced glucose intolerance, hepatic steatosis, mitochondrial oxidative stress and related damage in the adult offspring. Maternal MitoQ supplementation reduced hepatic mitochondrial oxidative stress and improved markers of mitophagy and mitochondrial biogenesis. This may restore hepatic mitochondrial health and was associated with an amelioration of glucose intolerance, hepatic steatosis and pathological changes induced by maternal SE. MitoQ supplementation may potentially prevent metabolic dysfunction and hepatic pathology induced by intrauterine SE.
Silva, MH, Berry, IJ, Strange, N, Djordjevic, SP & Padula, MP 2019, 'Terminomics methodologies and the completeness of reductive dimethylation: A meta-analysis of publicly available datasets', Proteomes, vol. 7, no. 2.View/Download from: Publisher's site
© 2019 by the authors. Methods for analyzing the terminal sequences of proteins have been refined over the previous decade; however, few studies have evaluated the quality of the data that have been produced from those methodologies. While performing global N-terminal labelling on bacteria, we observed that the labelling was not complete and investigated whether this was a common occurrence. We assessed the completeness of labelling in a selection of existing, publicly available N-terminomics datasets and empirically determined that amine-based labelling chemistry does not achieve complete labelling and potentially has issues with labelling amine groups at sequence-specific residues. This finding led us to conduct a thorough review of the historical literature that showed that this is not an unexpected finding, with numerous publications reporting incomplete labelling. These findings have implications for the quantitation of N-terminal peptides and the biological interpretations of these data.
Johnson, L, Cameron, M, Waters, L, Padula, MP & Marks, DC 2019, 'The impact of refrigerated storage of UVC pathogen inactivated platelet concentrates on in vitro platelet quality parameters.', Vox sanguinis, vol. 114, no. 1, pp. 47-56.View/Download from: Publisher's site
BACKGROUND AND OBJECTIVES:Refrigeration (cold-storage) of pathogen inactivated (PI) platelet components may increase the shelf-life and safety profile of platelet components, compared to conventional room-temperature (RT) storage. Whilst there is substantial knowledge regarding the impact of these individual treatments on platelets, the combined effect has not been assessed. MATERIALS AND METHODS:Using a pool-and-split study design, paired buffy-coat derived platelets in 70% platelet additive solution (SSP+; MacoPharma) were left untreated or PI-treated using the THERAFLEX UV-Platelets System (UVC; MacoPharma). Units from each pair were split and stored at room temperature (20-24°C) or cold-stored (2-6°C) to yield RT, cold, RT-UVC and cold-UVC study groups (n = 8 in each group). In vitro quality and function was tested over 9 days. RESULTS:Cold-storage of UVC-treated platelets reduced glycolytic metabolism (glucose consumption and lactate production) compared to RT-UVC units. Cold-UVC platelets demonstrated complete abrogation of HSR by day 5, increased externalisation of phosphatidylserine (annexin-V binding) and activation of the GPIIb/IIIa receptor (PAC-1 binding) above the levels observed with the individual treatments. Aggregation responses (ADP and collagen) were enhanced in the cold-UVC platelets compared to both RT groups, but this was primarily mediated by cold-storage. Haemostatic function, as measured using TEG, was similar between the groups. CONCLUSION:Cold-storage of UVC-treated platelets reduced PI-induced acceleration of glycolytic metabolism. However, combining cold-storage and UVC-treatment resulted in additional phenotypic changes compared to each treatment individually. Further work is required to understand the impact of these changes in clinical efficacy.
BACKGROUND:Extending the platelet (PLT) shelf life and enhancing product safety may be achieved by combining cryopreservation and pathogen inactivation (PI). Although studied individually, limited investigations into combining these treatments has been performed. The aim of this study was to investigate the effect of PI treating PLTs before cryopreservation on in vitro PLT quality and function. STUDY DESIGN AND METHODS:ABO-matched buffy coat-derived PLTs in PLT additive solution (SSP+; Macopharma) were pooled and split to form matched pairs (n = 8). One unit remained untreated and the other was treated with the THERAFLEX UV-Platelets System (UVC; Macopharma). For cryopreservation, 5% to 6% dimethyl sulfoxide was added to the PLTs, and they were frozen at -80°C. After being thawed, untreated cryopreserved PLTs (CPPs) and UVC-treated CPPs (UVC-CPPs) were resuspended in plasma. In vitro quality was assessed immediately after thawing and after 24 hours of room temperature storage. RESULTS:UVC-CPPs had lower in vitro recovery compared to CPPs. By flow cytometry, PLTs demonstrated a similar abundance of GPIX (CD42a), GPIIb (CD41a), and GPIbα (CD42b-HIP1), while the activation of GPIIb/IIIa (PAC-1) was increased in UVC-CPPs compared to CPPs. UVC-CPPs demonstrated greater phosphatidylserine exposure (annexin V) and microparticle shedding but similar P-selectin (CD62P) abundance compared to CPPs. UVC-CPPs displayed similar functionality to CPPs when assessed using aggregometry, thromboelastography, and thrombin generation. CONCLUSIONS:This study demonstrates the feasibility of cryopreserving UVC-PI-treated PLT products. UVC-PI treatment may increase the susceptibility of PLTs to damage caused during cryopreservation, but this is more pronounced during postthaw storage at room temperature.
Bogema, DR, Micallef, ML, Liu, M, Padula, MP, Djordjevic, SP, Darling, AE & Jenkins, C 2018, 'Analysis of Theileria orientalis draft genome sequences reveals potential species-level divergence of the Ikeda, Chitose and Buffeli genotypes.', BMC genomics, vol. 19, no. 1, pp. 298-298.View/Download from: Publisher's site
BACKGROUNDTheileria orientalis (Apicomplexa: Piroplasmida) has caused clinical disease in cattle of Eastern Asia for many years and its recent rapid spread throughout Australian and New Zealand herds has caused substantial economic losses to production through cattle deaths, late term abortion and morbidity. Disease outbreaks have been linked to the detection of a pathogenic genotype of T. orientalis, genotype Ikeda, which is also responsible for disease outbreaks in Asia. Here, we sequenced and compared the draft genomes of one pathogenic (Ikeda) and two apathogenic (Chitose, Buffeli) isolates of T. orientalis sourced from Australian herds.RESULTSUsing de novo assembled sequences and a single nucleotide variant (SNV) analysis pipeline, we found extensive genetic divergence between the T. orientalis genotypes. A genome-wide phylogeny reconstructed to address continued confusion over nomenclature of this species displayed concordance with prior phylogenetic studies based on the major piroplasm surface protein (MPSP) gene. However, average nucleotide identity (ANI) values revealed that the divergence between isolates is comparable to that observed between other theilerias which represent distinct species. Analysis of SNVs revealed putative recombination between the Chitose and Buffeli genotypes and also between Australian and Japanese Ikeda isolates. Finally, to inform future vaccine studies, dN/dS ratios and surface location predictions were analysed. Six predicted surface protein targets were confirmed to be expressed during the piroplasm phase of the parasite by mass spectrometry.CONCLUSIONSWe used whole genome sequencing to demonstrate that the T. orientalis Ikeda, Chitose and Buffeli variants show substantial genetic divergence. Our data indicates that future researchers could potentially consider disease-associated Ikeda and closely related genotypes as a separate species from non-pathogenic Chitose and Buffeli.
O'Rourke, MB, Djordjevic, SP & Padula, MP 2018, 'The Quest for Improved Reproducibility In MALDI Mass Spectrometry', Mass Spectrometry Reviews, vol. 37, no. 2, pp. 217-228.View/Download from: Publisher's site
Reproducibility has been one of the biggest hurdles faced when attempting to develop quantitative protocols for MALDI mass spectrometry. The heterogeneous nature of sample recrystallization has made automated sample acquisition somewhat “hit and miss” with manual intervention needed to ensure that all sample spots have been analyzed. In this review, we explore the last 30 years of literature and anecdotal evidence that has attempted to address and improve reproducibility in MALDI MS. Though many methods have been attempted, we have discovered a significant publication history surrounding the use of nitrocellulose as a substrate to improve homogeneity of crystal formation and therefore reproducibility. We therefore propose that this is the most promising avenue of research for developing a comprehensive and universal preparation protocol for quantitative MALDI MS analysis
O'Rourke, MB, Padula, MP, Smith, C, Youssef, P, Cordwell, S, Witting, P, Sutherland, G & Crossett, B 2018, 'Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows.', Journal of Visualized Experiments, no. 131.View/Download from: Publisher's site
The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host of biomolecules from drugs and lipids to N-glycans. Although various sample preparation techniques exist, detecting peptides from formaldehyde preserved tissues remains one of the most difficult challenges for this type of mass spectrometric analysis. For this reason, we have created and optimized a robust methodology that preserves the spatial information contained within the sample, while eliciting the greatest number of ionizable peptides. We have also aimed to achieve this in a cost effective and simple way, thereby eliminating potential bias or preparation error, which can occur when using automated instrumentation. The end result is a reproducible and inexpensive protocol.
O'Rourke, MB, Raymond, BBA, Djordjevic, SP & Padula, MP 2018, 'The Effect of Collimating Lens Focusing on Laser Beam Shape in Matrix Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS)', JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY, vol. 29, no. 3, pp. 512-515.View/Download from: Publisher's site
Roediger, B, Lee, Q, Tikoo, S, Cobbin, JCA, Henderson, JM, Jormakka, M, O'Rourke, MB, Padula, MP, Pinello, N, Henry, M, Wynne, M, Santagostino, SF, Brayton, CF, Rasmussen, L, Lisowski, L, Tay, SS, Harris, DC, Bertram, JF, Dowling, JP, Bertolino, P, Lai, JH, Wu, W, Bachovchin, WW, Wong, JJ-L, Gorrell, MD, Shaban, B, Holmes, EC, Jolly, CJ, Monette, S & Weninger, W 2018, 'An Atypical Parvovirus Drives Chronic Tubulointerstitial Nephropathy and Kidney Fibrosis.', Cell, vol. 175, no. 2, pp. 530-543.e24.View/Download from: Publisher's site
The occurrence of a spontaneous nephropathy with intranuclear inclusions in laboratory mice has puzzled pathologists for over 4 decades, because its etiology remains elusive. The condition is more severe in immunodeficient animals, suggesting an infectious cause. Using metagenomics, we identify the causative agent as an atypical virus, termed "mouse kidney parvovirus" (MKPV), belonging to a divergent genus of Parvoviridae. MKPV was identified in animal facilities in Australia and North America, is transmitted via a fecal-oral or urinary-oral route, and is controlled by the adaptive immune system. Detailed analysis of the clinical course and histopathological features demonstrated a stepwise progression of pathology ranging from sporadic tubular inclusions to tubular degeneration and interstitial fibrosis and culminating in renal failure. In summary, we identify a widely distributed pathogen in laboratory mice and establish MKPV-induced nephropathy as a new tool for elucidating mechanisms of tubulointerstitial fibrosis that shares molecular features with chronic kidney disease in humans.
Waters, L, Cameron, M, Padula, MP, Marks, DC & Johnson, L 2018, 'Refrigeration, cryopreservation and pathogen inactivation: an updated perspective on platelet storage conditions.', Vox sanguinis, vol. 113, no. 4, pp. 317-328.View/Download from: Publisher's site
Conventional storage of platelet concentrates limits their shelf life to between 5 and 7 days due to the risk of bacterial proliferation and the development of the platelet storage lesion. Cold storage and cryopreservation of platelets may facilitate extension of the shelf life to weeks and years, and may also provide the benefit of being more haemostatically effective than conventionally stored platelets. Further, treatment of platelet concentrates with pathogen inactivation systems reduces bacterial contamination and provides a safeguard against the risk of emerging and re-emerging pathogens. While each of these alternative storage techniques is gaining traction individually, little work has been done to examine the effect of combining treatments in an effort to further improve product safety and minimize wastage. This review aims to discuss the benefits of alternative storage techniques and how they may be combined to alleviate the problems associated with conventional platelet storage.
Wong, SL, To, J, Santos, J, Allam, VSRR, Dalton, JP, Djordjevic, SP, Donnelly, S, Padula, MP & Sukkar, MB 2018, 'Proteomic Analysis of Extracellular HMGB1 Identifies Binding Partners and Exposes Its Potential Role in Airway Epithelial Cell Homeostasis.', Journal of Proteome Research, vol. 17, no. 1, pp. 33-45.View/Download from: Publisher's site
The release of damage-associated molecular patterns (DAMPs) by airway epithelial cells is believed to play a crucial role in the initiation and development of chronic airway conditions such as asthma and chronic obstructive pulmonary disease (COPD). Intriguingly, the classic DAMP high-mobility group box-1 (HMGB1) is detected in the culture supernatant of airway epithelial cells under basal conditions, indicating a role for HMGB1 in the regulation of epithelial cellular and immune homeostasis. To gain contextual insight into the potential role of HMGB1 in airway epithelial cell homeostasis, we used the orthogonal and complementary methods of high-resolution clear native electrophoresis, immunoprecipitation, and pull-downs coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) to profile HMGB1 and its binding partners in the culture supernatant of unstimulated airway epithelial cells. We found that HMGB1 presents exclusively as a protein complex under basal conditions. Moreover, protein network analysis performed on 185 binding proteins revealed 14 that directly associate with HMGB1: amyloid precursor protein, F-actin-capping protein subunit alpha-1 (CAPZA1), glyceraldehyde-3 phosphate dehydrogenase (GAPDH), ubiquitin, several members of the heat shock protein family (HSPA8, HSP90B1, HSP90AA1), XRCC5 and XRCC6, high mobility group A1 (HMGA1), histone 3 (H3F3B), the FACT (facilitates chromatin transcription) complex constituents SUPT1H and SSRP1, and heterogeneous ribonucleoprotein K (HNRNPK). These studies provide a new understanding of the extracellular functions of HMGB1 in cellular and immune homeostasis at the airway mucosal surface and could have implications for therapeutic targeting.
Main, BJ, Bowling, LC, Padula, MP, Bishop, DP, Mitrovic, SM, Guillemin, GJ & Rodgers, KJ 2018, 'Detection of the suspected neurotoxin β-methylamino-l-alanine (BMAA) in cyanobacterial blooms from multiple water bodies in Eastern Australia.', Harmful algae, vol. 74, pp. 10-18.View/Download from: Publisher's site
The emerging toxin β-methylamino-l-alanine (BMAA) has been linked to the development of a number of neurodegenerative diseases in humans including amyotrophic lateral sclerosis (ALS), Alzheimer's disease, and Parkinson's disease. BMAA has been found to be produced by a range of cyanobacteria, diatoms, and dinoflagellates worldwide, and is present in freshwater, saltwater, and terrestrial ecosystems. Surface scum samples were collected from waterways in rural and urban New South Wales, Australia and algal species identified. Reverse phase liquid chromatography-tandem mass spectrometry was used to analyse sixteen cyanobacterial scum for the presence of BMAA as well as its toxic structural isomer 2,4-diaminobutyric acid (2,4-DAB). BMAA was detected in ten of the samples analysed, and 2,4-DAB in all sixteen. The presence of these toxins in water used for agriculture raises concerns for public health and food security in Australia.
Raymond, BBA, Madhkoor, R, Schleicher, I, Uphoff, CC, Turnbull, L, Whitchurch, CB, Rohde, M, Padula, MP & Djordjevic, SP 2018, 'Extracellular Actin Is a Receptor for Mycoplasma hyopneumoniae.', Frontiers in cellular and infection microbiology, vol. 8, no. Feb.View/Download from: Publisher's site
Mycoplasma hyopneumoniae, an agriculturally important porcine pathogen, disrupts the mucociliary escalator causing ciliostasis, loss of cilial function, and epithelial cell death within the porcine lung. Losses to swine production due to growth rate retardation and reduced feed conversion efficiency are severe, and antibiotics are used heavily to control mycoplasmal pneumonia. Notably, little is known about the repertoire of host receptors that M. hyopneumoniae targets to facilitate colonization. Here we show, for the first time, that actin exists extracellularly on porcine epithelial monolayers (PK-15) using surface biotinylation and 3D-Structured Illumination Microscopy (3D-SIM), and that M. hyopneumoniae binds to the extracellular β-actin exposed on the surface of these cells. Consistent with this hypothesis we show: (i) monoclonal antibodies that target β-actin significantly block the ability of M. hyopneumoniae to adhere and colonize PK-15 cells; (ii) microtiter plate binding assays show that M. hyopneumoniae cells bind to monomeric G-actin in a dose dependent manner; (iii) more than 100 M. hyopneumoniae proteins were recovered from affinity-chromatography experiments using immobilized actin as bait; and (iv) biotinylated monomeric actin binds directly to M. hyopneumoniae proteins in ligand blotting studies. Specifically, we show that the P97 cilium adhesin possesses at least two distinct actin-binding regions, and binds monomeric actin with nanomolar affinity. Taken together, these observations suggest that actin may be an important receptor for M. hyopneumoniae within the swine lung and will aid in the future development of intervention strategies against this devastating pathogen. Furthermore, our observations have wider implications for extracellular actin as an important bacterial receptor.
Aili, SR, Touchard, A, Petitclerc, F, Dejean, A, Orivel, J, Padula, MP, Escoubas, P & Nicholson, GM 2017, 'Combined Peptidomic and Proteomic Analysis of Electrically Stimulated and Manually Dissected Venom from the South American Bullet Ant Paraponera clavata', JOURNAL OF PROTEOME RESEARCH, vol. 16, no. 3, pp. 1339-1351.View/Download from: Publisher's site
Berry, IJ, Jarocki, VM, Tacchi, JL, Raymond, BBA, Widjaja, M, Padula, MP & Djordjevic, SP 2017, 'N-terminomics identifies widespread endoproteolysis and novel methionine excision in a genome-reduced bacterial pathogen.', Scientific Reports, vol. 7, no. 1, pp. 1-17.View/Download from: Publisher's site
Proteolytic processing alters protein function. Here we present the first systems-wide analysis of endoproteolysis in the genome-reduced pathogen Mycoplasma hyopneumoniae. 669 N-terminal peptides from 164 proteins were identified, demonstrating that functionally diverse proteins are processed, more than half of which 75 (53%) were accessible on the cell surface. Multiple cleavage sites were characterised, but cleavage with arginine in P1 predominated. Putative functions for a subset of cleaved fragments were assigned by affinity chromatography using heparin, actin, plasminogen and fibronectin as bait. Binding affinity was correlated with the number of cleavages in a protein, indicating that novel binding motifs are exposed, and protein disorder increases, after a cleavage event. Glyceraldehyde 3-phosphate dehydrogenase was used as a model protein to demonstrate this. We define the rules governing methionine excision, show that several aminopeptidases are involved, and propose that through processing, genome-reduced organisms can expand protein function.
O'Rourke, MB & Padula, MP 2017, 'A new standard of visual data representation for imaging mass spectrometry.', PROTEOMICS - Clinical Applications, vol. 11, no. 3-4.View/Download from: Publisher's site
PURPOSE: MALDI imaging MS (IMS) is principally used for cancer diagnostics. In our own experience with publishing IMS data, we have been requested to modify our protocols with respect to the areas of the tissue that are imaged in order to comply with the wider literature. In light of this, we have determined that current methodologies lack effective controls and can potentially introduce bias by only imaging specific areas of the targeted tissue EXPERIMENTAL DESIGN: A previously imaged sample was selected and then cropped in different ways to show the potential effect of only imaging targeted areas. RESULTS: By using a model sample, we were able to effectively show how selective imaging of samples can misinterpret tissue features and by changing the areas that are acquired, according to our new standard, an effective internal control can be introduced. CONCLUSIONS AND CLINICAL RELEVANCE: Current IMS sampling convention relies on the assumption that sample preparation has been performed correctly. This prevents users from checking whether molecules have moved beyond borders of the tissue due to delocalization and consequentially products of improper sample preparation could be interpreted as biological features that are of critical importance when encountered in a visual diagnostic.
O'Rourke, MB, Raymond, BBA & Padula, MP 2017, 'The Characterization of Laser Ablation Patterns and a New Definition of Resolution in Matrix Assisted Laser Desorption Ionization Imaging Mass Spectrometry (MALDI-IMS).', Journal of The American Society for Mass Spectrometry, vol. 28, no. 5, pp. 895-900.View/Download from: Publisher's site
Matrix assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) is a technique that has seen a sharp rise in both use and development. Despite this rapid adoption, there have been few thorough investigations into the actual physical mechanisms that underlie the acquisition of IMS images. We therefore set out to characterize the effect of IMS laser ablation patterns on the surface of a sample. We also concluded that the governing factors that control spatial resolution have not been correctly defined and therefore propose a new definition of resolution. Graphical Abstract ᅟ.
Padula, MP, Berry, IJ, O Rourke, MB, Raymond, BBA, Santos, J & Djordjevic, SP 2017, 'A Comprehensive Guide for Performing Sample Preparation and Top-Down Protein Analysis.', Proteomes, vol. 5, no. 2.View/Download from: Publisher's site
Methodologies for the global analysis of proteins in a sample, or proteome analysis, have been available since 1975 when Patrick O'Farrell published the first paper describing two-dimensional gel electrophoresis (2D-PAGE). This technique allowed the resolution of single protein isoforms, or proteoforms, into single 'spots' in a polyacrylamide gel, allowing the quantitation of changes in a proteoform's abundance to ascertain changes in an organism's phenotype when conditions change. In pursuit of the comprehensive profiling of the proteome, significant advances in technology have made the identification and quantitation of intact proteoforms from complex mixtures of proteins more routine, allowing analysis of the proteome from the 'Top-Down'. However, the number of proteoforms detected by Top-Down methodologies such as 2D-PAGE or mass spectrometry has not significantly increased since O'Farrell's paper when compared to Bottom-Up, peptide-centric techniques. This article explores and explains the numerous methodologies and technologies available to analyse the proteome from the Top-Down with a strong emphasis on the necessity to analyse intact proteoforms as a better indicator of changes in biology and phenotype. We arrive at the conclusion that the complete and comprehensive profiling of an organism's proteome is still, at present, beyond our reach but the continuing evolution of protein fractionation techniques and mass spectrometry brings comprehensive Top-Down proteome profiling closer.
Santos, J, Milthorpe, BK, Herbert, BR & Padula, MP 2017, 'Proteomic Analysis of Human Adipose Derived Stem Cells during Small Molecule Chemical Stimulated Pre-neuronal Differentiation.', International journal of stem cells, vol. 10, no. 2, pp. 193-217.View/Download from: Publisher's site
Adipose derived stem cells (ADSCs) are acquired from abdominal liposuction yielding a thousand fold more stem cells per millilitre than those from bone marrow. A large research void exists as to whether ADSCs are capable of transdermal differentiation toward neuronal phenotypes. Previous studies have investigated the use of chemical cocktails with varying inconclusive results.Human ADSCs were treated with a chemical stimulant, beta-mercaptoethanol, to direct them toward a neuronal-like lineage within 24 hours. Quantitative proteomics using iTRAQ was then performed to ascertain protein abundance differences between ADSCs, beta-mercaptoethanol treated ADSCs and a glioblastoma cell line.The soluble proteome of ADSCs differentiated for 12 hours and 24 hours was significantly different from basal ADSCs and control cells, expressing a number of remodeling, neuroprotective and neuroproliferative proteins. However toward the later time point presented stress and shock related proteins were observed to be up regulated with a large down regulation of structural proteins. Cytokine profiles support a large cellular remodeling shift as well indicating cellular distress.The earlier time point indicates an initiation of differentiation. At the latter time point there is a vast loss of cell population during treatment. At 24 hours drastically decreased cytokine profiles and overexpression of stress proteins reveal that exposure to beta-mercaptoethanol beyond 24 hours may not be suitable for clinical application as our results indicate that the cells are in trauma whilst producing neuronal-like morphologies. The shorter treatment time is promising, indicating a reducing agent has fast acting potential to initiate neuronal differentiation of ADSCs.
Stroud, LJ, Slapeta, J, Padula, MP, Druery, D, Tsiotsioras, G, Coorssen, JR & Stack, CM 2017, 'Comparative proteomic analysis of two pathogenic Tritrichomonas foetus genotypes: there is more to the proteome than meets the eye', INTERNATIONAL JOURNAL FOR PARASITOLOGY, vol. 47, no. 4, pp. 203-213.View/Download from: Publisher's site
Waters, L, Padula, MP, Marks, DC & Johnson, L 2017, 'Cryopreserved platelets demonstrate reduced activation responses and impaired signaling after agonist stimulation', TRANSFUSION, vol. 57, no. 12, pp. 2845-2857.View/Download from: Publisher's site
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.
Jiang, Z, Kumar, M, Padula, MP, Pernice, M, Kahlke, T, Kim, M & Ralph, PJ 2017, 'Development of an Efficient Protein Extraction Method Compatible with LC-MS/MS for Proteome Mapping in Two Australian Seagrasses Zostera muelleri and Posidonia australis.', Frontiers in Plant Science, vol. 8, pp. 1-14.View/Download from: Publisher's site
The availability of the first complete genome sequence of the marine flowering plant Zostera marina (commonly known as seagrass) in early 2016, is expected to significantly raise the impact of seagrass proteomics. Seagrasses are marine ecosystem engineers that are currently declining worldwide at an alarming rate due to both natural and anthropogenic disturbances. Seagrasses (especially species of the genus Zostera) are compromised for proteomic studies primarily due to the lack of efficient protein extraction methods because of their recalcitrant cell wall which is rich in complex polysaccharides and a high abundance of secondary metabolites in their cells. In the present study, three protein extraction methods that are commonly used in plant proteomics i.e., phenol (P); trichloroacetic acid/acetone/SDS/phenol (TASP); and borax/polyvinyl-polypyrrolidone/phenol (BPP) extraction, were evaluated quantitatively and qualitatively based on two dimensional isoelectric focusing (2D-IEF) maps and LC-MS/MS analysis using the two most abundant Australian seagrass species, namely Zostera muelleri and Posidonia australis. All three tested methods produced high quality protein extracts with excellent 2D-IEF maps in P. australis. However, the BPP method produces better results in Z. muelleri compared to TASP and P. Therefore, we further modified the BPP method (M-BPP) by homogenizing the tissue in a modified protein extraction buffer containing both ionic and non-ionic detergents (0.5% SDS; 1.5% Triton X-100), 2% PVPP and protease inhibitors. Further, the extracted proteins were solubilized in 0.5% of zwitterionic detergent (C7BzO) instead of 4% CHAPS. This slight modification to the BPP method resulted in a higher protein yield, and good quality 2-DE maps with a higher number of protein spots in both the tested seagrasses. Further, the M-BPP method was successfully utilized in western-blot analysis of phosphoenolpyruvate carboxylase (PEPC-a key enzyme for carbon metabolism). ...
Kumar, M, Padula, MP, Davey, P, Pernice, M, Jiang, Z, Sablok, G, Contreras-Porcia, L & Ralph, PJ 2017, 'Proteome Analysis Reveals Extensive Light Stress-Response Reprogramming in the Seagrass Zostera muelleri (Alismatales, Zosteraceae) Metabolism.', Frontiers in Plant Science, vol. 7, pp. 1-19.View/Download from: Publisher's site
Seagrasses are marine ecosystem engineers that are currently declining in abundance at an alarming rate due to both natural and anthropogenic disturbances in ecological niches. Despite reports on the morphological and physiological adaptations of seagrasses to extreme environments, little is known of the molecular mechanisms underlying photo-acclimation, and/or tolerance in these marine plants. This study applies the two-dimensional isoelectric focusing (2D-IEF) proteomics approach to identify photo-acclimation/tolerance proteins in the marine seagrass Zostera muelleri. For this, Z. muelleri was exposed for 10 days in laboratory mesocosms to saturating (control, 200 μmol photons m-2 s-1), super-saturating (SSL, 600 μmol photons m-2 s-1), and limited light (LL, 20 μmol photons m-2 s-1) irradiance conditions. Using LC-MS/MS analysis, 93 and 40 protein spots were differentially regulated under SSL and LL conditions, respectively, when compared to the control. In contrast to the LL condition, Z. muelleri robustly tolerated super-saturation light than control conditions, evidenced by their higher relative maximum electron transport rate and minimum saturating irradiance values. Proteomic analyses revealed up-regulation and/or appearances of proteins belonging to the Calvin-Benson and Krebs cycle, glycolysis, the glycine cleavage system of photorespiration, and the antioxidant system. These proteins, together with those from the inter-connected glutamate-proline-GABA pathway, shaped Z. muelleri photosynthesis and growth under SSL conditions. In contrast, the LL condition negatively impacted the metabolic activities of Z. muelleri by down-regulating key metabolic enzymes for photosynthesis and the metabolism of carbohydrates and amino acids, which is consistent with the observation with lower photosynthetic performance under LL condition. This study provides novel insights into the underlying molecular photo-acclimation mechanisms in Z. muelleri, in addition to identify...
Tran, N-AT, Padula, MP, Evenhuis, CR, Commault, AS, Ralph, PJ & Tamburic, B 2016, 'Proteomic and biophysical analyses reveal a metabolic shift in nitrogen deprived Nannochloropsis oculata', ALGAL RESEARCH-BIOMASS BIOFUELS AND BIOPRODUCTS, vol. 19, pp. 1-11.View/Download from: Publisher's site
Aili, SR, Touchard, A, Koh, JMS, Dejean, A, Orivel, J, Padula, MP, Escoubas, P & Nicholson, GM 2016, 'Comparisons of protein and peptide complexity in poneroid and formicoid ant venoms', Journal of Proteome Research, vol. 15, pp. 3039-3054.View/Download from: Publisher's site
Animal venom peptides are currently being
developed as novel drugs and bioinsecticides. Because ants use
venoms for defense and predation, venomous ants represent
an untapped source of potential bioactive toxins. This study
compared the protein and peptide components of the
poneroid ants Neoponera commutata, Neoponera apicalis, and
Odontomachus hastatus and the formicoid ants Ectatomma
tuberculatum, Ectatomma brunneum, and Myrmecia gulosa. 1D
and 2D PAGE revealed venom proteins in the mass range <10 to >250 kDa. NanoLC-ESI-QTOF MS/MS analysis of tryptic
peptides revealed the presence of common venom proteins and also many undescribed proteins. RP-HPLC separation followed
by MALDI-TOF MS of the venom peptides also revealed considerable heterogeneity. It was found that the venoms contained
between 144 and 1032 peptides with 5−95% of peptides in the ranges 1−4 and 1−8 kDa for poneroid and formicoid ants,
respectively. By employing the reducing MALDI matrix 1,5-diaminonapthalene, up to 28 disulfide-bonded peptides were also
identified in each of the venoms. In particular, the mass range of peptides from poneroid ants is lower than peptides from other
venoms, indicating possible novel structures and pharmacologies. These results indicate that ant venoms represent an enormous, untapped source of novel therapeutic and bioinsecticide leads.
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.
Lu, JF, Pokharel, D, Padula, MP & Bebawy, M 2016, 'A novel method to detect translation of membrane proteins following microvesicle intercellular transfer of nucleic acids.', Journal of biochemistry, vol. 160, no. 5, pp. 281-289.View/Download from: Publisher's site
Microvesicles (MVs) serve as vectors of nucleic-acid dissemination and are important mediators of intercellular communication. However, the functionality of packaged nucleic acids on recipient cells following transfer of MV-cargo has not been clearly elucidated. This limitation is attributed to a lack of methodology available in assessing protein translation following homotypic intercellular transfer of nucleic-acids. Using surface peptide shaving we have demonstrated that MVs derived from human leukaemic cells transfer functional P-glycoprotein transcripts, conferring drug-efflux capacity to recipient cells. We demonstrate expression of newly synthesized protein using Western blot. Furthermore, we show functionality of translated P-gp protein in recipient cells using Calcein-AM dye exclusion assays on flow cytometry. Newly synthesized 170 kDa P-gp was detected in recipient cells after co-culture with shaven MVs and these proteins were functional, conferring drug-efflux. This is the first demonstration of functionality of transferred nucleic-acids between human homotypic cells as well as the translation of the cancer multidrug-resistance protein in recipient cells following intercellular transfer of its transcript. This study supports the significant role of MV's in the transfer of deleterious traits in cancer populations and describes a new paradigm in mechanisms governing the acquisition of traits in cancer cell populations.
O'Rourke, M, Djordjevic, SP & Padula, MP 2016, 'The Renaissance of Microbiology: The Necessary Future for Matrix AssistedLaser Desorption Ionisation Mass Spectrometry Based Bio Typing', Journal of Microbial & Biochemical Technology, vol. 8, no. 4, pp. 373-374.View/Download from: Publisher's site
O'Rourke, MB & Padula, MP 2016, 'Analysis of formalin-fixed, paraffin-embedded (FFPE) tissue via proteomic techniques and misconceptions of antigen retrieval', BIOTECHNIQUES, vol. 60, no. 5, pp. 229-+.View/Download from: Publisher's site
Pokharel, D, Padula, MP, Lu, JF, Jaiswal, R, Djordjevic, SP & Bebawy, M 2016, 'The Role of CD44 and ERM Proteins in Expression and Functionality of P-glycoprotein in Breast Cancer Cells.', Molecules (Basel, Switzerland), vol. 21, no. 3, pp. 1-14.View/Download from: Publisher's site
Multidrug resistance (MDR) is often attributed to the over-expression of P-glycoprotein (P-gp), which prevents the accumulation of anticancer drugs within cells by virtue of its active drug efflux capacity. We have previously described the intercellular transfer of P-gp via extracellular vesicles (EVs) and proposed the involvement of a unique protein complex in regulating this process. In this paper, we investigate the role of these mediators in the regulation of P-gp functionality and hence the acquisition of MDR following cell to cell transfer. By sequentially silencing the FERM domain-binding proteins, Ezrin, Radixin and Moesin (ERM), as well as CD44, which we also report a selective packaging in breast cancer derived EVs, we have established a role for these proteins, in particular Radixin and CD44, in influencing the P-gp-mediated MDR in whole cells. We also report for the first time the role of ERM proteins in the vesicular transfer of functional P-gp. Specifically, we demonstrate that intercellular membrane insertion is dependent on Ezrin and Moesin, whilst P-gp functionality is governed by the integrity of all ERM proteins in the recipient cell. This study identifies these candidate proteins as potential new therapeutic targets in circumventing MDR clinically.
Reynolds, OL, Padula, MP, Zeng, R & Gurr, GM 2016, 'Silicon: Potential to Promote Direct and Indirect Effects on Plant Defense Against Arthropod Pests in Agriculture.', Frontiers in Plant Science, vol. 7, pp. 1-13.View/Download from: Publisher's site
Silicon has generally not been considered essential for plant growth, although it is well recognized that many plants, particularly Poaceae, have substantial plant tissue concentrations of this element. Recently, however, the International Plant Nutrition Institute [IPNI] (2015), Georgia, USA has listed it as a "beneficial substance". This reflects that numerous studies have now established that silicon may alleviate both biotic and abiotic stress. This paper explores the existing knowledge and recent advances in elucidating the role of silicon in plant defense against biotic stress, particularly against arthropod pests in agriculture and attraction of beneficial insects. Silicon confers resistance to herbivores via two described mechanisms: physical and biochemical/molecular. Until recently, studies have mainly centered on two trophic levels; the herbivore and plant. However, several studies now describe tri-trophic effects involving silicon that operate by attracting predators or parasitoids to plants under herbivore attack. Indeed, it has been demonstrated that silicon-treated, arthropod-attacked plants display increased attractiveness to natural enemies, an effect that was reflected in elevated biological control in the field. The reported relationships between soluble silicon and the jasmonic acid (JA) defense pathway, and JA and herbivore-induced plant volatiles (HIPVs) suggest that soluble silicon may enhance the production of HIPVs. Further, it is feasible that silicon uptake may affect protein expression (or modify proteins structurally) so that they can produce additional, or modify, the HIPV profile of plants. Ultimately, understanding silicon under plant ecological, physiological, biochemical, and molecular contexts will assist in fully elucidating the mechanisms behind silicon and plant response to biotic stress at both the bi- and tri-trophic levels.
Tacchi, JL, Raymond, BBA, Haynes, PA, Berry, IJ, Widjaja, M, Bogema, DR, Woolley, LK, Jenkins, C, Minion, FC, Padula, MP & Djordjevic, SP 2016, 'Post-translational processing targets functionally diverse proteins in Mycoplasma hyopneumoniae', OPEN BIOLOGY, vol. 6, no. 2.View/Download from: Publisher's site
Wood, B, Padula, MP, Marks, DC & Johnson, L 2016, 'Refrigerated storage of platelets initiates changes in platelet surface marker expression and localization of intracellular proteins', TRANSFUSION, vol. 56, no. 10, pp. 2548-2559.View/Download from: Publisher's site
Lao, W, Jin, X, Tan, Y, Xiao, L, Padula, M, Bishop, D, Reedy, B, Ong, M, Kamal, M & Qu, X 2016, 'Characterisation of Bone Beneficial Components from Australian Wallaby Bone', Medicines, vol. 3, pp. 1-13.View/Download from: Publisher's site
Background: Osteoporosis is a condition in which the bones become brittle, increasing the risk of fractures. Complementary medicines have traditionally used animal bones for managing bone disorders, such as osteoporosis. This study aimed to discover new natural products for these types of conditions by determining mineral and protein content of bone extracts derived from the Australian wallaby. Methods: Inductively coupled plasma-mass spectrometry and Fourier transform infrared spectroscopic analysis were used for mineral tests, proteome analysis was using LC/MS/MS and the effects of wallaby bone extracts (WBE)s on calcium deposition and alkaline phosphatase activity were evaluated in osteogenic cells derived from adipose tissue-derived stem cells (ADSCs). Results: Concentrations of calcium and phosphorus were 26.21% and 14.72% in WBE respectively. Additionally, minerals found were wide in variety and high in concentration, while heavy metal concentrations of aluminium, iron, zinc and other elements were at safe levels for human consumption. Proteome analysis showed that extracts contained high amounts of bone remodelling proteins, such as osteomodulin, osteopontin and osteoglycin. Furthermore, in vitro evaluation of WBEs showed increased deposition of calcium in osteoblasts with enhanced alkaline phosphatase activity in differentiated adipose-derived stem cells. Conclusion: Our results demonstrate that wallaby bone extracts possess proteins and minerals beneficial for bone metabolism. WBEs may therefore be used for developing natural products for conditions such as osteoporosis and further investigation to understand biomolecular mechanism by which WBEs prevent osteoporosis is warranted.
Raymond, BBA, Jenkins, C, Seymour, LM, Tacchi, JL, Widjaja, M, Jarocki, VM, Deutscher, AT, Turnbull, L, Whitchurch, CB, Padula, MP & Djordjevic, SP 2015, 'Proteolytic processing of the cilium adhesin MHJ_0194 (P123(J)) in Mycoplasma hyopneumoniae generates a functionally diverse array of cleavage fragments that bind multiple host molecules', CELLULAR MICROBIOLOGY, vol. 17, no. 3, pp. 425-444.View/Download from: Publisher's site
Campbell, LT, Padula, MP, Harry, E & Carter, DA 2015, 'You are what you secrete: extracellular proteins and virulence in Cryptococcus', Microbiology Australia, vol. 36, no. 2, pp. 93-95.View/Download from: Publisher's site
Fungal organisms secrete a wide range of biomolecules,
including degradative enzymes that are essential for nutrition,
toxins, effectors and secondary compounds that modulate
interactions with host animals and plants, and a variety
of signaling and stress-related proteins1
. As these are likely
to be key determinants of virulence and may also be useful
diagnostic and therapeutic targets, we investigated the
secretome of different strains of the fungal pathogen Cryptococcus.
Virulent strains secreted predominantly hydrolytic
and proteolytic enzymes, while the least virulent strain
secreted a range of additional non-degradative proteins
including many that lacked secretion signals, some that
appear to be ‘moonlighting’, and a number that are known
to be allergenic. It appears that in Cryptococcus, the secretome
may influence virulence both through the presence of
harmful enzymes and through the absence of proteins that
alert the host defence mechanisms.
Campbell, LT, Simonin, AR, Chen, C, Ferdous, J, Padula, MP, Harry, E, Hofer, M, Campbell, IL & Carter, DA 2015, 'Cryptococcus Strains with Different Pathogenic Potentials Have Diverse Protein Secretomes', EUKARYOTIC CELL, vol. 14, no. 6, pp. 554-563.View/Download from: Publisher's site
Jarocki, VM, Padula, MP & Djordjevic, S 2015, 'Mycoplasmal surface-associated aminopeptidases are multifunctional moonlighting proteins', Inflammation and Cell Signaling, vol. 2, no. 3, pp. 1-4.View/Download from: Publisher's site
Many bacterial pathogens require adhesion to the mucosal epithelium to establish colonisation and employ numerous strategies to then avoid clearance by the host immune system. One such strategy involves expressing plasminogen receptors on the cell surface. Recently we showed that Mycoplasma hyopneumoniae is adept at capturing porcine plasminogen onto cell surface adhesins. This interaction promotes the conversion of bound plasminogen to plasmin where it plays an important role in regulating lung inflammation. Cell surface plasmin triggers a proteolytic cascade that is thought to promote dissemination of the pathogen from the initial site of colonisation. M. hyopneumoniae is a genome-reduced pathogen that has lost the genes required to synthesise amino acids and is thus reliant on the host for amino acids for growth. We have shown M. hyopneumoniae expresses a glutamyl-aminopeptidase (MHJ_0125) and a leucyl-aminopeptidase (MHJ_0461) on the extracellular surface of the cell membrane and both are perceived as playing a key role in the generation of a pool of free amino acids for growth during pathogenesis. MHJ_0461 displays a catalytic preference for leucine, phenylalanine, and methionine, whilst MHJ_0125 demonstrates a preference for glutamic acid and alanine. In addition to their catalytic functions as aminopeptidases, both enzymes bind porcine plasminogen, promoting its conversion to plasmin by tPA, and display an affinity for highly sulphated glycosaminoglycans. MHJ_0461 was also shown to bind extracellular DNA. These studies highlight the multifunctional properties of surface proteins in M. hyopneumoniae and the increasing pool of evidence that moonlighting proteins play important roles during microbial pathogenesis.
Jarocki, VM, Santos, J, Tacchi, JL, Raymond, BBA, Deutscher, AT, Jenkins, C, Padula, MP & Djordjevic, SP 2015, 'MHJ_0461 is a multifunctional leucine aminopeptidase on the surface of Mycoplasma hyopneumoniae', Open Biology, vol. 5, no. 1, pp. 1-13.View/Download from: Publisher's site
Aminopeptidases are part of the arsenal of virulence factors produced by bacterial pathogens that inactivate host immune peptides. Mycoplasma hyopneumoniae is a genome-reduced pathogen of swine that lacks the genetic repertoire to synthesize amino acids and relies on the host for availability of amino acids for growth. M. hyopneumoniae recruits plasmin(ogen) onto its cell surface via the P97 and P102 adhesins and the glutamyl aminopeptidase MHJ_0125. Plasmin plays an important role in regulating the inflammatory response in the lungs of pigs infected with M. hyopneumoniae. We show that recombinant MHJ_0461 (rMHJ_0461) functions as a leucine aminopeptidase (LAP) with broad substrate specificity for leucine, alanine, phenylalanine, methionine and arginine and that MHJ_0461 resides on the surface of M. hyopneumoniae. rMHJ_0461 also binds heparin, plasminogen and foreign DNA. Plasminogen bound to rMHJ_0461 was readily converted to plasmin in the presence of tPA. Computational modelling identified putative DNA and heparin-binding motifs on solvent-exposed sites around a large pore on the LAP hexamer. We conclude that MHJ_0461 is a LAP that moonlights as a multifunctional adhesin on the cell surface of M. hyopneumoniae.
O'Rourke, MB, Djordjevic, SP & Padula, MP 2015, 'A non-instrument-based method for the analysis of formalin-fixed paraffin-embedded human spinal cord via matrix-assisted laser desorption/ionisation imaging mass spectrometry', RAPID COMMUNICATIONS IN MASS SPECTROMETRY, vol. 29, no. 19, pp. 1836-1840.View/Download from: Publisher's site
O'Rourke, MB, Raymond, BBA, Djordjevic, SP & Padula, MP 2015, 'A versatile cost-effective method for the analysis of fresh frozen tissue sections via matrix-assisted laser desorption/ionisation imaging mass spectrometry', RAPID COMMUNICATIONS IN MASS SPECTROMETRY, vol. 29, no. 7, pp. 637-644.View/Download from: Publisher's site
Raynel, S, Padula, MP, Marks, DC & Johnson, L 2015, 'Cryopreservation alters the membrane and cytoskeletal protein profile of platelet microparticles', TRANSFUSION, vol. 55, no. 10, pp. 2422-2432.View/Download from: Publisher's site
Widjaja, M, Berry, I, Pont, E, Padula, M & Djordjevic, S 2015, 'P40 and P90 from Mpn142 are Targets of Multiple Processing Events on the Surface of Mycoplasma pneumoniae', Proteomes, vol. 3, no. 4, pp. 512-537.View/Download from: Publisher's site
Mycoplasma pneumoniae is a significant cause of community acquired pneumonia globally. Despite having a genome less than 1 Mb in size, M. pneumoniae presents a structurally sophisticated attachment organelle that (i) provides cell polarity, (ii) directs adherence to receptors presented on respiratory epithelium, and (iii) plays a major role in cell motility. The major adhesins, P1 (Mpn141) and P30 (Mpn453), are localised to the tip of the attachment organelle by the surface accessible cleavage fragments P90 and P40 derived from Mpn142. Two events play a defining role in the formation of P90 and P40; removal of a leader peptide at position 26 (23SLA↓NTY28) during secretion to the cell surface and cleavage at amino acid 455 (452GPL↓RAG457) generating P40 and P90. Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) analysis of tryptic peptides generated by digesting size-fractionated cell lysates of M. pneumoniae identified 15 cleavage fragments of Mpn142 ranging in mass from 9–84 kDa. Further evidence for the existence of cleavage fragments of Mpn142 was generated by mapping tryptic peptides to proteins recovered from size fractionated eluents from affinity columns loaded with heparin, fibronectin, fetuin, actin, plasminogen and A549 surface proteins as bait. To define the sites of cleavage in Mpn142, neo-N-termini in cell lysates of M. pneumoniae were dimethyl-labelled and characterised by LC-MS/MS. Our data suggests that Mpn142 is cleaved to generate adhesins that are auxiliary to P1 and P30.
Aili, SR, Touchard, A, Escoubas, P, Padula, MP, Orivel, J, Dejean, A & Nicholson, GM 2014, 'Diversity of peptide toxins from stinging ant venoms', Toxicon, vol. 92, pp. 166-178.View/Download from: Publisher's site
Ants (Hymenoptera: Formicidae) represent a taxonomically diverse group of arthropods comprising nearly 13,000 extant species. Sixteen ant subfamilies have individuals that possess a stinger and use their venom for purposes such as a defence against predators, competitors and microbial pathogens, for predation, as well as for social communication. They exhibit a range of activities including antimicrobial, haemolytic, cytolytic, paralytic, insecticidal and pain-producing pharmacologies. While ant venoms are known to be rich in alkaloids and hydrocarbons, ant venoms rich in peptides are becoming more common, yet remain understudied. Recent advances in mass spectrometry techniques have begun to reveal the true complexity of ant venom peptide composition. In the few venoms explored thus far, most peptide toxins appear to occur as small polycationic linear toxins, with antibacterial properties and insecticidal activity. Unlike other venomous animals, a number of ant venoms also contain a range of homodimeric and heterodimeric peptides with one or two interchain disulfide bonds possessing pore- forming, allergenic and paralytic actions. However, ant venoms seem to have only a small number of monomeric disulfide-linked peptides. The present review details the structure and pharmacology of known ant venom peptide toxins and their potential as a source of novel bioinsecticides and therapeutic agents.
Moxon, JV, Liu, D, Moran, CS, Crossman, DJ, Krishna, SM, Yonglitthipagon, P, Emeto, TI, Morris, DR, Padula, M, Mulvenna, JP, Rush, CM & Golledge, J 2014, 'Proteomic and genomic analyses suggest the association of Apolipoprotein C1 with abdominal aortic aneurysm.', PROTEOMICS - Clinical Applications, vol. 8, no. 9-10, pp. 762-772.View/Download from: Publisher's site
Abdominal aortic aneurysm (AAA) is an important cause of mortality in the elderly. Mouse models are widely used to investigate AAA pathogenesis but their suitability for biomarker discovery is unexplored.
We conducted a three-phase study. Phase 1: Aortas from angiotensin-II-infused apolipoprotein E deficient (ApoE−/−) mice with and without AAA were assessed via iTRAQ and analyzed in silico to identify potential circulating markers. Microarray data from ApoE−/− mice and human patients were analyzed in parallel. Phase 2: Putative markers were compared between datasets to shortlist common candidates. Phase 3: The relationship of two shortlisted markers and AAA presence was assessed.
iTRAQ identified eight proteins with biomarker potential. Microarray data identified 72 and 96 potential biomarkers from ApoE−/− mice and human patients, respectively. All three datasets suggested apolipoprotein C1 (ApoC1) as a marker for AAA; microarray data identified matrix metalloproteinase 9 (MMP9) as a second potential marker. Plasma ApoC1 and MMP9 concentrations positively correlated with AAA diameter in ApoE−/− mice.
Padula, MP, Wright, EP, Partridge, MA, Gauci, VJ, Malladi, CS & Coorssen, JR 2014, 'Top-down proteomics: Enhancing 2D gel-electrophoresis from tissue processing to high sensitivity protein detection', Proteomics, vol. 14, no. 7-8, pp. 872-889.View/Download from: Publisher's site
Pendarvis, K, Padula, MP, Tacchi, JL, Petersen, AC, Djordjevic, SP, Burgess, SC & Minion, F 2014, 'Proteogenomic mapping of Mycoplasma hyopneumoniae virulent strain 232', BMC Genomics, vol. 15, no. 1, pp. 1-8.View/Download from: Publisher's site
Pokharel, D, Padula, MP, Lu, JF, Tacchi, JL, Luk, F, Djordjevic, SP & Bebawy, M 2014, 'Proteome analysis of multidrug-resistant, breast cancer-derived microparticles.', Journal of Extracellular Vesicles, vol. 3, no. 1, pp. 1-10.View/Download from: Publisher's site
Cancer multidrug resistance (MDR) occurs when cancer cells evade the cytotoxic actions of chemotherapeutics through the active efflux of drugs from within the cells. Our group have previously demonstrated that multidrug-resistant breast cancer cells spontaneously shed microparticles (MPs) and that these MPs can transfer resistance to drug-responsive cells and confer MDR on those cells in as little as 4 h. Furthermore, we also showed that, unlike MPs derived from leukaemia cells, breast cancer-derived MPs display a tissue selectivity in the transfer of P-glycoprotein (P-gp), transferring the resistance protein only to malignant breast cells. This study aims to define the proteome of breast cancer-derived MPs in order to understand the differences in protein profiles between those shed from drug-resistant versus drug-sensitive breast cancer cells. In doing so, we detail the protein cargo required for the intercellular transfer of MDR to drug-sensitive recipient cells and the factors governing the transfer selectivity to malignant breast cells. We describe the first proteomic analysis of MPs derived from human breast cancer cells using SDS PAGE and liquid chromatography-tandem mass spectrometry (LC/MS/MS), in which we identify 120 unique proteins found only in drug-resistant, breast cancer-derived MPs. Our results demonstrate that the MP-mediated transfer of P-gp to recipient cells occurs alongside CD44; the Ezrin, Radixin and Moesin protein family (ERM); and cytoskeleton motor proteins within the MP cargo.
Tacchi, JL, Raymond, BB, Jarocki, VM, Berry, IJ, Padula, M & Djordjevic, SP 2014, 'Cilium Adhesin P216 (MHJ_0493) Is a Target of Ectodomain Shedding and Aminopeptidase Activity on the Surface of Mycoplasma hyopneumoniae', Journal of Proteome Research, vol. Epub.View/Download from: Publisher's site
MHJ_0493 (P216) is a highly expressed cilium adhesin in Mycoplasma hyopneumoniae. P216 undergoes cleavage at position 1074 in the S/T-X-F?-X-D/E-like motif 1072TN-F?Q-E1076 generating N-terminal and C-terminal fragments of 120 kDa (P120) and 85 kDa (P85) on the surface of M. hyopneumoniae. Here we show that several S/T-X-F?X-D/E-like motifs exist in P216 but only 1072TN-F?Q-E1076 and 1344I-T-F?A-D-Y1349 were determined to be bona fide processing sites by identifying semitryptic peptides consistent with cleavage at the phenylalanine residue. The location of S/T-X-F?-X-D/E-like motifs within or abutting regions of protein disorder greater than 40 consecutive amino acids is consistent with our hypothesis that site access influences the cleavage efficiency. Approximately 20 cleavage fragments of P216 were identified on the surface of M. hyopneumoniae by LCMS/MS analysis of biotinylated proteins and 2D SDS-PAGE. LCMS/MS analysis of semitryptic peptides within P216 identified novel cleavage sites. Moreover, detection of a series of overlapping semitryptic peptides that differed by the loss a single amino acid at their N-terminus is consistent with aminopeptidase activity on the surface of M. hyopneumoniae. P120 and P85 and their cleavage fragments bind heparin and cell-surface proteins derived from porcine epithelial-like cells, indicating that P216 cleavage fragments retain the ability to bind glycosaminoglycans.
Wright, E, Prasad, KAG, Padula, M & Coorsen, JR 2014, 'Deep Imaging: How Much of the Proteome Does Current Top-Down Technology Already Resolve?', PLoS One, vol. 9, no. 1, pp. e86058-e86058.View/Download from: Publisher's site
The main obstacle to effective and comprehensive proteome
analysis has ostensibly been resolution. A variety of methods have
been investigated in order to resolve ever smaller quantities of
protein and detect them quantitatively [1–4]. One of the original
and most powerful methods has been two-dimensional gel
electrophoresis (2DE) . Not only does this yield a position in a
gel indicating isoelectric point (pI) and molecular weight, it does so
with high reproducibility, and also resolves protein variants
including isoforms and post-translationally modified forms (i.e.
protein species ). While much dogma was associated with this
method for a number of years, many of the suggested resolution
issues have been addressed, enabling the full spectrum of proteins to
be resolved by a refined, standardized protocol for sample
preparation and 2DE [7–8]. This was largely achieved through
the introduction of commercial immobilized pH gradient (IPG)
strips [9–11], fine tuning of buffer, reducing and detergent
components, and the use of fractionation to improve proteome
Wright, EP, Padula, MP, Higgins, VJ, Aldrich-Wright, JR & Coorssen, JR 2014, 'A Systems Biology Approach to Understanding the Mechanisms of Action of an Alternative Anticancer Compound in Comparison to Cisplatin', PROTEOMES, vol. 2, no. 4.View/Download from: Publisher's site
Robinson, MW, Buchtmann, KA, Jenkins, C, Tacchi, JL, Raymond, BBA, To, J, Chowdhury, PR, Woolley, LK, Labbate, M, Turnbull, L, Whitchurch, CB, Padula, MP & Djordjevic, SP 2013, 'MHJ_0125 is an M42 glutamyl aminopeptidase that moonlights as a multifunctional adhesin on the surface of Mycoplasma hyopneumoniae', OPEN BIOLOGY, vol. 3.View/Download from: Publisher's site
Gel electrophoresis, particularly one- (1DE) and two-dimensional electrophoresis (2DE), remain among the most widely used top-down methods for resolving and analysing proteomes. Detection of the resulting protein maps relies on staining (i.e. colloidal c
Green, DW, Padula, M, Santos, J, Chou, J, Milthorpe, BK & Ben-Nissan, B 2013, 'A Therapeutic Potential for Marine Skeletal Proteins in Bone Regeneration.', Marine Drugs, vol. 11, no. 4, pp. 1203-1220.View/Download from: Publisher's site
A vital ingredient for engineering bone tissue, in the culture dish, is the use of recombinant matrix and growth proteins to help accelerate the growth of cultivated tissues into clinically acceptable quantities. The skeletal organic matrices of calcifying marine invertebrates are an untouched potential source of such growth inducing proteins. They have the advantage of being ready-made and retain the native state of the original protein. Striking evidence shows that skeleton building bone morphogenic protein-2/4 (BMP) and transforming growth factor beta (TGF-ß) exist within various marine invertebrates such as, corals. Best practice mariculture and the latest innovations in long-term marine invertebrate cell cultivation can be implemented to ensure that these proteins are produced sustainably and supplied continuously. This also guarantees that coral reef habitats are not damaged during the collection of specimens. Potential proteins for bone repair, either extracted from the skeleton or derived from cultivated tissues, can be identified, evaluated and retrieved using chromatography, cell assays and proteomic methods. Due to the current evidence for bone matrix protein analogues in marine invertebrates, together with the methods established for their production and retrieval there is a genuine prospect that they can be used to regenerate living bone for potential clinical use.
Raymond, B, Tacchi, JL, Jarocki, VM, Minion, F, Padula, M & Djordjevic, SP 2013, 'P159 from Mycoplasma hyopneumoniae binds porcine cilia and heparin and is cleaved in a manner akin to ectodomain shedding', Journal of Proteome Research, vol. 12, no. 12, pp. 5891-5903.View/Download from: Publisher's site
Mycoplasma hyopneumoniae colonizes the ciliated epithelial lining of the upper respiratory tract of swine and results in chronic infection. Previously, we have observed that members of P97 and P102 paralog families of cilium adhesins undergo endoproteolytic processing on the surface of M. hyopneumoniae. We show that P159 (MHJ_0494), an epithelial cell adhesin unrelated to P97 and P102 paralog families, is a cilium adhesin that undergoes dominant cleavage events at S/T-X-F?X-D/E-like motifs located at positions 233F?Q234 and 981F?Q982, generating P27, P110, and P52. An unrelated cleavage site 738L-K-V?G-A-A743 in P110 shows sequence identity with a cleavage site (L-N-V?A-V-S) identified in the P97 paralog, Mhp385, and generates 76 (P76) and 35 kDa (P35) fragments. LCMS/MS analysis of biotinylated surface proteins identified six peptides with a biotin moiety on their N-terminus indicating novel, low abundance neo-N-termini. LCMS/MS of proteins separated by 2D-PAGE, 2D immunoblotting using monospecific antiserum raised against recombinant fragments spanning P159 (F1P159-F4P159), and proteins that bound to heparin-agarose were all used to map P159 cleavage fragments. P159 is the first cilium adhesin not belonging to the P97/P102 paralog families and is extensively processed in a manner akin to ectodomain shedding in eukaryotes.
Bogema, DR, Deutscher, AT, Woolley, LK, Seymour, LM, Raymond, BB, Tacchi, JL, Padula, M, Dixon, NE, Minion, F, Jenkins, C, Walker, MJ & Djordjevic, SP 2012, 'Characterization of cleavage events in the multifunctional cilium adhesin Mhp684 (P146) reveals a mechanism by which Mycoplasma hyopneumoniae regulates surface topography', mBio, vol. 3, no. 2, pp. 1-11.View/Download from: Publisher's site
Mycoplasma hyopneumoniae causes enormous economic losses to swine production worldwide by colonizing the ciliated epithelium in the porcine respiratory tract, resulting in widespread damage to the mucociliary escalator, prolonged inflammation, reduced weight gain, and secondary infections. Protein Mhp684 (P146) comprises 1,317 amino acids, and while the N-terminal 400 residues display significant sequence identity to the archetype cilium adhesin P97, the remainder of the molecule is novel and displays unusual motifs. Proteome analysis shows that P146 preprotein is endogenously cleaved into three major fragments identified here as P50(P146), P40(P146), and P85(P146) that reside on the cell surface. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) identified a semitryptic peptide that delineated a major cleavage site in Mhp684. Cleavage occurred at the phenylalanine residue within sequence (672)ATEF down arrow QQ(677), consistent with a cleavage motif resembling S/T-X-F down arrow X-D/E recently identified in Mhp683 and other P97/P102 family members. Biotinylated surface proteins recovered by avidin chromatography and separated by two-dimensional gel electrophoresis (2-D GE) showed that more-extensive endoproteolytic cleavage of P146 occurs. Recombinant fragments F1(P146)-F3(P146) that mimic P50(P146), P40(P146), and P85(P146) were constructed and shown to bind porcine epithelial cilia and biotinylated heparin with physiologically relevant affinity. Recombinant versions of F3(P146) generated from M. hyopneumoniae strain J and 232 sequences strongly bind porcine plasminogen, and the removal of their respective C-terminal lysine and arginine residues significantly reduces this interaction. These data reveal that P146 is an extensively processed, multifunctional adhesin of M. hyopneumoniae. Extensive cleavage coupled with variable cleavage efficiency provides a mechanism by which M. hyopneumoniae regulates protein topography.
Chong, H, Campbell, L, Padula, M, Hill, C, Harry, L, Li, SS, Wilkins, MR, Herbert, BR & Carter, DA 2012, 'Time-Course Proteome Analysis Reveals the Dynamic Response of Cryptococcus gattii Cells to Fluconazole', PLoS One, vol. 7, no. 8, pp. 1-10.View/Download from: Publisher's site
Cryptococcus gattii is an encapsulated fungus capable of causing fatal disease in immunocompetent humans and animals. As current antifungal therapies are few and limited in efficacy, and resistance is an emerging issue, the development of new treatment strategies is urgently required. The current study undertook a time-course analysis of the proteome of C. gattii during treatment with fluconazole (FLC), which is used widely in prophylactic and maintenance therapies. The aims were to analyze the overall cellular response to FLC, and to find fungal proteins involved in this response that might be useful targets in therapies that augment the antifungal activity of FLC. During FLC treatment, an increase in stress response, ATP synthesis and mitochondrial respiratory chain proteins, and a decrease in most ribosomal proteins was observed, suggesting that ATP-dependent efflux pumps had been initiated for survival and that the maintenance of ribosome synthesis was differentially expressed. Two proteins involved in fungal specific pathways were responsive to FLC. An integrative network analysis revealed co-ordinated processes involved in drug response, and highlighted hubs in the network representing essential proteins that are required for cell viability. This work demonstrates the dynamic cellular response of a typical susceptible isolate of C. gattii to FLC, and identified a number of proteins and pathways that could be targeted to augment the activity of FLC
Couttas, TA, Raffery, MJ, Padula, M, Herbert, BR & Wilkins, MR 2012, 'Methylation of translation-associated proteins in Saccharomyces cerevisiae: Identification of methylated lysines and their methyltransferases', Proteomics, vol. 12, no. 7, pp. 960-972.View/Download from: Publisher's site
This study aimed to identify sites of lysine methylation in Saccharomyces cerevisiae and the associated methyltransferases. Hexapeptide ligand affinity chromatography was used to normalize the abundance levels of proteins in whole cell lysate. MS/MS, in association with antibody-based detection, was then used to identify lysine methylated proteins and the precise sites of modification. Lysine methylation was found on the proteins elongation factor (EF) 1-alpha, 2, and 3A, as well as ribosomal proteins 40S S18-A/B, 60S L11-A/B, L18-A/B, and L42-A/B. Precise sites were mapped in all cases. Single-gene knockouts of known and putative methyltransferase(s), in association with MS/MS, showed that EF1-alpha is monomethylated by Efm1 at lysin 30 and dimethylated by See1 at lysine 316. Methyltransferase Rkm1 was found to monomethylate 40S ribosomal protein S18-A/B at lysine 48. Knockout analysis also revealed that putative methyltransferase YBR271W affects the methylation of proteins EF2 and 3A; this was detected by Western blotting and immunodetection. This methyltransferase shows strong interspecies conservation and a tryptophan-containing motif associated with its active site. We suggest that enzyme YBR271W is named EF methyltransferase 2 (Efm2), in line with the recent naming of YHL039W as Efm1
Deutscher, AT, Tacchi, JL, Minion, F, Padula, M, Crossett, B, Bogema, DR, Jenkins, C, Kuit, TA, Walker, MJ & Djordjevic, SP 2012, 'Mycoplasma hyopneumoniae surface proteins Mhp385 and Mhp384 bind host cilia and glycosaminoglycans and are endoproteolytically processed by proteases that recognize different cleavage motifs', Journal of Proteome Research, vol. 11, no. 3, pp. 1924-1936.View/Download from: Publisher's site
P97 and P102 paralogues occur as endoproteolytic cleavage fragments on the surface of Mycoplasma hyopneumoniae that bind glycosaminoglycans, plasminogen, and fibronectin and perform essential roles in colonization of ciliated epithelia. We show that the P102 paralogue Mhp384 is efficiently cleaved at an S/T-X-F down arrow X-D/E-like site, creating P60(384) and P50(384). The P97 paralogue Mhp385 is inefficiently cleaved, with tryptic peptides from a 115 kDa protein (P115(385)) and 88 kDa (P88(385)) and 27 kDa (P27(385)) cleavage fragments identified by LC-MS/MS. This is the first time a preprotein belonging to the P97 and P102 paralogue families has been identified by mass spectrometry. The semitryptic peptide (752)IQFELEPISLNV(763) denotes the C-terminus of P88(385) and defines the novel cleavage site L-761-N-V down arrow A-V-S-766 in Mhp385. P115(385), P88(385), P27(385), P60(384), and P50(384) were shown to reside extracellularly, though it is unknown how the fragments remain attached to the cell surface. Heparin- and cilium-binding sites were identified within P60(384), P50(384), and P88(385). No primary function was attributed to P27(385); however, this molecule contains four tandem R1 repeats with similarity to porcine collagen type VI (alpha 3 chain). P97 and P102 paralogue families are adhesins targeted by several proteases with different cleavage efficiencies, and this process generates combinatorial complexity on the surface of M. hyopneumoniae.
Packer, JM, Irish, JL, Herbert, BR, Hill, C, Padula, M, Blair, S, Carter, D & Harry, L 2012, 'Specific Non-Peroxide Antibacterial Effect Of Manuka Honey On The Staphylococcus Aureus Proteome', International Journal Of Antimicrobial Agents, vol. 40, no. 1, pp. 43-50.View/Download from: Publisher's site
Manuka honey, derived from the New Zealand flowering plant Leptospermum scoparium, shows promise as a topical antibacterial agent and effective chronic wound dressing. The aim of this study was to determine the non-peroxide antibacterial effects of this
Seymour, LM, Jenkins, C, Deutscher, AT, Raymond, BB, Padula, M, Tacchi, JL, Bogema, DR, Eamens, GJ, Woolley, LK, Dixon, NE, Walker, MJ & Djordjevic, SP 2012, 'Mhp182 (P102) binds fibronectin and contributes to the recruitment of plasmin(ogen) to the Mycoplasma hyopneumoniae cell surface', Cellular Microbiology, vol. 14, no. 1, pp. 81-94.View/Download from: Publisher's site
Mycoplasma hyopneumoniae is a major, economically damaging respiratory pathogen. Although M. hyopneumoniae cells bind plasminogen, the identification of plasminogen-binding surface proteins and the biological ramifications of acquiring plasminogen requires further investigation. mhp182 encodes a highly expressed 102 kDa protein (P102) that undergoes proteolytic processing to generate surface-located N-terminal 60 kDa (P60) and C-terminal 42 kDa (P42) proteins of unknown function. We show that recombinant P102 (rP102) binds plasminogen at physiologically relevant concentrations (KD similar to 76 nM) increasing the susceptibility of plasmin(ogen) to activation by tissue-specific plasminogen activator (tPA). Recombinant proteins constructed to mimic P60 (rP60) and P42 (rP42) also bound plasminogen at physiologically significant levels. M. hyopneumoniae surface-bound plasminogen was activated by tPA and is able to degrade fibrinogen, demonstrating the biological functionality of M. hyopneumoniae-bound plasmin(ogen) upon activation. Plasmin(ogen) was readily detected in porcine ciliated airways and plasmin levels were consistently higher in bronchoalveolar lavage fluid from M. hyopneumoniae-infected animals. Additionally, rP102 and rP42 bind fibronectin with KDs of 26 and 33 nM respectively and recombinant P102 proteins promote adherence to porcine kidney epithelial-like cells. The multifunctional binding ability of P102 and activation of M. hyopneumoniae-sequestered plasmin(ogen) by an exogenous activator suggests P102 plays an important role in virulence.
Bogema, DR, Scott, NE, Padula, M, Tacchi, JL, Raymond, B, Jenkins, C, Cordwell, SJ, Minion, F, Walker, MJ & Djordjevic, SP 2011, 'Sequence TTKF | QE defines the site of proteolytic cleavage in Mhp683, a novel glycosaminoglycan and cilium adhesin of Mycoplasma hyopneumoniae', Journal Of Biological Chemistry, vol. 286, no. 48, pp. 41217-41229.View/Download from: Publisher's site
Mycoplasma hyopneumoniae colonizes the ciliated respiratory epithelium of swine, disrupting mucociliary function and inducing chronic inflammation. P97 and P102 family members are major surface proteins of M. hyopneumoniae and play key roles in colonizing cilia via interactions with glycosaminoglycans and mucin. The p102 paralog, mhp683, and homologs in strains from different geographic origins encode a 135-kDa pre-protein (P135) that is cleaved into three fragments identified here as P45683, P48683, and P50683. A peptide sequence (TTKF?QE) was identified surrounding both cleavage sites in Mhp683. N-terminal sequences of P48683 and P50683, determined by Edman degradation and mass spectrometry, confirmed cleavage after the phenylalanine residue. A similar proteolytic cleavage site was identified by mass spectrometry in another paralog of the P97/P102 family. Trypsin digestion and surface biotinylation studies showed that P45683, P48683, and P50683 reside on the M. hyopneumoniae cell surface. Binding assays of recombinant proteins F1683F5683, spanning Mhp683, showed saturable and dose-dependent binding to biotinylated heparin that was inhibited by unlabeled heparin, fucoidan, and mucin. F1683F5683 also bound porcine epithelial cilia, and antisera to F2683 and F5683 significantly inhibited cilium binding by M. hyopneumoniae cells. These data suggest that P45683, P48683, and P50683 each display cilium- and proteoglycan-binding sites. Mhp683 is the first characterized glycosaminoglycan-binding member of the P102 family.
Moxon, J, Padula, M, Clancy, P, Emeto, T, Herbert, BR, Norman, P & Golledge, J 2011, 'Proteomic Analysis Of Intra-arterial Thrombus Secretions Reveals A Negative Association Of Clusterin And Thrombospondin-1 With Abdominal Aortic Aneurysm', Atherosclerosis, vol. 219, no. 2, pp. 432-439.View/Download from: Publisher's site
Objective: Abdominal aortic aneurysm (AAA) is usually accompanied by the formation of a large volume of intra-luminal thrombus (ILT). ILT-derived proteins have been suggested as circulating markers for AAA. We conducted a proteomic study screening whole
Robinson, MW, Corvo, I, Jones, PM, George, AM, Padula, M, To, J, Cancela, M, Rinaldi, G, Tort, JF, Roche, L & Dalton, JP 2011, 'Collagenolytic Activities of the Major Secreted Cathepsin L Peptidases Involved in the Virulence of the Helminth Pathogen, Fasciola hepatica', Plos Neglected Tropical Diseases, vol. 5, no. 4, pp. 1-13.View/Download from: Publisher's site
Background: The temporal expression and secretion of distinct members of a family of virulence-associated cathepsin L cysteine peptidases (FhCL) correlates with the entry and migration of the helminth pathogen Fasciola hepatica in the host. Thus, infective larvae traversing the gut wall secrete cathepsin L3 (FhCL3), liver migrating juvenile parasites secrete both FhCL1 and FhCL2 while the mature bile duct parasites, which are obligate blood feeders, secrete predominantly FhCL1 but also FhCL2. Methodology/Principal Findings: Here we show that FhCL1, FhCL2 and FhCL3 exhibit differences in their kinetic parameters towards a range of peptide substrates. Uniquely, FhCL2 and FhCL3 readily cleave substrates with Pro in the P2 position and peptide substrates mimicking the repeating Gly-Pro-Xaa motifs that occur within the primary sequence of collagen. FhCL1, FhCL2 and FhCL3 hydrolysed native type I and II collagen at neutral pH but while FhCL1 cleaved only non-collagenous (NC, non-Gly-X-Y) domains FhCL2 and FhCL3 exhibited collagenase activity by cleaving at multiple sites within the alpha 1 and alpha 2 triple helix regions (Col domains). Molecular simulations created for FhCL1, FhCL2 and FhCL3 complexed to various seven-residue peptides supports the idea that Trp67 and Tyr67 in the S2 subsite of the active sites of FhCL3 and FhCL2, respectively, are critical to conferring the unique collagenase-like activity to these enzymes by accommodating either Gly or Pro residues at P2 in the substrate. The data also suggests that FhCL3 accommodates hydroxyproline (Hyp)-Gly at P3-P2 better than FhCL2 explaining the observed greater ability of FhCL3 to digest type I and II collagens compared to FhCL2 and why these enzymes cleave at different positions within the Col domains. Conclusions/Significance: These studies further our understanding of how this helminth parasite regulates peptidase expression to ensure infection, migration and establishment in host tissues.
Seymour, LM, Falconer, L, Deutscher, A, Minion, F, Padula, M, Dixon, NE, Djordjevic, SP & Walker, M 2011, 'Mhp 107 is a member of the multifunctional adhesin family of Mycoplasma hyopneumoniae', Journal Of Biological Chemistry, vol. 286, no. 12, pp. 10097-10104.View/Download from: Publisher's site
Mycoplasma hyopneumoniae is the causative pathogen of porcine enzootic pneumonia, an economically significant disease that disrupts the mucociliary escalator in the swine respiratory tract. Expression of Mhp107, a P97 paralog encoded by the gene mhp107, was confirmed using ESI-MS/MS. To investigate the function of Mhp107, three recombinant proteins, F1Mhp107, F2Mhp107, and F3Mhp107, spanning the N-terminal, central, and C-terminal regions of Mhp107 were constructed. Colonization of swine by M. hyopneumoniae requires adherence of the bacterium to ciliated cells of the respiratory tract. Recent studies have identified a number of M. hyopneumoniae adhesins that bind heparin, fibronectin, and plasminogen. F1Mhp107 was found to bind porcine heparin (KD ~90 nm) in a dose-dependent and saturable manner, whereas F3Mhp107 bound fibronectin (KD ~180 nm) at physiologically relevant concentrations. F1Mhp107 also bound porcine plasminogen (KD = 24 nm) in a dose-dependent and physiologically relevant manner. Microspheres coated with F3Mhp107 mediate adherence to porcine kidney epithelial-like (PK15) cells, and all three recombinant proteins (F1Mhp107-F3Mhp107) bound swine respiratory cilia. Together, these findings indicate that Mhp107 is a member of the multifunctional M. hyopneumoniae adhesin family of surface proteins and contributes to both adherence to the host and pathogenesis.
Deutscher, A, Jenkins, C, Minion, F, Seymour, LM, Padula, M, Dixon, N, Walker, MJ & Djordjevic, SP 2010, 'Repeat regions R1 and R2 in the P97 paralogue Mhp271 of Mycoplasma hyopneumoniae nind heparin fibronectin and procine cilia', Molecular Microbiology, vol. 78, no. 2, pp. 444-458.View/Download from: Publisher's site
Mycoplasma hyopneumoniae, the causative agent of porcine enzootic pneumonia, adheres to ciliated respiratory epithelia resulting in ciliostasis and epithelial cell death. The cilium adhesin P97 (Mhp183) contains two repeat regions, designated R1 and R2, that play key roles in adherence. Eight pentapeptide repeats in R1 are sufficient to bind porcine cilia; however, both R1 and R2 are needed to bind heparin. Mhp271, a paralogue of P97, is the only other M. hyopneumoniae protein to contain both R1 and R2 repeats. These repeats are arranged as a set of three pentapeptide repeats (designated R1A271), two decapeptide repeats (designated R2271), and a second set of six pentapeptide repeats (designated R1B271). To determine their function, recombinant proteins containing R1A271 (F1271) and R2271-R1B271 (F2271) were constructed and used in in vitro binding assays. F2271, but not F1271, bound heparin (KD = 8.1 ± 0.4 nM), fibronectin (KD = 174 ± 13 nM) and porcine cilia. Pre-incubation of F2271 with 100 µM heparin blocked cilium binding by ?69%. Cell surface shaving with trypsin combined with two-dimensional liquid chromatography coupled to tandem mass spectrometry analysis identified Mhp271 as surface-exposed. Our data suggest that both R1 and R2 in Mhp271 are involved in binding to host molecules
Jobbins, S, Hill, C, D'Souza-Basseal, J, Padula, M, Herbert, BR & Krockenberger, M 2010, 'Immunoproteomic Approach To Elucidating The Pathogenesis Of Cryptococcosis Caused By Cryptococcus Gattii', Journal Of Proteome Research, vol. 9, no. 8, pp. 3832-3841.View/Download from: Publisher's site
Cryptococcosis caused by Cryptococcus gattii is a devastating disease of immunocompetent hosts with an incompletely understood pathogenesis. Utilizing an immunoproteomic approach in a naturally occurring koala model of disease, a number of key proteins a
Seymour, LM, Deutscher, A, Jenkins, C, Kuit, TA, Falconer, L, Minion, F, Crossett, B, Padula, M, Dixon, N, Djordjevic, SP & Walker, M 2010, 'A Processed Multidomain Mycoplasma hyopneumoniae Adhesin Binds Fibronectin, Plasminogen, and Swine Respiratory Cilia', Journal Of Biological Chemistry, vol. 285, no. 44, pp. 33971-33978.View/Download from: Publisher's site
orcine enzootic pneumonia is a chronic respiratory disease that affects swine. The etiological agent of the disease, Mycoplasma hyopneumoniae, is a bacterium that adheres to cilia of the swine respiratory tract, resulting in loss of cilia and epithelial cell damage. A M. hyopneumoniae protein P116, encoded by mhp108, was investigated as a potential adhesin. Examination of P116 expression using proteomic analyses observed P116 as a full-length protein and also as fragments, ranging from 17 to 70 kDa in size. A variety of pathogenic bacterial species have been shown to bind the extracellular matrix component fibronectin as an adherence mechanism. M. hyopneumoniae cells were found to bind fibronectin in a dose-dependent and saturable manner. Surface plasmon resonance was used to show that a recombinant C-terminal domain of P116 bound fibronectin at physiologically relevant concentrations (KD 24 ± 6 nm). Plasmin(ogen)-binding proteins are also expressed by many bacterial pathogens, facilitating extracellular matrix degradation. M. hyopneumoniae cells were found to also bind plasminogen in a dose-dependent and saturable manner; the C-terminal domain of P116 binds to plasminogen (KD 44 ± 5 nm). Plasminogen binding was abolished when the C-terminal lysine of P116 was deleted, implicating this residue as part of the plasminogen binding site. P116 fragments adhere to the PK15 porcine kidney epithelial-like cell line and swine respiratory cilia. Collectively these data suggest that P116 is an important adhesin and virulence factor of M. hyopneumoniae
Szczepanek, SM, Frasca Jr, S, Schumacher, VL, Padula, M, Djordjevic, SP & Geary, SJ 2010, 'Identification of lipoprotein MslA as a neoteric virulence factor of Mycoplasma gallisepticum', Infection And Immunity, vol. 78, no. 8, pp. 3475-3483.View/Download from: Publisher's site
Many lipoproteins are expressed on the surfaces of mycoplasmas, and some have been implicated as playing roles in pathogenesis. Family 2 lipoproteins of Mycoplasma plleumoniae have a conserved "mycoplasma liM poprotein X" central domain and a "mycoplasma lipoprotein 10" CMterminal domain and are differentially expressed in response to environmental conditions. Homologues of family 2 lipoproteins are Mycoplasma specific and include the lipoprotein of Mycoplasma gaIlisepticum, encoded by the MGA0674 gene. Comparative transcriptomic analysis of the M. gaUisepticum live attenuated vaccine strain F and the virulent strain Rio"",, reported in this study, indicated that MGA0674 is one of several differentially expressed genes. The MGA0674M encoded lipoprotein is a proteolytically processed, immunogenic, TXM114 detergentMphase protein which apM pears to have antigenic divergence between field strains R10w and S6. We examined the virulence of an R10w AMGA0674 mutant (PIH9) in vivo and observed reduced recovery and attenuated virulence in the tracheas of experimentally infected chickens. The virulence of two additional R10w AMGA0674 mutants, 2162 and 2204, was assessed in a second ill vivo virulence experiment. These mutants exhibited partial to complete attenuation in vivo, but recovery was observed more frequently. Since only Mycoplasma species harbor homologues of MGA0674, the gene product has been renamed "AlycoplasmaMspecific lipoprotein A" (MslA). Collectively, these data indicate that MslA is an immunogenic lipoprotein exhibiting reduced expression in an attenuated strain and plays a role in M. gallisepticum virulence.
Huang, K, Filarsky, M, Padula, M, Raftery, MJ, Herbert, BR & Wilkins, MR 2009, 'Micropreparative fractionation of the complexome by blue native continuous elution electrophoresis.', Proteomics, vol. 9, no. 9, pp. 2494-2502.View/Download from: Publisher's site
The large-scale analysis of protein complexes is an emerging challenge in the field of proteomics. Currently, there are few methods available for the fractionation of protein complexes that are compatible with downstream proteomic techniques. Here, we describe the technique of blue native continuous elution electrophoresis (BN-CEE). It combines the features of blue native PAGE (BN-PAGE) and continuous elution electrophoresis (CEE), generating liquid-phase fractions of protein complexes of up to 800 kDa. The resulting complexes can be further analysed by BN-PAGE, by SDS-PAGE and/or by MS. This can help define the constituent proteins of many complexes and their stoichiometry. As BN-CEE is also micropreparative, with a capacity to separate milligram quantities of protein complexes, it will assist the study of proteins of lower abundance. In this regard, the acrylamide concentration and elution rate during separation can be controlled to help `zoom in on particular high mass regions and thus complexes of interest. We illustrate the utility of the technique in the analysis of Saccharomyces cerevisiae cellular lysate.
Padula, M 2009, 'Challenges, current status and future perspectives of proteomics in improving understanding, diagnosis and treatment of vascular disease', European Journal of Vascular and Endovascular Surgery, vol. 38, pp. 346-355.View/Download from: Publisher's site
Herbert, BR, Hill, C, Padula, MP, DSouza-Bassea, JM, Krockenberger, MB, Malik, R, Ngamskulrungroj, P, Chong, HS, Meyer, W, Harry, EJ & Carter, DA 2008, 'Fungal Lung Infection : Understanding Cryptococcus Gattii Infection and the Challenges of Mixed Proteomes', Journal of Proteomics & Bioinformatics, vol. S2, no. 01, pp. 097-098.View/Download from: Publisher's site
Padula, M, Herbert, BR & Broady, KW 2008, 'Identification of Proteins, Enzymes and Potential Vaccine Candidates from the Aust Paralysis Tick, Ixodes Holocyclus Using 2D-PAGE and Equalizer Technology', Journal of Proteomics & Bioinformatics, vol. S2, no. 01, pp. 208-209.View/Download from: Publisher's site
Robinson, MW, Tort, JF, Lowther, J, Donnelly, SM, Wong, E, Xu, W, Stack, CM, Padula, M, Herbert, BR & Dalton, JP 2008, 'Proteomics and phylogenetic analysis of the cathepsin L protease family of the helminth pathogen Fasciola hepatica', Molecular & Cellular Proteomics, vol. 7, no. 6, pp. 1111-1123.View/Download from: Publisher's site
Cathepsin L proteases secreted by the helminth pathogen Fasciola hepatica have functions in parasite virulence including tissue invasion and suppression of host immune responses. Using proteomics methods alongside phylogenetic studies we characterized th
Smith, CC, O'Rourke, MB, Padula, M, De La Monte, SM, Sheedy, DL, Kril, JJ & Sutherland, GT 2018, 'ACCURATE MASS MALDI-TOF/ TOF LIPID IMAGING OF HUMAN BRAIN TISSUE', ALCOHOLISM-CLINICAL AND EXPERIMENTAL RESEARCH, 19th World Congress of International-Society-for-Biomedical-Research-on-Alcoholism (ISBRA), WILEY, Kyoto, JAPAN, pp. 38A-38A.
Rodgers, K, Steele, J & Padula, M 2017, 'A novel approach to detect the presence of levodopa (lDOPA) in the polypeptide chains of proteins', JOURNAL OF NEUROCHEMISTRY, Biennial Meeting of the International-Society-for-Neurochemistry and European-Society-for-Neurochemistry (ISN-ESN), WILEY, Paris, FRANCE, pp. 164-164.
Moeke, CH, Padula, MP, Beck, HC, Hansen, ML, Rasmussen, LM & Davies, MJ 2016, 'Detection of Pyruvate Kinase Isoform M2 (PKM2) in Arterial Wall Cells, Intact Arteries and Human Atherosclerotic Lesions', FREE RADICAL BIOLOGY AND MEDICINE, ELSEVIER SCIENCE INC, pp. S139-S139.View/Download from: Publisher's site
Wood, BW, Padula, MP, Marks, DC & Johnson, L 2016, 'Characterization and Comparison of the Surface Phenotype of Refrigerated and Cryopreserved Platelets', Transfusion, AABB Annual Meeting, Wiley: 12 months, pp. 37A-38A.
Berry, IJ, Tacchi, JL, Jarocki, VM, Raymond, BBA, Padula, MP & Djordjevic, SP 2014, 'The Significance of Post-Translational Proteolysis in the Model Pathogen, Mycoplasma hyopneumoniae.', 2nd “Proteomics & Beyond” Symposium, Proteomics & Beyond Symposium, Australian Proteomics Analysis Facility.
Proteolytic cleavage is one of the most ubiquitous post-translational modifications to proteins, responsible for protein signalling, activation, localisation and ultimately degradation. Due to a variety of experimental limitations this important physiological process has been largely understudied, particularly in prokaryotes and archaea. In order determine the scale and functional roles of protein processing in bacteria and to explore the underlying mechanisms in the production of mature proteins, a high-throughput systems wide approach is needed..
The primary focus of this project is to sequence the N-terminal sequences of mature protein products of prokaryotic pathogens. Using this data, we can identify true protein start sites and any downstream post-translational processing. The genome-reduced agriculturally-important pathogen Mycoplasma hyopneumoniae, was selected as a model organism for these studies due to the large body of literature demonstrating the extensive proteolytic processing of many highly expressed surface proteins of this organism, which are critical for pathogenesis.
Using a high-throughput methodology called Terminal Amine Isotopic Labelling of Substrates (TAILS), the data obtained from the analysis of M. hyopneumoniae has provided evidence confirming true start sites of protein translation and complementary data pinpointing the precise sites of proteolytic cleavage. Our data indicates that an unprecedented number of cell surface proteins are targets for posttranslational processing.
Johnson, L, Raynel, S, Padula, MP & Marks, DC 2014, 'Phenotypic Characterization of Platelet Microparticles Formed by Cryopreservation', TRANSFUSION, Annual Meeting of the American-Association-of-Blood-Banks (AABB), WILEY-BLACKWELL, Philadelphia, PA, pp. 86A-87A.
Green, D, Padula, M, Santos, J, Chou, J, Milthorpe, BK & Ben-Nissan, B 2012, 'A new role for marine skeletal proteins in regenerative orthopaedics', Key Engineering Materials (Volumes 529 - 530) bioceramics 24, Bioceramics, Scientific.net, Fukuoka, Japan, pp. 654-659.View/Download from: Publisher's site
Use of ready-made marine skeletons is one of the simplest possible remedies to major problems hindering the future development of regenerative orthopaedics- such as, providing a richness of framework designs and now a potentially rich, accessible source
Campbell, LT, Simonin, AR, Padula, MP, Harry, E, Herbert, BR & Carter, DA 2012, 'The fungal secretome and virulence: Analysis of the proteins secreted by Cryptococcus gattii strains with different virulence profiles', MYCOSES, WILEY-BLACKWELL, pp. 163-163.
Chong, HS, Campbell, LT, Padula, M, Harry, E, Hill, C, Li, S, Herbert, B, Wilkins, MR & Carter, DA 2012, 'The dynamic response of Cryptococcus gattii cells to fluconazole: a time-course analysis of the proteome and interactome', MYCOSES, WILEY-BLACKWELL, pp. 115-116.
dos Remedios, CG, Estigoy, C, Cameron, D, Ho, JWK, Herbert, B, Padula, M, Pickford, R, Guilhaus, M, Odeberg, J & Ponten, F 2010, 'Proteomics of the Human Cardiac Intercalated Disc: A More Complex Multi-Functional Structure than was Previously Thought', BIOPHYSICAL JOURNAL, CELL PRESS, pp. 755A-756A.View/Download from: Publisher's site
Graudins, A, Sung, K, Hains, PG, Padula, M, Broady, KW & Nicholson, GM 2001, 'Partial protein and DNA sequences of Latrodectus hasselti, L. hesperus and L. mactans latrotoxins: are they homologous?', 6th Asia Pacific Congress on Animal, Plant and Microbial Toxins, Cairns.
Gunning, SJ, Khalife, A, Padula, M, Smith, R, Broady, KW & Nicholson, GM 2001, 'Modulation of sodium channel gating and kinetics by Î´ missulenatoxin Mb1a from the Australian eastern mouse spider Missulena bradleyi.', 6th Asia Pacific Congress on Animal, Plant and Microbial Toxins, Cairns.
Graudins, A, Padula, M, Broady, KW & Nicholson, GM 2000, 'Evidence for red back spider antivenom efficacy in the prevention of envenomation by other widow spiders (genus Latrodectus)', Journal of Toxicology-Clinical Toxicology, Taylor & Francis, XX International Congress of the European Association of Poisons Centres and Clinical Toxicologists, pp. 205-206.