Thermo Scientific Annual Users' Meeting

Modern MS-based proteomics is beginning to fulfill its long held promise in biology, chemical biology and medicine. Fast, high performance LC-MS/MS instruments, combined with recent advances in quantitative sample preparation workflows and computational analysis, are providing information of unprecedented detail. We can now define the content, relative abundance, modification states and interaction partners of proteins in a dynamic and temporal manner on a near-global basis in organelles, whole cells and clinical samples. At the Broad Institute we are employing these technologies in a wide array of studies including delineating the genetic underpinnings of mitochondrial disorders, connecting cancer genotype to molecular phenotype, unraveling the basis of the innate-immune response, identifying the mechanism of action of drug-like molecules and to discover and verify protein biomarkers of disease. A new era of quantitative biology is on the horizon, enabled by MS-based assays to screen and quantify proteins and their modifications in any biological context. A representative set of project vignettes will be presented to convey a sense of the breadth and depth of application of modern proteomics to biology and medicine.

We will present recent developments by using both dried blood spot samples and high-resolution accurate-mass spectrometry in clinical research (newborn screening, metabolic profiling of small molecules). The minimal invasiveness of sampling, the relative ease of handling and storing these materials are expected to offer additional opportunities to measure and analyze future biomarkers. The combination with high-throughput online-sample preparation - multidimensional chromatography using TurboFlow technology- advantages of the Orbitrap configuration like high resolving power, precision and accuracy, and novel bioinformatics software, allow to meet the analytical challenges in future clinical implications.

The massive increase in throughput of quantitative mass spectrometry-based proteomics through multiplexed data acquisition using isobaric labeling strategies, such as iTRAQ or TMT, has the potential to fundamentally change the type of questions we can ask about biological systems at the proteome level. In this presentation we show how initial shortcomings of the technology regarding accuracy and precision of quantitative results - the interference problem - were resolved using gas phase purification in an MS3 method. We now present that the reporter ion intensities in MS3 spectra are increased up to 8 fold by simultaneous co-isolation of multiple MS2-fragment ions using isolation waveforms with multiple frequency notches (multi-notch strategy). We show how a new generation of isobaric labeling reagents allows comparing up to 18 samples in one experiment. First applications of the methods will be shown including protein expression profiles across yeast deletion strains for 17 deubiquitinating enzymes as well as across multiple cancer cell lines.

Elucidation of endogenous cellular protein-protein interactions and their networks is most desirable for biological studies. Here we report our study of endogenous human protein complex networks obtained from integrative mass spectrometry-based analysis of ~4,000 affinity purifications. By preserving weak protein interactions during complex isolation and utilizing high levels of reciprocity in the large dataset we identified many unreported protein associations, such as a transcriptional network formed by ZMYND8, ZNF687 and ZNF592. Furthermore, our work revealed a tiered interplay within networks that share common proteins, providing a conceptual organization of a cellular proteome composed of minimal endogenous modules (MEMOs), functional uniCOREs and regulatory complex-complex interaction networks (CCIs). This resource will effectively fill a void in linking correlative genomic studies with an understanding of transcriptional regulatory protein functions within the proteome for formulation and testing of future hypotheses. I will also discuss a similar approach with mouse tissues.

Pathways and complexes can be considered fundamental units of cell biology, but their relationship to each other is difficult to define. Comprehensive tagging and purification experiments have generated networks of interactions that represent most stable protein complexes. We describe this work in various organisms, including budding yeast and in infectious organisms like HIV and TB, and show how the analysis of pairwise epistatic relationships between genes complements the physical interaction data, and furthermore can be used to classify gene products into parallel and linear pathways.

Genome-wide association studies (GWAS) have identified many risk loci for complex diseases, but because of the multiple genes involved in many diseases and/or conditions the effect size is usually quite small. We recently published a large genome-wide association study measuring over 500 metabolites in approximately 3000 blood samples using a global metabolomics technology (Nature, 477, pg. 37-41, 2011). 37 highly-significant genetic loci were associated with blood metabolite concentrations, of which 25 showed effect sizes that are unusually high for GWAS and account for 10-60% differences in metabolite levels per allele copy. These metabolite associations, and potentially many others, provide new functional insights for many disease-related associations that have been reported in previous studies, including those for cardiovascular and kidney disorders, type 2 diabetes, cancer, gout, venous thromboembolism and Crohn’s disease.

Multi-dimension mass spectrometry-based shotgun lipidomics developed by using triple quadrupole mass spectrometers is a mature technology platform in lipidomics. At its current stage of development, this platform allows us to identify and quantify over 30 lipid classes, hundreds to thousands of individual lipid molecular species, and > 95% of the content of a cellular lipidome. In the presentation, the principles of this technology platform will be summarized. Very recently, bearing these principles, we have developed a novel, ultra high-throughput technology platform by using an ultra high resolution/ accurate mass instrument (i.e., Thermo Scientific Q Exactive mass spectrometer) for identification and quantification of over 15 lipid classes. In the presentation, this novel platform will be introduced. It can be anticipated that applying one of these platforms should greatly aid the rapidly spreading lipidomics for biological and biomedical research.

This study was designed to evaluate the quantitation of 19 bile acids and salts with poor fragmentation characteristics using a Thermo Scientific Q Exactive hybrid quadrupole-Orbitrap high-resolution mass spectrometer (HRMS) coupled with UHPLC separation with minimal instrument optimization for high throughput bioanalysis. Because of poor fragmentation, MRM based analysis results in low sensitivity and extended chromatographic separation. Initial quantitative findings indicate that comparable/or better results were achieved on the HRMS system for all 19 bile acids/bile salts tested with Internal standard (IS) peak areas yielding an average within-run precision of ~7.7 % RSD. Quantitation results of the endogenous levels of bile acids in each matrix obtained from the HRMS method are compared with these obtained by the traditional MRM method.

Metabolite identification allows identification of metabolic soft spots of chemical candidates during drug discovery process. Such data enable chemists to design safer molecules with acceptable PK by blocking a metabolic route to reduce clearance or avoid a substructure that results in a potentially reactive metabolite. However, the fast turnaround time for this data is demanding. Cassette analysis strategies for metabolite identification provide the analytical advantage of higher throughput, but require greater detection sensitivity and selectivity. This presentation reports our evaluation of a post-incubation cassette analysis for metabolite identification that employed the latest technology advancement in high resolution mass spectrometry (HRMS) with fast acquisition and polarity switching coupled with UHPLC.

Pushing mass spectrometry into the world of high throughput screening (HTS) is a daunting task. MS is inherently serial while HTS relies on a high degree of parallelism and affords analysis rates in the range of 10⁶ to 10⁷ samples per year. Yet the lure of using a label-free technology that can directly measure changes in an enzyme substrate or product are sufficiently attractive that we have undertaken the effort to deploy it in our HTS labs. There are several analysis modes that have been explored in our attempts to push the limits of assay speed; currently approximately 10⁶ samples per year. Many practical considerations have been addressed to achieve this level of throughput but new technologies continue to afford opportunities for improvements in speed.

Protein HDX, followed by protease digestion and LC/MS analysis has become a method of choice to study protein conformation and dynamics in the academic world. However, two main factors limit its wide application in pharmaceutical industry. First, the difficulty to control the deuterium back exchange during analysis and second, the time consuming data processing step. This presentation describes an Orbitrap™-based platform, with strategies to deal with both problems. First, back exchange is controlled by introducing a set of internal standards. Second, a comprehensive HDX model is developed to simulate the entire deuterium labeling and back exchange process. The HDX model is implemented into the in-house developed software MassAnalyzer, and enables fully unattended processing of the entire HDX MS data set starting from ion feature detection and peptide identification to the final output of protection factors, typically within one day. The utility of the HDX MS platform is demonstrated by exploring the conformational transition of a monoclonal antibody by increasing concentrations of guanidine.

Advances in mass spectrometry proteomics have transformed the fields of biochemistry and cell biology, establishing the biological rationale required for development of therapeutics against novel cellular processes. One field that has particularly benefitted has been the study of ubiquitin, a small protein that functions as a post-translational modification and a central regulator of mammalian cell biology. Alterations in the ubiquitin system are implicated in a wide range of in human diseases ranging from cancer and viral infection to neurodegeneration and metabolic disorders. This talk will focus on the use of high resolution tandem mass spectrometry for characterizing the enzymes, substrates and cellular signals of the ubiquitin system.

The EU Commission stipulates sampling levels and frequencies for residues in vegetable and animal products and matrices in order to check for illegitimate used substances. Compared to time and effort to fulfil these requirements the number of used sample preparation and instrument methods increased. This indicates that there is a clear need for quick and simple screening and quantification methods to routinely and accurately control levels of residues. The presentation demonstrate that with the Thermo Scientific Exactive Plus, a high-resolution mass spectrometer, it is possible to screen and quantify in one chromatographic run with reliable results in determination and quantification.

The identification of new and emerging contaminants in environmental media has long been hindered by the need to utilize targeted mass spectrometric methodologies, which rely on the availability of analytical standards for each analyte. The emergence of high-resolution mass spectrometry (HRMS) and its application to the field of environmental chemistry has for the first time made possible identification of emerging contaminants in complex environmental mixtures without a priori knowledge of contaminant identity or occurrence. In this study, we present a strategy and analytical workflow based on a Thermo Scientific LTQ Orbitrap HRMS and MS/MS in conjunction with newly developed software tools (e.g. Thermo Scientific ExactFinder software) and informatics tools (e.g. spectral library searching, literature data) for identification of emerging environmental contaminants in the aquatic environment. We have utilized this approach to identify novel organic microcontaminants in wastewater and wastewater-impacted surface waters on the coast of South Carolina. Results illustrate our ability to identify a broad range of organic contaminants in water, including several compounds that have not been previously reported to occur in environmental systems.

The field of metabolomics, aiming at global analysis of numerous targeted or non-targeted low molecular compounds (metabolites) in a biological sample, has recently found its application in diverse research areas including food quality and authenticity ("foodomics"). Rapid growth of metabolomics has been enabled by substantial advances in analytical techniques such as mass spectrometry (MS) and nuclear magnetic resonance (NMR), which facilitate analysis of a wide range of metabolites with diverse physicochemical properties, occurring at different concentration levels. One of the most recent advances has been the introduction of ambient desorption ionization techniques finding the use in many areas, including food safety and authenticity testing. Contrary to "classic" mass spectrometric measurements, it allows high-throughput analysis of various samples under ambient conditions of the laboratory with minimal sample preparation requirements. Nowadays, direct analysis in real time (DART) represents probably the most widespread ambient ionization technique, which can be coupled to almost any of commercially available LC-MS systems. The potential of DART ion source equipped with various autosampler systems, coupled to high-resolution mass spectrometry (utilizing either time-of-flight or Orbitrap technology) in both targeted and non-targeted metabolomics-based authentication / quality assessment of various food commodities, including as oils and fats, milk-based products, wine and honey, will be presented. To process and interpret the complex data obtained within the metabolomic workflow, multivariate chemometric analysis will be employed.

With the introduction of performing high-resolution mass spectrometers (HRMS), such as the Thermo Scientific Exactive Plus instrument, a shift of paradigm is taking place: HRMS will reshape clinical analyses by allowing simultaneous quantitative and qualitative analyses. We will present a few examples of LC-MS analyses that can strongly benefit from the Exactive PlusTM HRMS.

In order for peptide and protein quantification by MS to make its way into routine use in clinical research laboratories, standardized methods for assay development and verification must be identified and deployed across different laboratories by different operators. Unfortunately, most enrichment methods (bead or plate-based) cannot deliver the requisite enrichment of low abundance analytes to allow detection using robust microspray methods due to capacity and physical limitations. Recently, immunoenrichment using MSIA (mass spectrometry immunoassay) microcolumn technology coupled to a robust, automated platform including microflow SRM was demonstrated for picomolar concentrations of parathyroid hormone in clinical samples. The monolithic microcolumn in the MSIA tips allows for greater capacity and larger sample volumes which enables the detection of very low abundance analytes in blood and other fluids. In addition, immunoenrichment with MSIA tips can be coupled with MS analysis of intact clinical analytes (before SRM assay development) enabling the identification of truncated forms, sequence isoforms and post translational modifications. Protein microheterogeneity has been demonstrated in a variety of clinically important analytes such as, BNP, PTH, PSA and others. The combination of MSIA with SRM assays provides a platform and workflow that streamlines the steps involved in developing SRM assays and facilitates rapid deployment in clinical research laboratories due to its simplicity, reproducibility and robustness.