Introduction

‘Omics’ research is the non-targeted and non-biased analysis of a specific biological sample, the findings of which may give rise to hypotheses that can then be tested by further investigations. It incorporates a range of disciplines and sophisticated analytical technologies. Together, they provide a holistic view of the molecules that make up a cell, tissue or organism and how they respond to environmental stimuli.

Genomics is the systematic study of an organism’s genome, which contains the DNA that dictates how a cell develops and regulates cell function. When a gene receives the excitatory signal, a template for a specific protein will be produced in the form of mRNA. The study of all mRNA in a cell is called transcriptomics and provides a picture of the genes that are actively being expressed at a given moment.

Proteomics is the study of the resultant proteins, and metabolomics is the study of global metabolite profiles, which can detail how protein expression and cellular pathways are affected by different stressors, such as toxins and disease states.

Although all areas of ‘omics’ research are closely related, each provides different information, which can be pieced together to give a more complete picture of cell function. For example, although an organism has one genome and the proteome is the direct product of a genome, the proteome is ever-changing in response to current environmental demands.

Furthermore, the number of proteins can exceed the number of genes. This is possible through alternative gene splicing and post-translational modifications. Without the study of both gene sequences and the complement and structure of protein, the full story could not be unraveled.

‘Omics’ research is continually evolving as enhancement of analytical technologies and the development of novel methodologies make more and more in-depth investigations possible. Mass spectrometry and nuclear magnetic resonance have revolutionized the detection and quantification of analytes in proteomic and metabolomic research.

Similarly, advances in microarray technology have allowed DNA analyses to become widespread and enabled large-scale sequencing to be conducted more rapidly and with greater accuracy.

However, requirements are continually changing, and collaboration between researchers and the manufacturers of analytical equipment allow analytical methodologies and instrumentation capabilities to be refined to meet specific needs, be they in the laboratory of a research team, hospital, or forensic department.

Sensitivity and resolution are being steadily enhanced to reduce the impact of limits of detection. However, it remains important that the increasing sophistication of analytical technologies does not breed complacency. Due attention must still be given to proper sample collection and preparation and the interpretation of data obtained.

The increasing capabilities of analytical technologies mean that a huge amount of data is now obtained in ‘omics’ research, necessitating powerful hardware and software to undertake the complex data analysis. In addition, extremely rare events are now being captured for which numerous measurements may not be possible, reducing the robustness of any conclusions drawn.

This article will provide an overview of recent research in genomics, proteomics, metabolomics, and metallomics, which studies the role of metals within a cell. It will also highlight some of the latest technologies that have made the research possible. Presenters and exhibitors at Pittcon 2018 will elaborate on the examples presented here and cover myriad additional ingenious advances that are helping to improve the scope and accuracy of ‘omics’ research.

This includes Professor Jeremy Nicholson, Head of the Department of Surgery and Cancer and Director of the MRC-NIHR National Phenome Centre Faculty of Medicine, who will be presenting the Wallace H. Coulter Lecture at Pittcon 2018 “Analytical Science in Precision Medicine: Facing the Challenges of the 21st Century Healthcare”. By using patient specific details at the genome, proteome and metabolome level, Prof. Nicholson will explore how the analytical technologies described throughout this article can deliver improved healthcare solutions.

Chapter 1 – Proteomics: where we are today

The study of protein structure, its relationship to function and the effect of external stimuli on protein structure has massively furthered the understanding of multiple systems in the body and the etiology of many diseases.

With advances in biotechnology and the compilation of databases defining DNA and protein sequences, proteins can now be investigated in greater detail than ever before. Consequently, there is an entire branch of science dedicated to the large-scale analysis of proteins—proteomics.

The vast array of molecular biology and imaging techniques now available are applied to analyzing and defining the structure, function, and interactions of proteins expressed by a particular cell or tissue.

It is possible to evaluate changes in protein content and protein structure in response to external cues, giving an insight into how cells modify the way they function to meet current requirements dictated by their immediate environment.

The findings are correlated with existing databases and potential applications of the new-found knowledge are explored. Proteomics has proved particularly valuable in elucidating the mechanisms of disease and identifying potential targets for novel therapies.

Sessions at Pittcon 2018 will provide details of the latest in proteomics research, the development of novel methodologies and new uses for existing technologies in the analysis of proteins, and how proteomics research can be applied to address ongoing biological conundrums.

The growth of proteomics

Proteomics has shown significant growth over the last decade and it is predicted that this will continue for several years to come; the global proteomics market is projected to be worth USD 21.87 billion by 2021.

Numerous innovative technologies have been used to provide a wide range of sophisticated techniques for use in the study of proteins. The analysis of highly complex mixtures is now commonplace, but researchers continue to adapt, combine and enhance the available technologies in their quest to answer more and more complex biological questions.

The speed with which these new technologies have been developed and utilized to address crucial questions and support hypotheses has led to guidelines being developed to ensure a high level of stringency and accuracy in all proteomic research methodologies.

The proteomics rush, however, has recently been quelled in several countries as a result of the economic crises, which has meant that there are fewer funds available for the purchase of high cost equipment and research grants. Such economic restraints, however, have not hit Brazil.

As Daniel Martins-de-Souza explains in his presentation at Pittcon 2018 entitled “Proteomics in Brazil: Current Status and Perspectives”, proteomics in Brazil continues to grow both quantitatively and qualitatively. Brazil is home to over one hundred mass spectrometers dedicated to proteomics and several of the leading names in the field, making it a powerful center for proteomics investigation.

The role of mass spectrometry

Mass spectrometry is a sophisticated tool used across a broad range of disciplines and applications. Its combination with stable isotopic labeling has been particularly valuable in proteomics. Mass spectrometry studies have provided the basis for numerous hypotheses relating to biochemical mechanisms and their involvement in disease states. Indeed, mass spectrometry is one of the most important developments in protein identification and quantification.

In the last decade, the sensitivity of analyses and accuracy of results for protein analyses by mass spectrometry have increased by several orders of magnitude. Mass spectrometry can simultaneously analyze hundreds of peptides, allowing investigation of changes in expression and modification of proteins involved in several pathways and networks which can then be related to function.

At Pittcon 2018, in a presentation entitled “Structural Proteomics: Mass Spectrometry as a Tool for Structural Biology”, Daniel Martins-de-Souza explains how mass spectrometry has been pivotal in gaining knowledge of protein structure, function and modification. Various modifications and adaptations have been applied to the basic concept of the mass spectrometry technology tailoring it to specific proteomic studies.

Emerging mass spectrometry methodologies

In his seminar at Pittcon 2018 “Structural Mass Spectrometry and Top Down Proteomics of Proteoforms and Their Complexes: Mass Spectrometry”, Professor Neil Kelleher of Northwestern University will be exploring the latest cutting-edge methodologies using mass spectrometry to study whole proteins.

Broadly speaking, mass spectrometry studies of proteins follow either a bottom up or top-down strategy. Typically, bottom-up proteomics have been used in which proteases are used to break the protein and the resultant mixture of short peptides is analyzed by mass spectrometry. However, it became apparent that a single gene can produce a range of different proteins due to polymorphisms. Furthermore, expressed proteins can be modified by a range of intracellular processes that change the molecular weight of the protein. Consequently, analysis and interpretations of such a complex heterogeneous mixture of peptides is not straightforward.

Mass spectrometry of intact proteins, without previous proteolytic digestion—top-down proteomics—thus became increasingly common in the characterization of protein primary structures. The top-down approach shows which protein forms are present and their relative quantities before the protein is broken down for complete characterization. However, it is not suitable for the study of entire proteomes.

A top-down/bottom-up hybrid methodology has been developed to take advantage of the best features from the two strategies. This hybrid strategy is known as middle-down proteomics and analyses larger peptide fragments (>3 kDa) that contain multiple post-translational modifications. It is used to facilitate the identification of biomarkers and to characterize recombinant proteins.

New technologies are being developed to facilitate the successful implementation of a top-down proteomics. Native electrospray mass spectrometry is a particularly promising technology for the identification and characterization of whole protein complexes. Although it can currently assess proteins of 100 kDa to 1 MDa, it is likely to soon be capable of complete proteome compositional analysis.

Mass spectrometry hardware

A range of different, high specification mass spectrometers, each designed to optimize the results of particular types of analyses, are needed to undertake quality proteomics studies. Representatives from world-leading producers of mass spectrometry equipment will be at Pittcon 2018, showcasing the latest technologies that are advancing the capabilities of proteomics research.

Thermo Fisher Scientific, the worldwide leader in liquid chromatography mass spectrometer solutions for protein research, will be demonstrating their high-resolution accurate mass spectrometers, such as Orbitrap Fusion™ and Q Exactive™, which are designed for the identification of potential biomarkers.

Bruker, another provider of high-performance systems for proteomics research, continues to build on its innovative and extensive range of state-of-the-art technologies and solutions. Their latest addition, Impact II™, an ultra-high-resolution benchtop Q-TOF platform will be on display at Pittcon 2018.

This mass spectrometer provides market-leading full-sensitivity resolution for analysis of multiple molecules in a single process. The highest proteome coverage reported to date was achieved with Impact II™ (11,257 proteins were identified in single measurements of cerebellum).

Bruker have also been working to facilitate the advance of top-down proteomics and have developed a solution to help address some of the challenges of achieving a stable and robust nanoflow during electrospray mass spectrometry.

Bruker will be demonstrating the CaptiveSpray™ source and the new nanoBooster™, a revolutionary ion source with a patented design that provides easy operation and great flexibility.

Glass Expansion manufactures high-precision components for mass spectrometers capable of achieving high accuracy even within the narrowest of analytical specifications. At Pittcon 2018, they will be presenting the most recent addition to their achievements, the glass expansion helix spray chamber™ that improves transport efficiency, precision, and washout to optimize sample introduction.

References

• Graves PR, Haystead TAJ. Molecular Biologist’s Guide to Proteomics. Microbiol Mol Biol Rev. 2002; 66(1): 39–63
• Liebler DC, Zimmermann LJ. Targeted Quantitation of Proteins by Mass Spectrometry. Biochemistry 2013;52:3797−3806
• Wilkins MR, et al. Guidelines for the Next 10 Years of Proteomics. Proteomics 2006;6 Issue 1:4–8
• Kelleher NL. Top-down Proteomics. Analytical Chemistry 2004; June1:199 203
• Siuti N, Kelleher NL. Decoding protein modifications using top-down mass spectrometry. Nat Methods 2007;4(10):817–821
• Catherman AD, et al. Top Down proteomics: Facts and perspectives. Biochemical and Biophysical Research Communications 2104;445(4):683 693
• Beck S, et al. The impact II, a very high resolution quadrupole time-of-flight instrument for deep shotgun proteomics. Mol Cell Proteomics. 2015;14(7):2014 2029.Chapter 2 – Mass Spectrometry Dominates the “Omics”

Pittcon Tracks

Chapter 2

Metabolomics

Living cells comprise a complex, ever-changing solution of small-molecule endogenous metabolites, intermediates and metabolism products. These are the substrates and products of numerous inter-related pathways that are activated or inactivated according to requirements dictated by their immediate environment.

The unique chemical fingerprint of a cell consequently changes as the result of specific cellular processes caused by external stressors.

The study of the relative quantities of endogenous metabolites is called metabolomics. It provides a comprehensive snapshot of the biochemistry of a biological system and gives an insight into time-dependent metabolic responses to changes in the environment.

These changes in metabolism can lead to changes in gene transcription and protein expression or changes in protein structure and function. Metabolomics is therefore commonly combined with genomic, transcriptomic and proteomic studies to fully elucidate the mechanisms of action through which drugs or other contaminants cause toxicity.

The latest metabolomics research, including novel analytical techniques to improve selectivity and sensitivity whilst minimising bias, will be presented at Pittcon 2018.

Mass spectrometry and metabolomics

Metabolomic studies aim to identify and quantify a huge variety of different metabolites in complex mixtures, which is a huge challenge to standard analytical techniques. High-frequency nuclear magnetic resonance (1H NMR) and mass spectrometry are the techniques most suited to metabolomics, as they can simultaneously measure numerous endogenous metabolites.
Mass spectrometry has the added benefit of also allowing quantitative assessments. The capabilities of mass spectrometry are further increased by using it in combination with other separation and analytical technologies. A selection of these are discussed below.

Ultra performance liquid chromatography – mass spectrometry

Ultra Performance Liquid Chromatography (UPLC) is one of the most significant developments in separations science in recent years. The re-development of high-specification equipment, including the column, injectors, pumps, and detectors has reduced dispersion, allowing sharper and more concentrated peaks to be achieved.

Using UPLC in conjunction with mass spectrometry combines the physical separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry. UPLC-mass spectrometry provides vastly improved sensitivity and spectral quality in the analyses of complex solutions.

Evaluation of the system in the evaluation of 1300 samples of human urine showed an overall variation in spectral peak area response of 8%, and the mean variation in retention time reproducibility was only 1%. It has also provided good separation of amino acids and amines from human plasma with a rapid throughput and high sensitivity and precision.

Waters Corporation will be available at Pittcon 2018 to discuss their ACQUITY UPLC system with the Xevo G2-XS QTof mass spectrometer.

Ion mobility-mass spectrometry

Ion mobility-mass spectrometry (IM-MS) has become an important analytical tool for ‘omics’ studies, since it has a much greater peak capacity than mass spectrometry alone, so a greater number of compounds can be identified.

Ion-mobility spectrometry separates gas phase ions on a millisecond timescale then the separated components are identified using mass spectrometry on a microsecond timescale. It has proved especially valuable for detailed structural analysis of large and heterogeneous protein complexes. Although proteins are renowned for their precise structures, around 40% of human proteins lack a regular structure. Furthermore, many intrinsically disordered proteins play important roles in disease, such as cancer and Parkinson’s disease.

It is thought that their lack of ordered structure is vital for them to function correctly. There has been extensive research into developing a mass spectrometry-based approach capable of determining the exact structures of intrinsically disordered proteins.

In his presentation at Pittcon 2018 entitled “Development of an Ion Mobility-Orbitrap Mass Spectrometer with a Variable Temperature Nano-ESI Source”, David Russell, Professor of Applied Biosystems at Texas A&M University, will be discussing the development of IM-MS instrumentation with high mobility resolution that is capable of analyzing larger proteins and protein complexes.

Liquid chromatography-mass spectrometry

Liquid chromatography mass spectrometry (LC-MS) is another key tool in metabolomics as it offers the broadest coverage of metabolites. This is largely because it is possible to use different column chemistries, as in reversed phase liquid chromatography (RPLC).

Furthermore, metabolites separated by liquid chromatography do not generally need derivatization and do not need to be volatile. Consequently, LC-MS has wide applicability in both untargeted and targeted metabolomic analyses.

The Thermo Fisher Scientific™ Exactive™ Plus EMR mass spectrometer LC-MS System provides a new level of accuracy in the study of native tertiary and quaternary protein structures. This high-resolution technology is also ideally suited to screening complex mixtures, providing accurate-mass full-scan mass spectra. Thermo Fisher Scientific will be available at Pittcon 2018 to discuss this technology in more detail.

Secondary-ion mass spectrometry

Secondary-ion mass spectrometry (SIMS) allows analysis of the composition of solid surfaces and thin films. A focused primary ion beam is directed at the surface and the mass/charge ratios of the secondary ions reflected back are analyzed by mass spectrometry. This provides data on the elemental, isotopic, or molecular composition of the surface.

Pittcon 2018 exhibitor CAMECA will be presenting their NanoSIMS 50L, which simultaneously delivers a range of key performance metrics that previously required had to be executed individually using a range of technologies. It has been used to quantitatively measure a subcellular slow protein turnover inside the cochlea of bullfrogs.

Mass spectrometry analysis of glycosylation

Glycosylation is one of the most common covalent modifications of proteins. It is frequently the means by which external stimuli cause physiological responses within a cell. It is thus essential for cell survival, and glycosylation errors are the cause of many human diseases, including cancer and infectious diseases.

Study of glycoproteins can therefore provide important information about cellular development and disease states. However, due to their heterogeneity, it is exceptionally difficult to thoroughly analyze glycoproteins in complex biological samples. Recently, a range of chemical and enzymatic methods have been developed to facilitate the study of protein glycosylation using mass spectrometry. Such advances in mass spectrometry -based proteomics have provided a unique opportunity to systematically study protein glycosylation.

At Pittcon 2018, in a presentation entitled “Novel Mass Spectrometry-Based Methods to Globally and Site-Specifically Analyze Glycoproteins”, Ronghu Wu of the Georgia Institute of Technology will be describing the latest technique for studying surface glycoproteins.

Since glycoproteins are key players in many important biological processes, including disease, they are prime targets for identifying biomarkers. Biomarkers are a valuable tool for facilitating early detection of disease and more accurate prognoses, and individually tailored treatment plans. Investigation of site-specific protein glycosylation, enabled by advances in mass spectrometry technologies, is essential for the functional analyses of complex glycoproteins. In his talk at Pittcon 2018, “Ultrahigh Resolution FTICR-MS for Clinical Glycomics Applications”, Yuri van der Burgt of Leiden University Medical Center, will describe clinical glycomics studies indicating the potential prognostic value of total serum N-glycome analysis.

The high resolution of Thermo Fisher Scientific’s Orbitrap Elite™ Hybrid Ion Trap-Orbitrap Mass Spectrometer facilitates analysis of low-abundance glycoproteins in complex samples. In addition, their UltiMate™ Well Plate Autosamplers delivers injections of even the smallest sample volumes with high reproducibility, reducing delay times and improving accuracy and peak resolution.

Fourier transform ion-cyclotron resonance mass spectrometry

Fourier transform ion-cyclotron resonance (FT-ICR) mass spectrometry is a type of mass spectrometry particularly suited to identifying heavy molecules, providing the highest resolving power and mass accuracy among all types of mass spectrometers. It differs from other mass spectrometry techniques in that the ions are identified according to the frequency with which they pass a detector when moving in a circular motion in a strong magnetic field.

Since this is determined by their mass, it can be used to determine their mass-to-charge ratio. The technique can be used on intact proteins without damaging them, making it an important tool proteomics and metabolomics.

Pittcon 2018 exhibitor Bruker offers the SolariXXR™ system, that provides mass resolution greater than 10 million. This represents an increase of an entire order of magnitude, opening up the possibility for molecular biology investigations at a totally new level.

References

• Nicholson J, et al. Metabonomics: a platform for studying drug toxicity and gene function. Nat Rev Drug Discov 2002;1:153–161
• Waters Corporation. Stability of the Acquity UPLC system with Xevo G2-XS QTof Mass Spectrometer for metabolic phenotyping of human urine in large cohort studies. Technology Brief 2016. Available at http://www.waters.com/webassets/cms/library/docs/720005798en.pdf
• Wilson ID, Plumb RS. An Validated Bioanalytical Method for the Quantification of Biogenic Amines using UPLC-MS/MS. Application Note 2016. Available at http://www.waters.com/webassets/cms/library/docs/720005882en.pdf
• Chen SH, Russell DHJ. Am. Soc. Mass Spectrom. 2015;26:1433
• Corey DP. Movement and Growth of Hair-Cell Stereocilia. Presentation at “Molecular Cell Dynamics” conference, June 1, 2007, University of Münster, Germany. Available at http://www.cameca.com/markets/life-science/cell-biology-protein-turnover-nanosims

Chapter 3 – The Importance of other Analytical Technologies

In addition to the advances in mass spectrometry instrumentation and methodologies, state-of-the-art sample preparation techniques and other analytical technologies have played a key role in increasing analytical capabilities.

Sessions at Pittcon 2018 will describe how qualitative and quantitative analyses have evolved to meet the growing complexities of proteomics studies and detail the current scope of technologies available to support research initiatives.

Technology supporting research

Mass spectrometry is a sophisticated tool used across a broad range of applications and has been pivotal in gaining knowledge of protein structure, function, modification and global protein dynamics. However, the more we learn, the more complex the hypotheses become, and increasingly sophisticated technologies are needed to test them.

As researchers voice their needs for better separation, better resolution or better reproducibility, biotechnology companies respond with various modifications and enhancements that tailor their technologies to meet the specific needs of a particular research application.

The successful collaboration of scientists and industry to optimize existing techniques and devise novel analytical solutions will be clearly evident at Pittcon 2018, both in presentations and on the exhibition stands.

Many of the market-leading producers of analytical equipment, including Bruker, Thermo Fisher Scientific, and Waters Corporation, will be exhibiting at Pittcon 2018 to showcase the latest additions to their capabilities and discuss additional analytical requirements.

Bruker have steadily improved the resolution, mass accuracy, and dynamic range of their high-resolution quadrupole time-of-flight mass spectrometers, facilitating more in-depth proteomics research. The latest addition, Impact II™, combines several state-of-the-art technologies to provide accurate and reproducible protein quantification.

The adapted heated liquid chromatography system enables very narrow peptide peaks, the CaptiveSpray™ nanoBooster™ ensures high reproducibility, while the new collision cell more than doubles ion extraction at high tandem mass spectrometer frequencies and an improved detector increases resolving power by up to 80%. Furthermore, spectral quality is preserved, allowing a high degree of sensitivity, by inclusion of a data-dependant acquisition method.

Proteomics workflows

Proteomics research is so complex, and has such a broad scope, that a vast number of different technologies are used to achieve the required results with the necessary degree of quality. However, it is not only the instrumentation used in the analysis that is important for consistent, high-quality results. Sample preparation techniques and analytical software can both also significantly impact the data and therefore must be carefully considered in order for research to be successful.

Sample preparation

Proper sample preparation is a critical step in accurate mass spectroscopy analysis, since the quality and reproducibility of sample extraction and preparation will significantly impact the data obtained. Sample preparation protocols differ according to the abundance, complexity and cellular location of the proteins to be analysed, experimental goals and analytical method to be used.

For example, labelling may be required to facilitate the identification of post-translational modifications, such as phosphorylation and glycosylation.

Optimized cellular lysis, subcellular fractionation, depletion of high abundant proteins or enrichment of select proteins and mass tagging tools are all important in the accurate quantification of global protein expression in complex samples.

Thermo Fisher Scientific, who will be exhibiting at Pittcon 2018, produce a wide range of protein digestion products, labelling technologies and apparatus to facilitate sample preparation and enhance the quality of data in proteomics studies.

Data acquisition and analysis

To optimise outcomes from time-consuming investigations, it is essential that all the data obtained by sophisticated analytical technologies are accurately and completely collected and analysed. In order to achieve this, the mass spectrometer and chromatographic instrumentation must be linked to computer hardware loaded with sufficiently powerful software.

Representatives from Waters Corporation will be available at Pittcon 2018 to discuss their novel data acquisition mode, SONAR™. This technology, designed to support the Xevo® G2-XS quadrupole time-of-flight mass spectrometer, provides tandem mass spectroscopy data from data-independent acquisitions (DIA).

The quantification and identification of lipids, metabolites, and proteins in complex samples is possible from a single injection without additional method development, thereby increasing laboratory throughput.

Similarly, JEOL Ltd have developed ‘ALICE2 for Metabolome’, a multivariate data processing software that automatically analyses data acquired using nuclear magnetic resonance.

Combining technologies

Using chromatography and mass spectrometry technologies together is common practice in metabolomic studies. The combination of chromatographic separation and mass spectrometric detection enables rapid, high-resolution identification of many peaks. Gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry are thus powerful tools in metabolomics.

Waters have developed a comprehensive analytical workflow incorporating state-of-the-art chromatography, ionization sources, and mass spectrometry technology combined with simple, scalable, yet powerful informatics solutions. Furthermore, their data analysis software, Progenesis QI™ incorporates a metabolite database of 240,00 compounds to facilitate rapid and accurate compound identification using mass spectrometry spectra.

Retention time is a key measurement for discovering changed metabolites in metabolomics research. Consequently, reproducibility of retention time is critical for accurate results. The Shimadzu High Performance Liquid Chromatography Prominence Series is renowned for its exceptional performance. Its combination with high-speed mass spectrometry provides a wealth of accurate data required to enable complex pathways to be resolved.

To aid identification of the metabolites isolated, Shimadzu have compiled a metabolite database of retention times and software that compares the structures of metabolites in pre- and post-metabolic data—MetID Solution™.

Metallomics

Metallomics is the study of biomolecules within a cell that bind a metal atom or ion, or are affected in some way by the presence of a metal. It incorporates an incredibly broad range of potential chemistries, masses and complexes that often occur only in trace amounts.

The analytical technique most commonly employed to meet the exacting demands of metallomic studies is inductively coupled plasma mass spectrometry (ICP-MS), which is capable of detecting metal at concentrations as low as one part in 1015. ICP-MS is often coupled with some form of chromatographic separation.

Data from ICP-MS typically need to be integrated into parallel analyses, which are usually not purely parallel in time, place, or methods of sample preparation/separation. In his talk at Pittcon 2018 entitled ‘Extreme Versatility: Coupling of the Liquid Sampling-Atmospheric Pressure Glow Discharge (LS-APGD) Microplasma and Diverse “Organic” Mass Analyzers for Metallomics Applications’, Kenneth Marcus of Clemson University will describe a technique using microplasma as an ionization source for metallomics analyses.

The LS-APGD is mounted in place of the normal electrospray ionization source of a mass spectrometer system facilitating direct coupling with chromatographic technologies. The capacity for the LS-APGD ionization source to affect both atomic mass spectra and structurally significant spectra for organometallic complexes is a unique and potentially powerful combination.

Microfluidic sequencing

Single-cell sequencing to characterize the genome of individual cells is used to investigate scarce and/or precious cells for which large samples are not available. It is also valuable in the study of genomic variations in a heterogeneous population of cells. Single-cell whole genome analysis has proved particularly important for cutting-edge clinical diagnoses, such as molecular sub-typing of single tumor cells.

Single-cell sequencing is typically conducted in microfluidic devices that require very small volumes, thereby increasing the efficiency of reactions. Microfluidic devices enable high-throughput analyses of multiple single cells in parallel with minimum risk of contamination.

Yanyi Huang of Peking University will be giving a presentation at Pittcon 2018, ‘Microfluidic Single Cell Sequencing’, in which he describes a microfluidic device developed in his laboratory to perform multiplex single-cell whole-genome amplification using multiple annealing and looping-based amplification cycles.

Unlike traditional methods, this emulsion whole-genome amplification method gives constant amplification yield along the genome allowing detection of copy number variations and single-nucleotide variations. The automated device is simple to use and associated with minimal risk of contamination, making it suitable for potential applications in medical diagnoses.

Representatives from Microfluidic ChipShop will be onsite at Pittcon 2018 to discuss their range of affordable microfluidic systems as well as the option to design a bespoke system tailored to meet a specific research need.

References

• Beck S, et al. The Impact II, a very high resolution quadrupole time-of-flight instrument for deep shotgun proteomics. Mol Cell Proteomics. 2015;14(7):2014-2029
• Fu Y, et al. Uniform and accurate single-cell sequencing based on emulsion whole-genome amplification Proc Natl Acad Sci 2015;112(38):11923 11928
• Gethings LA, et al. identifying and quantifying peptides, proteins, and posttranslational modifications (PTMs) such as phosphorylation and glycosylation. Waters Corporation Application Note 720006072en. Available at http://www.waters.com/webassets/cms/library/docs/720006072en.pdf
• Kaspar S, et al. High quantification efficiency in plasma targeted proteomics with a full-capability discovery Q-TOF platform. Bruker Application Note # LCMS-89. Available at https://www.bruker.com/fileadmin/user_upload/8-PDF-Docs/Separations_MassSpectrometry/Literature/ApplicationNotes/LCMS-89_Targeted_Proteomics_ebook.pdf
• King A, et al. Increased Specificity, Improved Lipidomic Clarity, and Identification Confidence with SONAR. Waters Application note 720006036EN. Available at http://www.waters.com/waters/library.htm?locale=en_GB&cid=514526&lid=134944356
• Marcus RK, et al. Liquid Sampling-Atmospheric Pressure Glow Discharge Ionization Source for Elemental Mass Spectrometry. Anal Chem. 2011;83(7):2425–2429
• Ogura T, Sakamoto Y. Application of Metabolomics Techniques using LC/MS and GC/MS Profiling Analysis of Green Tea Leaves. Shimadzu Application note No.10 (Lifescience). Available at http://www.ssi.shimadzu.com/products/literature/gcms,lcms/laancxxe011.pdf
• Yu Z, et al. Microfluidic Whole Genome Amplification Device for Single Cell Sequencing. Anal Chem. 2014;86(19):9386–9390
• Zhang LX, et al. Preliminary Assessment of Potential for Metal–Ligand Speciation in Aqueous Solution via the Liquid Sampling–Atmospheric Pressure Glow Discharge (LS-APGD) Ionization Source: Uranyl Acetate. Anal Chem. 2015;87(14):7218–7225

Chapter 4 – Real-world applications of ‘Omics’ research

The preceding chapters have focused on the development of technologies and techniques to facilitate our understanding of cellular processes through proteomic and metabolomic research. However, they also have important applications that are directly relevant to everyday life. The instrumentation and methodologies used in research have become valuable tools for a range of real-world assessments.

These include aiding decisions regarding medical treatment strategies, determining toxicity, providing quality control, and detecting unscrupulous activity.

Pittcon 2018 will be providing details of a range of both current and potential future real-world applications of ‘omics’ research.

Metabolomics in oncology

Cancer affects normal cellular metabolism, and so tumor cells can be readily distinguished from healthy cells by their unique metabolic phenotype. Since metabolomics provides a global assessment of a cellular state, it is ideally suited to detecting and characterising cancer cells. Metabolomics also provides valuable data for informing treatment decisions and the development of novel therapeutics.

With the increasing sensitivity and specificities of nuclear magnetic resonance and mass spectrometry technologies, they are becoming important tools in the clinical management of cancer. The benefits of metabolomic analysis have already been proven with the use of metabolite imaging in the screening and diagnosis of breast and prostate cancers. Biomarkers also hold great promise for enabling earlier cancer diagnosis and predicting which treatments will be most effective for a given patient.

Elevated tissue choline levels are a reliable indicator of breast cancer. Similarly, metabolomic determination of lipid metabolic profiles by NMR identifies the presence of tumour cells with 83% accuracy. This could reduce the need for tissue biopsy. Metabolic profiling is also key to sub-typing breast cancer, providing valuable prognostic and predictive information regarding long-term outcomes for a given patient.

It is also becoming clear that metabolomic analysis can predict how likely a surgically excised tumour is to recur. If it is known that a tumour is likely to regrow quickly surgery, and its associated complications, could be avoided in patients for whom it is not likely to be curative.

In cases where it is decided that clinical excision is the best option, it is often necessary to pharmacologically reduce the size of the tumour before surgery. Although pharmacological treatment can elicit a strong metabolic response in all patients, a good clinical response is not achieved in many cases. Metabolomic NMR analysis has helped determine which metabolic profile is predictive of survival for more than 5 years.

At Pittcon 2018, Facundo Ferrnandez of Georgia Institute of Technology will be giving a presentation entitled “Fused MS and NMR Metabolic Signatures of Prostate Cancer Recurrence Following Radical Prostatectomy” in which he will describe the use of nuclear magnetic resonance and liquid chromatography – mass spectrometry to make an a priori prediction of the likelihood of prostate cancer recurrence prior to prostatectomy.

Nuclear magnetic resonance metabolomics is thus making it possible to provide clinical cancer care that is tailored on an individual patient level so that patients are provided with the treatment that is most likely to be successful against the particular cancer that they have.

Modern spectrometers readily provide an accuracy of ±5%, assuming relaxation issues are handled properly. With attention to potential sources of error, such as baseline distortions, poorly tuned instrumentation, signal to noise, accuracy can be increased to <1%.

Genomics

Proteomics research is so complex, and has such a broad scope, that a vast number of different technologies are used to achieve the required results with the necessary degree of quality. However, it is not only the instrumentation used in the analysis that is important for consistent, high-quality results. Sample preparation techniques and analytical software can both also significantly impact the data and therefore must be carefully considered in order for research to be successful.

Epigenetic profiling

A new epigenetic identification technique using bisulfite modification, polymerase chain reaction (PCR) and pyrosequencing has been developed and validated. It determines the specific pattern of DNA methylation in the sample and this can be matched with the patterns found in semen, saliva, and blood. Semen has been identified using this technique in samples containing as little as 1ng of genomic DNA.

The procedure will be described at Pittcon 2018 by Bruce McCord of Florida International University, in his presentation entitled ‘Forensic Epigenetics, A Novel Method for Body Fluid Identification and Phenotyping’.

Thermo Fisher Scientific, who will be exhibiting at Pittcon 2018, produced the PSQ 96 system for the analysis of DNA using pyrosequencing. It allows real-time sequencing of large numbers of short- to medium-length DNA sequences. Preparation and analysis of a sample is easily automated and results are obtained in under 2 hours.

Extragene will also have a stand at Pittcon 2018 where their range of PCR equipment can be explored. Extragene PCR tubes, strips and tips are made from prime virgin polypropylenes that gives a perfect balance between transparency, softness, robustness, antistatic characteristics and gas tightness.

Short tandem repeat genotyping

Within the human genome, there are many areas of repeated DNA sequences. These repeated sequences varying in size and are classified according to the length of the repeating unit. Repeats 2-6 bases pairs long are called short tandem repeats (STRs).

STRs have several unique features that make them a valuable means of human identification. The number of repeats in STR markers varies markedly between individuals (an individual inherits an STR from each parent, which may or may not have similar repeat sizes) and have a low mutation rate. In addition, STRs are easily amplified by PCR and so have become popular DNA markers.

However, STRs can be challenging to analyze due to the propensity for errors to arise during amplification. A novel targeted sequencing technology that simultaneously genotypes thousands of STR loci and phase proximal SNPs with significantly higher accuracy than currently available methods will be described at Pittcon 2018.

In a presentation entitled ‘Population Haplotype Analysis of 2,543 STRs and their Flanking SNPs Using a Massively Parallel Next-Generation Sequencing Technology’ Giwon Shin from Stanford University School of Medicine will present data obtained using the new STR sequencing methodology.

Proteomics

Quantification of proteomes by mass spectrometry is commonly used in human pathology to study the effects of disease and treatments. Novel therapeutic agents are evaluated in animal models of disease before being tested in humans. A key aspect of such studies is to determine changes in protein expression.

Proteomic analysis may quantify thousands of proteins, making it a challenge to identify changes, particularly if only a small subset of proteins is affected. Furthermore, it may be difficult to distinguish which changes are in response to the treatment and which are a consequence of the disease.

It is also desirable to identify the changes in protein expression occurring at the earliest stages of the disease process since reversing these is more likely to change the course of the disease and thus have the potential to be successful drug targets.

To facilitate such studies, a new technique has been developed that enables in vivo quantitative proteomic analysis specifically of newly synthesized proteins. The PALM (Pulse AHA Labeling in Mammals) technique allows for the first time in vivo labeling of mouse tissues to differentiate protein synthesis rates at discrete time points.

This new technique will be presented at Pittcon 2018 by John Yates of the Georgia Institute of Technology in his presentation entitled “PALM (Pulse Azidohomoalanine Labeling in Mammals) Analysis for Global Analysis of Newly-Synthesized Proteins in Animal Models of Disease”.

References

• Antunes J, et al. High-resolution melt analysis of DNA methylation to discriminate semen in biological stains. Anal Biochem. 2016;494:40-45
• Madi T, et al. The determination of tissue-specific DNA methylation patterns in forensic biofluids using bisulfite modification and pyrosequencing. Electrophoresis. 2012;33(12):1736-1745
• McClatchy DB, et al. Pulsed Azidohomoalanine Labeling in Mammals (PALM) Detects Changes in Liver-Specific LKB1 Knockout Mice. J Proteome Res. 2015 Nov 6;14(11):4815-22. Available at http://pubs.acs.org/doi/ipdf/10.1021/acs.jproteome.5b00653
• Shin G, et al. High resolution STR analysis using next generation sequencing. Patent US 20160362751 A1. Available at https://www.google.com/patents/US20160362751
• Shin G, et al. CRISPR-Cas9-targeted fragmentation and selective sequencing enable massively parallel microsatellite analysis. Nat Commun. 2017;8:14291. Available at https://www.nature.com/articles/ncomms14291
• Silva DSBS, et al. Developmental validation studies of epigenetic DNA methylation markers for the detection of blood, semen and saliva samples. Forensic Sci Int Genet. 2016;23:55-63

Conclusion

Living cells comprise a complex, ever-changing solution of small-molecule endogenous metabolites that represent the substrates and products of numerous inter-related pathways. This chemical fingerprint can therefore give an indication of which specific cellular processes are activated.
Consequently, changes in the precise combination of species present, along with changes in protein structure, reflect metabolic modifications in a cell providing insight into how a cell responds to external stimuli.

Metabolomic studies assessing metabolite composition are commonly used in conjunction with studies evaluating genetic mutations (genomics) and protein content and modification (proteomics) to obtain a holistic view of cell metabolism for full elucidation of the effects of environmental change.

Such ‘omic’ assessments have many applications in addition to being a valuable research tool, such as screening for and diagnosis of disease processes and forensic investigations. ‘Omic’ techniques are also valuable for informing treatment decisions.

They can identify gene mutations and biomarkers that predict disease course and treatment response. Such analyses allow treatment strategies to be tailored on an individual patient basis. This has proved especially important in oncology, where unpredictable efficacy and response rates had previously meant that finding an effective treatment strategy involved an element of trial and error. For example, NMR metabolic profiling can identify those patients most likely to respond to pre-surgical chemotherapy, and combined mass spectrometry and NMR metabolic signatures have been used to predict post-surgical prostate cancer recurrence.

High-frequency NMR and mass spectrometry are the techniques most suited to metabolomic and proteomic analyses, as they can simultaneously measure numerous endogenous metabolites.

Although the analysis of highly complex mixtures is now commonplace, novel innovative uses and adaptations of these technologies continue to increase their capabilities. Ultra-high-resolution mass spectrometers, such as Orbitrap Fusion™ and Impact II™, enable accurate analysis of multiple molecules in a single process, facilitating biomarker identification.

Novel mass spectrometry based methods have also been developed to address current research needs, such as FTICR-MS for site-specific analysis of glycoproteins and non-damaging analysis of intact proteins, IM-MS for the analysis of intrinsically disordered proteins, and PALM for differentiation of protein synthesis rates at discrete time in vivo.

Mass spectrometry techniques are also used in combination with chromatographic separation, e.g.gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry, to increase resolution and facilitate peak identification.

In genomics, a novel polymerase chain reaction methodology has been developed that can identify a body fluid from a 1 ng sample of DNA, which will help evaluation of evidence collected from a crime scene. Another forensic tool used as a means of human identification—analysis of short tandem repeats—has also been improved by the development of a novel targeted sequencing technology.

Pittcon 2018 will see Prof. Nicholson discussing his research and expertise on how the omics can be combined with analytical technologies and computational modeling to develop a personalized healthcare approach, during this year’s Wallace H. Coulter Lecture.

The research and technologies highlighted here will be covered in more detail in the symposia, oral presentations, short courses, and poster sessions at Pittcon 2018, along with multiple additional cutting-edge developments.

In addition, many of the market-leading producers of analytical equipment, including Bruker, Thermo Fisher Scientific, and Waters Corporation, will be exhibiting at Pittcon 2018 to showcase the latest additions to their capabilities and discuss additional analytical requirements.

Pittcon is a world-leading annual forum for communicating the latest research technologies and instrumentations; the 2018 conference will take place in Orlando from 26 February to 1 March.