WORLD PROTEOMICS COMES TO WESTMEAD!
Dr. Michael Crouch
Director of Business Development, TGR BioSciences Pty Ltd
Very high throughput immunoassay systems for protein detection in research and drug discovery
TGR BioSciences, South Australia, Australia.
Protein detection and quantification systems vary according to the desired endpoint. Systems for detecting the presence of particular known proteins in a complex mixture generally rely on antibody-based methods, as these convey selectivity and sensitivity, combined with relatively inexpensive measurement techniques. However, such systems are generally quite slow (hours to days), can process few samples (up to hundreds), and usually have several operator handling steps. The needs of basic research and, even more so, drug discovery efforts are changing, and require analysis of thousands to millions of samples over quite short time-lines. These assay requirements include those of cellular systems, providing even greater emphasis on techniques that can utilise some degree of automation, allowing higher throughput and greater data generation.
To address such protein detection requirements, we have developed assay systems that are fully automatable, and can therefore immunoanalyse thousands of cellular samples fully robotically, or manually, as desired, with total assay times of hours. Further recent modifications to these systems have allowed the assay development and manufacturing processes to be greatly streamlined, reducing assay development time as well as costs.
Data will be presented on the assay format, detection modes, and automation capabilities. This will include demonstration of its adaptation to other assay platforms, including those incorporating multiplexed protein analysis.
Prof. Nicolle Packer
Director, MQ Biomolecular Frontiers Research Centre, Macquarie University
MALDI imaging mass spectrometry of N-linked glycans on formalin-fixed tissue: differentiating tissue types.
Arun V. Everest-Dass1
, Matthew T. Briggs2
, Peter Hoffmann2
, Nicolle H. Packer1
Biomolecular Frontiers Research Centre, ARC Centre for Nanocale Biophotonics, Macquarie University, Sydney, New South Wales, Australia.
Adelaide Proteomics Centre, School of Molecular and Biomedical Science, University of Adelaide, Adelaide, South Australia, Australia.
Recent developments in targeting protein distribution in tissue sections by spatial proteomics imaging have paved the way for retrospective in situ mass spectrometry (MS) analyses of formalin-fixed clinical tissue samples. This type of analysis is commonly referred to as matrix assisted laser desorption/ionization (MALDI) imaging. We have used in situ PNGase F mediated release and measurement of N-linked glycans from sections of formalin-fixed tissue to allow analyses of tissue-specific N-glycosylation profiles.
The presented data indicates that N-glycans can be cleaved from proteins from different areas within formalin-fixed tissue. The released glycans can then be characterised by extraction and compositional analysis by liquid chromatography coupled to electrospray ionization ion trap MS and the identified glycan masses can then be located in situ by MALDI imaging, which maps the tissue-specificity of individual N-glycans at high resolution.
Using this approach we have found that MALDI imaging of specific N-glycan structures released from proteins in stored formalin–fixed tissues in situ is able to differentiate tissue type, including the delineation of tumour from non-tumour tissue in ovarian cancer tissue samples.
Prof. Pall Thordarson
Director of Research, School of Chemistry, UNSW
Short peptides as building blocks for 3D in vitro cell cultures and other materials in nanomedicine
School of Chemistry, The University of New South Wales, Sydney, NSW 2052, Australia,
Australian Centre for Nanomedicine, The University of New South Wales, Sydney, NSW 2052, Australia,
ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, The University of New South Wales, Sydney, NSW 2052, Australia.
The field of bio-mimetic materials has until recently been dominated by approaches build on recombinantly created or otherwise reused biological materials such as collagen and elastin on one hand or by traditional polymer synthesis on the other – and occasionally a mixture of the two. Both have considerable limitations that largely stem from the lack of flexibility of the macromolecular network, and in the case of synthetic polymers in particular, the fact that these systems are not chemically well-defined with associated lack of batch-to-batch reproducibility.
As an example of an application of bio-mimetic materials are 3D in vitro cell culture materials. There is a significant interest in these as researchers have come to realise that the 3D environment of a cell is enormously important in various cellular process including growth, division and cell death. Several biological (e.g. Matrigel) and synthetic polymeric materials have been developed for 3D in vitro cell culture but most of these suffer from the above issues.
I will describe here our approach to making bio-mimetic materials which is based on self-assembly. Our materials are based on small short peptide molecules that interact with each other non-covalently to form fibrous networks not dissimilar to those found in cytosol and the extra cellular matrix. These fibrous-network are flexible and reversible and chemically well-defined. They are therefore excellent candidates for 3D in vitro cell cultures as well as other applications that require good bio-mimetic materials such as drug delivery and tissue engineering to take but two examples from the field of nanomedicine.
Prof. Richard J Payne
School of Chemistry, USyd
New ligation technologies for the rapid assembly and biological evolution of modified proteins.
, L. R. Malins1
, N. J. Mitchell1
, R. E. Thompson1
, R. J. Payne1
School of Chemistry, University of Sydney, New South Wales, Australia
Post-translational modifications (PTMs) are ubiquitous chemical alterations that are made to proteins after ribosomal synthesis.
1. The vast number of potential PTMs that can be installed on a specific protein leads to an explosion of structural diversity, with each isoform possessing potential variation in structure and function. This has led to significant interest in proteins bearing PTMs for use as pharmaceuticals.
2. Unfortunately, the non-templated nature of the PTM process in vivo leads to heterogeneous mixtures of isoforms that hinders the ability to study the role of a single modification in a meaningful way. We have developed a number of technologies for the efficient chemical assembly of peptides and proteins bearing homogeneous PTM patterns for the in depth interrogation of biological activity.
3. Highlights of our recent work will be discussed in this talk, including the application of these technologies in the synthesis of several homogeneously modified bioactive proteins.
Prof. Ronald J Quinn
Director, Eskitis Institute for Drug Discovery, Griffith University
Linking the Malaria and TB Proteomes using Bioaffinity Mass Spectrometry
Natural product diversity can be achieved using low MW natural products. A large number of scaffolds present in natural products are also present in low MW natural products. Chemoinformatic analysis has been used to analysis natural products and design a diversity set of natural products with molecular weight ≤ 250 Da that can be used in fragment-based screening. We used
• Atom type analysis using radial fingerprints
• Atom function analysis using pharmacophore fingerprint
• Ring scaffold analysis
• Consideration of non-flat molecules
Natural products enriched with biosynthetic intermediates or endogenous metabolites that have been exposed to a long selection process to interact with biological targets are excellent resources to search for protein binding partners. Fragment-based screening, in our laboratory, uses direct observation of ligand-protein complexes in the ICR cell of a FTMS. Multiple-drug resistance malaria and tuberculosis are a serious health problem. Rather than working from a single target, our approach has used multiple proteins within the proteome of the organisms that cause malaria and tuberculosis. The resultant Network Map has allowed a detailed analysis. Efforts to identify binding sites, to obtain accurate Kd and grow or link fragments will be presented.
Prof. Mark Baker
Translational Signatures of Colorectal Cancer
Current methods used for colorectal cancer (CRC) screening (e.g., FOBT, FIT and/or colonoscopy) are grossly inadequate on both sensitivity and specificity grounds. Here, we first report a program evaluating numerous (SWATH-MS and Proseek® Oncology) proteomics biomarker discovery methodologies. In addition, we discuss how proteomics allows identification of interacting membrane proteins (i.e., the metastasome) and demonstrate how proteins involved in the metastasome might regulate the cancer invasive phenotype and can be used a clinical biomarkers.
Expression of 92 potential plasma cancers biomarkers were measured in pooled CRC Dukes’ staged (i.e., A-D and controls) EDTA plasmas utilizing Olink’s PEA based Proseek® Multiplex Oncology I kit, where duplicate samples were analysed using Bio-Plex Pro™ human cytokine 27-plex immunoassays. Expression of CEA (a diagnostic biomarker for CRC) was found to be significantly high in malignant stages C and D, whilst IL 8 and prolactin expression changed significantly between control, benign and malignant stages. We also, employed SWATH-MS a data independent acquisition (DIA) method that allows a complete and permanent record of all fragment ions of detectable peptide precursors from pooled plasmas (n=20 per stage) that were previously immuno- (i.e., MARS-12) from the same Dukes’ stage A-D CRC patients with age-, sex- and other criteria matched control EDTA-plasmas. The results of these plasma biomarker studies aim for early detection of CRC and the differences between Dukes’ stages will be discussed.
In addition, shotgun proteomic studies suggest that integrin (v6) and protease receptor (uPAR) expression are changed in CRC, allowing us to understand changes associated with the metastatic phenotype. Analyses of the interaction between uPAR and v6 has allowed us to develop lead iPEPs that antagonise this metastasome. In addition, we report data analysing both proteins (uPAR; epithelially-restricted v6) as potential intra-stage tissue biomarkers of patient survival in a large retrospective 20 year rectal cancer FFPE tissue biomarker study.
Dr. Mark Baker
University of Newcastle, Australia
Choosing your man! It’s not all about his looks, think about his sperm!
Mark A. Baker1
University of Newcastle, Newcastle, New South Wales, Australia.
Male infertility is a very common condition, with reports suggesting that one in 15 men of reproductive age are affected. The diagnosis of male-factor infertility is difficult and involves discounting female infertility through hormone measurements, pelvic examination and invasive laparoscopy. A semen profile analysis can suggest male infertility, if sperm counts are <15-20 million/ml, or <50% of sperm possess forward progressive motility (and < 25% rapidly progressive sperm) or <4% good morphology sperm. However, for many couples (20-30%), infertility remains largely unexplained.
In addition, infertile men appear to have more than their share of problems. Not only are they dying younger, but on average demonstrate three times the average rates of cancer compared to the general population. As such, it appears that spermatozoa may give a “prophetic” insight into the overall health of men.
As such, we have used quantitative proteomics analysis to compare spermatozoa taken from healthy, fertile individuals and compared the proteome to that of an infertile male. Several proteins were found to be altered, including, the sperm specific protein, Outer Dense Fibre 1, which was virtually absent from the gametes of the infertile male. A second cohort of men, were missing the major chromatin compaction protein, MENT, together with Histone H2A Bbd and HSP4AL. Given these proteins are also expressed in somatic cells and regulate chromatin compaction, this represent the first biochemical insight as to how male infertility, may predict the future of a man’s health.
Prof. Marc Wilkins
University of New South Wales
Abstract coming soon
Dr. Jason Wong
Prince of Wales Clinical School and Lowy Cancer Research Centre, NSW
How accurate is quantitative proteomics? Assessing the need to improve mass spectrometry-based protein quantification.
Protein quantification has long been a paramount area in proteomics research. Stable isotopic and isobaric tag labelling has enabled accurate measurements of relative protein abundance between difference samples. On the other hand, the accuracy of measurements of the absolute or relative abundance of proteins within a sample remains difficult to assess on a proteome scale. The aim of this study was to assess existing computational methods of protein quantification and propose normalisation strategies to improve quantification accuracy.
We first assessed the accuracy of stable isotope labelling using a recent publically available dataset1. We found that the use of MaxLFQ provided the most accurate quantitative measure, however it lacked sensitivity as the method requires the detection of identical peptides across samples. Hi-3 provided a good compromise of accuracy and sensitivity and was found to be more accurate compared with iBAQ, but was less sensitive. We propose a normalisation method taking into account MS/MS spectral quality as a more general method for both accurate and sensitive method for relative protein quantification between samples.
We further assessed the accuracy of absolute protein quantification for comparison of abundance of proteins within a sample. Using published mRNA and protein copy number data from NIH3T3 cells3, we found that based on the iBAQ calculation, proteins that have substantially lower protein copy number in relation to mRNA copy number have significantly more theoretically detectable peptides compared with proteins that have substantially higher protein copy number in relation to mRNA copy number (p< 0.01). This difference was also found to present in 50 of the NCI-60 cell lines4, raising the possibility that iBAQ may lead to quantification bias of extremely large or small proteins. We discuss our efforts to resolve whether the difference is biological or is systematic in the iBAQ calculation.
A/Prof. Jean Yang
University of Sydney