The use of proteomics for the assessment of clinical samples in research

Proteomics, the analysis of expressed proteins, has been an important developing area of research for the past two decades [Anderson, NG, Anderson, NL. Twenty years of two-dimensional electrophoresis: past, present and future. Electrophoresis 1996;17:443-53]. Advances in technology have led to a rapid increase in applications to a wide range of samples; from initial experiments using cell lines, more complex tissues and biological fluids are now being assessed to establish changes in protein expression. A primary aim of clinical proteomics is the identification of biomarkers for diagnosis and therapeutic intervention of disease, by comparing the proteomic profiles of control and disease, and differing physiological states. This expansion into clinical samples has not been without difficulties owing to the complexity and dynamic range in plasma and human tissues including tissue biopsies. The most widely used techniques for analysis of clinical samples are surface-enhanced laser desorption/ionisation mass spectrometry (SELDI-MS) and 2-dimensional gel electrophoresis (2-DE) coupled to matrix-assisted laser desorption ionisation [Person, MD, Monks, TJ, Lau, SS. An integrated approach to identifying chemically induced posttranslational modifications using comparative MALDI-MS and targeted HPLC-ESI-MS/MS. Chem. Res. Toxicol. 2003;16:598-608]-mass spectroscopy (MALDI-MS). This review aims to summarise the findings of studies that have used proteomic research methods to analyse samples from clinical studies and to assess the impact that proteomic techniques have had in assessing clinical samples. © 2004 The Canadian Society of Clinical Chemists. All rights reserved.

Eukaryotic expression:Developments for structural proteomics

The production of sufficient quantities of protein is an essential prelude to a structure determination, but for many viral and human proteins this cannot be achieved using prokaryotic expression systems. Groups in the Structural Proteomics In Europe (SPINE) consortium have developed and implemented high-throughput (HTP) methodologies for cloning, expression screening and protein production in eukaryotic systems. Studies focused on three systems: yeast (Pichia pastoris and Saccharomyces cerevisiae), baculovirus-infected insect cells and transient expression in mammalian cells. Suitable vectors for HTP cloning are described and results from their use in expression screening and protein-production pipelines are reported. Strategies for co-expression, selenomethionine labelling (in all three eukaryotic systems) and control of glycosylation (for secreted proteins in mammalian cells) are assessed. © International Union of Crystallography, 2006.

Proteomics in the study of hippocampal plasticity

Synaptic plasticity is the dynamic regulation of the strength of synaptic communication between nerve cells. It is central to neuronal development as well as experience-dependent remodeling of the adult nervous system as occurs during memory formation. Aberrant forms of synaptic plasticity also accompany a variety of neurological and psychiatric diseases, and unraveling the biological basis of synaptic plasticity has been a major goal in neurobiology research. The biochemical and structural mechanisms underlying different forms of synaptic plasticity are complex, involving multiple signaling cascades, reconfigurations of structural proteins and the trafficking of synaptic proteins. As such, proteomics should be a valuable tool in dissecting the molecular events underlying normal and disease-related forms of plasticity. In fact, progress in this area has been disappointingly slow. We discuss the particular challenges associated with proteomic interrogation of synaptic plasticity processes and outline ways in which we believe proteomics may advance the field over the next few years. We pay particular attention to technical advances being made in small sample proteomics and the advent of proteomic imaging in studying brain plasticity.

Functional proteomics to dissect tyrosine kinase signalling pathways in cancer

Advances in the generation and interpretation of proteomics data have spurred a transition from focusing on protein identification to functional analysis. Here we review recent proteomics results that have elucidated new aspects of the roles and regulation of signal transduction pathways in cancer using the epidermal growth factor receptor (EGFR), ERK and breakpoint cluster region (BCR)-ABL1 networks as examples. The emerging theme is to understand cancer signalling as networks of multiprotein machines which process information in a highly dynamic environment that is shaped by changing protein interactions and post-translational modifications (PTMs). Cancerous genetic mutations derange these protein networks in complex ways that are tractable by proteomics.

Application of genomics, proteomics and metabolomics in drug discovery, development and clinic

Genomics, proteomics and metabolomics are three areas that are routinely applied throughout the drug-development process as well as after a product enters the market. This review discusses all three 'omics, reporting on the key applications, techniques, recent advances and expectations of each. Genomics, mainly through the use of novel and next-generation sequencing techniques, has advanced areas of drug discovery and development through the comparative assessment of normal and diseased-state tissues, transcription and/or expression profiling, side-effect profiling, pharmacogenomics and the identification of biomarkers. Proteomics, through techniques including isotope coded affinity tags, stable isotopic labeling by amino acids in cell culture, isobaric tags for relative and absolute quantification, multidirectional protein identification technology, activity-based probes, protein/peptide arrays, phage displays and two-hybrid systems is utilized in multiple areas through the drug development pipeline including target and lead identification, compound optimization, throughout the clinical trials process and after market analysis. Metabolomics, although the most recent and least developed of the three 'omics considered in this review, provides a significant contribution to drug development through systems biology approaches. Already implemented to some degree in the drug-discovery industry and used in applications spanning target identification through to toxicological analysis, metabolic network understanding is essential in generating future discoveries.

Application of genomics, proteomics and metabolomics in drug discovery, development and clinic

Genomics, proteomics and metabolomics are three areas that are routinely applied throughout the drug-development process as well as after a product enters the market. This review discusses all three 'omics, reporting on the key applications, techniques, recent advances and expectations of each. Genomics, mainly through the use of novel and next-generation sequencing techniques, has advanced areas of drug discovery and development through the comparative assessment of normal and diseased-state tissues, transcription and/or expression profiling, side-effect profiling, pharmacogenomics and the identification of biomarkers. Proteomics, through techniques including isotope coded affinity tags, stable isotopic labeling by amino acids in cell culture, isobaric tags for relative and absolute quantification, multidirectional protein identification technology, activity-based probes, protein/peptide arrays, phage displays and two-hybrid systems is utilized in multiple areas through the drug development pipeline including target and lead identification, compound optimization, throughout the clinical trials process and after market analysis. Metabolomics, although the most recent and least developed of the three 'omics considered in this review, provides a significant contribution to drug development through systems biology approaches. Already implemented to some degree in the drug-discovery industry and used in applications spanning target identification through to toxicological analysis, metabolic network understanding is essential in generating future discoveries.

Proteomics in vaccinology and immunobiology:an informatics perspective of the immunone

The postgenomic era, as manifest, inter alia, by proteomics, offers unparalleled opportunities for the efficient discovery of safe, efficacious, and novel subunit vaccines targeting a tranche of modern major diseases. A negative corollary of this opportunity is the risk of becoming overwhelmed by this embarrassment of riches. Informatics techniques, working to address issues of both data management and through prediction to shortcut the experimental process, can be of enormous benefit in leveraging the proteomic revolution.In this disquisition, we evaluate proteomic approaches to the discovery of subunit vaccines, focussing on viral, bacterial, fungal, and parasite systems. We also adumbrate the impact that proteomic analysis of host-pathogen interactions can have. Finally, we review relevant methods to the prediction of immunome, with special emphasis on quantitative methods, and the subcellular localization of proteins within bacteria.

Enhancing vaccine design strategies:applications for protein science, proteomics and adjuvants

Editorial

In vivo vitamin C supplementation increases phosphoinositol transfer protein expression in peripheral blood mononuclear cells from healthy individuals

Ascorbate can act as both a reducing and oxidising agent in vitro depending on its environment. It can modulate the intracellular redox environment of cells and therefore is predicted to modulate thiol-dependent cell signalling and gene expression pathways. Using proteomic analysis of vitamin C-treated T cells in vitro, we have previously reported changes in expression of five functional protein groups associated with signalling, carbohydrate metabolism, apoptosis, transcription and immune function. The increased expression of the signalling molecule phosphatidylinositol transfer protein (PITP) was also confirmed using Western blotting. Herein, we have compared protein changes elicited by ascorbate in vitro, with the effect of ascorbate on plasma potassium levels, on peripheral blood mononuclear cell (PBMC) apoptosis and PITP expression, in patients supplemented with vitamin C (0-2 g/d) for up to 10 weeks to investigate whether in vitro model systems are predictive of in vivo effects. PITP varied in expression widely between subjects at all time-points analysed but was increased by supplementation with 2 g ascorbate/d after 5 and 10 weeks. No effects on plasma potassium levels were observed in supplemented subjects despite a reduction of K+ channel proteins in ascorbate-treated T cells in vitro. Similarly, no effect of vitamin C supplementation on PBMC apoptosis was observed, whilst ascorbate decreased expression of caspase 3 recruitment domain protein in vitro. These data provide one of the first demonstrations that proteomics may be valuable in developing predictive markers of nutrient effects in vivo and may identify novel pathways for studying mechanisms of action in vivo.

Antioxidants, reactive oxygen and nitrogen species, gene induction and mitochondrial function

Redox-sensitive cell signalling Thiol groups and the regulation of gene expression Redox-sensitive signal transduction pathways Protein kinases Protein phosphatases Lipids and phospholipases Antioxidant (electrophile) response element Intracellular calcium signalling Transcription factors NF-?B AP-1 p53 Cellular responses to oxidative stress Cellular responses to change in redox state Proliferation Cell death Immune cell function Reactive oxygen and nitrogen species – good or bad? Reactive oxygen species and cell death Reactive oxygen species and inflammation Are specific reactive oxygen species and antioxidants involved in modulating cellular responses? Specific effects of dietary antioxidants in cell regulation Carotenoids Vitamin E Flavonoids Inducers of phase II enzymes Disease states affected Oxidants, antioxidants and mitochondria Introduction Mitochondrial generation of reactive oxygen and nitrogen species Mitochondria and apoptosis Mitochondria and antioxidant defences Key role of mitochondrial GSH in the defence against oxidative damage Mitochondrial oxidative damage Direct oxidative damage to the mitochondrial electron transport chain Nitric oxide and damage to mitochondria Effects of nutrients on mitochondria Caloric restriction and antioxidants Lipids Antioxidants Techniques and approaches Mitochondrial techniques cDNA microarray approaches Proteomics approaches Transgenic mice as tools in antioxidant research Gene knockout and over expression Transgenic reporter mice Conclusions Future research needs

The presence of ascorbate induces expression of brain derived neurotrophic factor in SH-SY5Y neuroblastoma cells after peroxide insult, which is associated with increased survival

Oxidative stress and free radical production have been implicated in Alzheimer's disease, where low levels of the antioxidant vitamin C (ascorbate) have been shown to be associated with the disease. In this study, neuroblastoma SH-SY5Y cells were treated with hydrogen peroxide in the presence of ascorbate in order to elucidate the me0chanism(s) of protection against oxidative stress afforded by ascorbate. Protein oxidation, glutathione levels, cell viability and the effects on the proteome and its oxidized counterpart were monitored. SH-SY5Y cells treated with ascorbate prior to co-incubation with peroxide showed increased viability in comparison to cells treated with peroxide alone. This dual treatment also caused an increase in protein carbonyl content and a decrease in glutathione levels within the cells. Proteins, extracted from SH-SY5Y cells that were treated with either ascorbate or peroxide alone or with ascorbate prior to peroxide, were separated by two-dimensional gel electrophoresis and analyzed for oxidation. Co-incubation for 24 hours decreased the number of oxidised proteins (e.g. acyl CoA oxidase 3) and induced brain derived neurotrophic factor (BDNF) expression. Enhanced expression of BDNF may contribute to the protective effects of ascorbate against oxidative stress in neuronal cells.

Alpha tocopherol supplementation elevates plasma apolipoprotein A1 isoforms in normal healthy subjects

Plasma α-tocopherol (AT) concentrations are inversely related to cardiovascular (CV) risk; however, intervention studies with AT have failed to show any consistent benefit against CV disease (CVD). Proteomics offers the opportunity to examine novel effects of AT supplementation on protein expression and therefore improve our understanding of the physiological roles of AT. Thus, to investigate the effects of AT supplementation on the plasma proteome of healthy subjects we have undertaken a double-blind, randomised, parallel design supplementation study in which healthy subjects (n = 32; 11 male and 21 female) consumed AT supplements (134 or 268 mg/day) or placebo capsules for up to 28 days. Plasma samples were obtained before supplementation and after 14 and 28 days of supplementation for analysis of changes in the plasma proteome using 2-DE and MALDI-MS. Using semiquantitative proteomics, we observed that proapolipoprotein A1 (identified by MS and Western blotting) was altered at least two-fold. Using quantitative ELISA techniques, we confirmed a significant increase in plasma apolipoprotein A1 concentration following supplementation with AT which was both time and dose dependent (p < 0.01 after 28 days supplementation with 268 mg AT/day). These data demonstrate the time and dose sensitivity of the plasma proteome to AT supplementation. © 2006 Wiley-VCH Verlag GmbH & Co. KGaA.

Performance of five different electrospray ionisation sources in conjunction with rapid monolithic column liquid chromatography and fast MS/MS scanning

The performances of five different ESI sources coupled to a polystyrene-divinylbenzene monolithic column were compared in a series of LC-ESI-MS/MS analyses of Escherichia coli outer membrane proteins. The sources selected for comparison included two different modifications of the standard electrospray source, a commercial low-flow sprayer, a stainless steel nanospray needle and a coated glass Picotip. Respective performances were judged on sensitivity and the number and reproducibility of significant protein identifications obtained through the analysis of multiple identical samples. Data quality varied between that of a ground silica capillary, with 160 total protein identifications, the lowest number of high quality peptide hits obtained (3012), and generally peaks of lower intensity; and a stainless steel nanospray needle, which resulted in increased precursor ion abundance, the highest-quality peptide fragmentation spectra (5414) and greatest number of total protein identifications (259) exhibiting the highest MASCOT scores (average increase in score of 27.5% per identified protein). The data presented show that, despite increased variability in comparative ion intensity, the stainless steel nanospray needle provides the highest overall sensitivity. However, the resulting data were less reproducible in terms of proteins identified in complex mixtures -- arguably due to an increased number of high intensity precursor ion candidates.

Protein and peptides in pictures:imaging with MALDI mass spectrometry

Imaging using MS has the potential to deliver highly parallel, multiplexed data on the specific localization of molecular ions in tissue samples directly, and to measure and map the variations of these ions during development and disease progression or treatment. There is an intrinsic potential to be able to identify the biomarkers in the same experiment, or by relatively simple extension of the technique. Unlike many other imaging techniques, no a priori knowledge of the markers being sought is necessary. This review concentrates on the use of MALDI-MS for MS imaging (MSI) of proteins and peptides, with an emphasis on mammalian tissue. We discuss the methodologies used, their potential limitations, overall experimental considerations and progress that has been made towards establishing MALDI-MSI as a routine technique for the spatially resolved measurement of peptides and proteins. As well as determining the local abundance of individual molecular ions, there is the potential to determine their identity within the same experiment using relatively simple extensions of the basic techniques. In this way MSI offers an important opportunity for biomarker discovery and identification.

Attenuated total reflection-FT-IR spectroscopic imaging of protein crystallization

Protein crystallization is of strategic and commercial relevance in the post-genomic era because of its pivotal role in structural proteomics projects. Although protein structures are crucial for understanding the function of proteins and to the success of rational drug design and other biotechnology applications, obtaining high quality crystals is a major bottleneck to progress. The major means of obtaining crystals is by massive-scale screening of a target protein solution with numerous crystallizing agents. However, when crystals appear in these screens, one cannot easily know if they are crystals of protein, salt, or any other molecule that happens to be present in the trials. We present here a method based on Attenuated Total Reflection (ATR)-FT-IR imaging that reliably identifies protein crystals through a combination of chemical specificity and the visualizing capability of this approach, thus solving a major hurdle in protein crystallization. ATR-FT-IR imaging was successfully applied to study the crystallization of thaumatin and lysozyme in a high-throughput manner, simultaneously from six different solutions. This approach is fast as it studies protein crystallization in situ and provides an opportunity to examine many different samples under a range of conditions.