The development of proteomic techniques to study the Australian paralysis Tick, Ixodes holocyclus : the application of proteomic technology to an organism with poor bioinformatic information

University of Technology, Sydney. Faculty of Science.The Australian paralysis tick, Ixodes holocyclus, is representative of the majority of organisms studied in biology in that the bioinformatic information available (genome sequence, annotated coding regions and protein sequences) are far from complete. The study of well characterised model organisms has shown that proteomics and its associated technologies are able to isolate, identify and characterise individual protein isoforms at femto to attomole amounts of sample. With these model organisms, this can be achieved in either unpurified or partially purified samples (shotgun proteomics) or by high resolution separations using isoelectric fractionation and two-dimensional gel electrophoresis. In a poorly characterised organism, this is not the case. The work presented in this thesis applies proteomic technologies to characterising the tick proteome in a hypothesis and non-hypothesis driven manner. In the non-hypothesis driven approaches, fractionation and separation methodologies were applied to determine which method or combination of methods provided the greatest number of protein identifications. The results of these studies showed that the resolution of protein isoforms provided by 2-DGE is invaluable for characterising proteins from I. holocyclus. This is because the homogenous protein spot can be excised from the gel and characterised by de novo sequencing of MS/MS spectra with the knowledge that all peptides are from the same protein. However, successful de novo sequencing is reliant on good quality MS/MS spectra, which is partly reliant on intensely stained gel spots, which is determined by the amount of sample loaded onto the gel. It is well documented and demonstrated in this study that overloading of 2-D gels with samples containing high abundance proteins, tick cytoskeletal proteins in this case, can cause spot resolution problems. Fractionation of the sample using a Multi-compartment Electrolyser and equalisation with Proteominer partially addresses this issue, but further refinement is necessary. The optimised sample preparation methods were then applied in hypothesis driven experiments to characterise specific protein subtypes using Western blots and a novel fluorescent zymogram approach. The analysis identified a number of proteins that will need further characterisation, using molecular biological and recombinant protein expression techniques, to determine their suitability as vaccine candidates

HPLC MS-MS Analysis Shows Measurement of Corticosterone in Egg Albumen Is Not a Valid Indicator of Chicken Welfare.

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

Analysis of formalin-fixed, paraffin-embedded (FFPE) tissue via proteomic techniques and misconceptions of antigen retrieval

© 2016, Eaton Publishing Company. All rights reserved. Since emerging in the late 19th century, formaldehyde fixation has become a standard method for preservation of tissues from clinical samples. The advantage of formaldehyde fixation is that fixed tissues can be stored at room temperature for decades without concern for degradation. This has led to the generation of huge tissue banks containing thousands of clinically significant samples. Here we review techniques for proteomic analysis of formalin-fixed, paraffin-embedded (FFPE) tissue samples with a specific focus on the methods used to extract and break formaldehyde crosslinks. We also discuss an error-of-interpretation associated with the technique known as “antigen retrieval.” We have discovered that this term has been mistakenly applied to two disparate molecular techniques; therefore, we argue that a terminology change is needed to ensure accurate reporting of experimental results. Finally, we suggest that more investigation is required to fully understand the process of formaldehyde fixation and its subsequent reversal

Special Issue "Top-down Proteomics: In Memory of Dr Alfred Yergey". Alfred Linwood Yergey, III, 17 September 1941-27 May 2018.

Please see J [...]

The quest for improved reproducibility in MALDI mass spectrometry

© 2016 Wiley Periodicals, Inc. 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. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 37:217–228, 2018

Deep imaging: How much of the proteome does current top-down technology already resolve?

Effective proteome analyses are based on interplay between resolution and detection. It had been claimed that resolution was the main factor limiting the use of two-dimensional gel electrophoresis. Improved protein detection now indicates that this is unlikely to be the case. Using a highly refined protocol, the rat brain proteome was extracted, resolved, and detected. In order to overcome the stain saturation threshold, high abundance protein species were excised from the gel following standard imaging. Gels were then imaged again using longer exposure times, enabling detection of lower abundance, less intensely stained protein species. This resulted in a significant enhancement in the detection of resolved proteins, and a slightly modified digestion protocol enabled effective identification by standard mass spectrometric methods. The data indicate that the resolution required for comprehensive proteome analyses is already available, can assess multiple samples in parallel, and preserve critical information concerning post-translational modifications. Further optimization of staining and detection methods promises additional improvements to this economical, widely accessible and effective top-down approach to proteome analysis. © 2014 Wright et al

A novel method to detect translation of membrane proteins following microvesicle intercellular transfer of nucleic acids

© 2016 The Authors. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved. 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 coculture 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

Silicon: Potential to promote direct and indirect effects on plant defense against arthropod pests in agriculture

© 2016 Reynolds, Padula, Zeng and Gurr. 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

A Novel Method for Creating a Synthetic L-DOPA Proteome and In Vitro Evidence of Incorporation

Proteinopathies are protein misfolding diseases that have an underlying factor that affects the conformation of proteoforms. A factor hypothesised to play a role in these diseases is the incorporation of non-protein amino acids into proteins, with a key example being the therapeutic drug levodopa. The presence of levodopa as a protein constituent has been explored in several studies, but it has not been examined in a global proteomic manner. This paper provides a proof-of-concept method for enzymatically creating levodopa-containing proteins using the enzyme tyrosinase and provides spectral evidence of in vitro incorporation in addition to the induction of the unfolded protein response due to levodop