Michael Schirm

Senior scientist, Protein chemistry, Caprion Proteomics, Montreal

Challenges of Plasma Proteomic Biomarker Discovery

New biomarkers are urgently needed to improve the diagnosis of diseases. The preferred diagnostic sample for biomarker discovery is human blood. It is easily accessible and, along with the common plasma proteins, it contains secreted proteins from all organs and tissues of the human body. Therefore, it may potentially allow the detection of any disease or disease state. However, discovery of biomarkers in plasma is very challenging. Plasma protein concentrations span a wide dynamic range >10 orders of magnitude with the top 10 most abundant proteins accounting for around 90% of the total plasma proteins. This makes the detection of low abundant proteins difficult.

We have developed highly multiplexed multiple reaction monitoring (MRM) assays allowing the accurate and precise quantification of hundreds of low and medium abundance plasma proteins within a single 30 min run. The lower limit of detection (LLOD) of these assays is in the low ng/mL range. The development of these MRM assays has been automated, with development for around 5,000 peptides within a week. These MRM assays were then applied to identify potential biomarkers for several diseases such as lung cancer.

Anne Marinier

Director, Medicinal Chemistry, IRIC, Université de Montréal

Mass Spectrometry as an enabling technology platform in Medicinal Chemistry

Mass spectrometry is playing an important role at different stages of the drug discovery pipeline to identify potential lead candidates for further validation and to profile metabolites and drug conjugates during clinical studies.  Although largely used for the ‘fingerprint’ analysis of organic compounds by the medicinal chemists, the availability of modern mass spectrometry instrumentation now expands the analytical potentials of this technique to the characterization of target-ligand interactions and to study the pharmacokinetic properties of specific compounds.  Since its inception in 2008, IRIC’s medicinal chemistry platform at UdeM made significant scientific contributions as part of different drug discovery projects of the Institute.  More specifically, molecules that expand hematopoietic stem cells have been developed as well as pharmacological chaperones for the MC4 receptor and these are presently evaluated in pre-clinical trials. This presentation will highlight different projects in which mass spectrometry played a key role in support to the medicinal chemistry platform.

Christiane Ayotte

Professor and Director, Doping control laboratory, INRS-Institut Armand-Frappier

The fight against doping in sports : progress and setbacks!

The INRS-Institut Armand-Frappier is involved in the drug testing control of athletes since the Montreal Olympics in 1976. The methods of collecting and analyzing samples have evolved, along with our understanding of doping in sports and the “political” issues at stake. Assays performed in our laboratory involve the development and validation of methods based on mass spectrometry, the use of stable isotopes and molecular biology techniques for the detection of glycoprotein hormones. The majority of urinary metabolites of doping substances are now detected and confirmed by CG-MS/MS and LC-MS/MS. The sensitivity of current instrumentation enables the detection of compounds that previously eluded drug testing controls, though the identification of new elicit drugs still remain problematic. Our studies have also identified farming practices used for raising slaughter animals which in some countries, result in the involuntary consumption of growth promoters residues.

Sébastien Sauvé, Ph.D.

Chemistry Department, Universite de Montreal

Ultrafast Environmental Monitoring of Emerging Contaminants using LDTD(APCI)-MS/MS

We have developed high throughput methods that use a laser diode thermal desorption (LDTD) – APCI interface to bypass liquid chromatography for the analysis of many emerging organic contaminants using tandem mass spectrometry, mainly steroid hormones, parabens, antiseptics, antibiotics and cyanotoxins.  The LDTD method uses heat generated from a laser diode to volatilize analytes and gaseous transfer for quantification directly into the APCI ionization chamber of the MS/MS with a sample turnover below 30 sec. We have so far adapted the LDTD method to the analysis of some pharmaceuticals, steroid hormones and parabens in water and milk (MDL in the range of 2 to 25 µg l-1 in liquids) but we have also had excellent results with solids such as contaminated soils, aquatic sediments and sewage sludge (MDL around 1 to 20 ng g-1 for solids). LDTD analyses allow some large resources savings, but have certain limitations, mainly a somewhat restricted number of analytes amenable to the LDTD/APCI method and lower performance for detection limits when compared to SPE-LC-MS/MS.

Dr Scott D. Tanner

Professor in the Department of Chemistry, University of Toronto, Canada and President and Co-Founder of DVS Sciences Inc.

Massively multi-parameter single cell data by Mass Cytometry: the technology of its acquisition and networks for its interpretation

Mass Cytometry brings the power, resolution, sensitivity and quantitative capabilities of atomic mass spectrometry to high throughput single cell analysis in order to address the challenges of multi-parameter, quantitative flow cytometry.  Individual cells that have been immunologically stained with stable isotope tags are injected into the analytical instrument that “reads” the tag elements.  The cells are vaporized, atomized and ionized in a high temperature plasma, and the atomic composition of each cell – including the metal tags – is measured by time of flight mass spectrometry.  Adapted from its long-time use in elemental analysis, the atomic mass spectrometer provides high sensitivity for many (up to 100) independent mass channels and offers the capability for absolute quantification.  At present, 35 stable isotopes of the metals are available as tags, and we expect that another 30 will be available in the foreseeable future with the eventual potential for 100.  The staining protocol is similar to that of flow cytometry, and the data output is in FCS format for porting into third party flow cytometry analysis software.  Because the detection channels are independent, and the sensitivity to each probe is similar, the selection of staining panels is trivial.  Accordingly, it is as easy to quantitatively analyze many parameters as a few, facilitated by the absence of need for compensation. A high level introductory tutorial on the technology of element-labeling and analysis will be given. We will use data from our laboratory and that of our collaborators in the Nolan group at Stanford University, notably on determining differential immune and drug responses across a human hematopoietic continuum using 31 simultaneous cell surface and intracellular probes, to assess the current art in multidimensional data analysis.

Richard M. Caprioli, Ph.D.

Vanderbilt Mass Spectrometry Research Center

Imaging Mass Spectrometry: Looking Beyond the Microscope

  The spatial and temporal aspects of molecular processes in cells and tissues play an enormous part in the biology that defines living systems both in health and disease.  Profiling and Imaging MALDI MS (matrix assisted laser desorption ionization mass spectrometry) provides an effective means to measure and assess these dimensions on a molecular basis, including peptides, proteins, lipids, metabolite as well as others.  The technology is extraordinarily high throughput with high molecular specificity and is an excellent discovery tool.  It provides the capability of mapping the location of specific molecules directly from fresh frozen and formalin fixed tissue sections.

  For example, utilization of this technology provides spatial information across a tissue section for a target protein expression or for a signature of multiple proteins and can be used to correlate changes in expression levels with specific disease states or drug response. Molecular patterns can be directly correlated to known histological regions within the tissue, a technique termed histology directed molecular profiling. In the imaging mode, high density laser ablation of an ordered array of spots over the entire tissue gives rise to a 2-dimensional ion density map (or image) with 20-40 µm lateral resolution in which location and relative abundance of a given analyte is displayed. From the analysis of a single section, images at virtually any molecular weight may be obtained.  Both fresh frozen and formalin fixed tissues can be analyzed.  The ease and multiplex nature of the technology for monitoring molecular markers capable of being monitored will provide a new platform for molecular pathology for enhanced diagnosis, prognosis and evaluation of therapy, assessed at the molecular level for individual patients

  This presentation will discuss applications of this technology, including examples of discovery of protein signatures in human tumors, characterizing protein differences between tumor grades, and for the creation of 3-D protein images.  MALDI ToF MS, MS/MS, ion mobility MS and FTICR MS for profiling and imaging of tissues will be discussed. The technology has also been applied to drug metabolism and efficacy in several animal model systems.

Dr. Graham Cooks

Department of Chemistry, Purdue University

Chemical reactions in Mass Spectrometry

Mass spectrometry has always depended on chemical reactions including the gas phase bimolecular reactions of chemical ionization and the unimolecular dissociations associated with collisional activation. However, bimolecular solution phase reactions are a recent component of the subject, made possible by the introduction of electrospray ionization and more recently the ambient ionization methods, especially desorption electrospray ionization (DESI).

This presentation covers a variety of homogeneous and heterogeneous phase chemical reactions that are associated with mass spectrometry.  The reactions are chosen to illustrate the degree to which MS is a chemical methodology as much as it is a form of spectroscopy.  Included are (i) in situ derivatization reactions that accompany the ambient ionization methods and increase ionization efficiency (ii) the use of desorption electrospray ionization (DESI) to sample reacting mixtures (iii) the accelerated reactions that occur in microdroplets in DESI (iv) the heterogeneous reactions that occur when mass selected ions are scattered from or are deposited on a surface (soft landed)  (v) the ambient reactions that occur when analytes in solution are heated in coiled tubes and the products sampled using mass spectrometry (atmospheric pressure thermal desorption) and (vi) the heterogeneous reactions that occur in chemical sputtering and the redox reactions that occur at organometallic interfaces prepared by ion soft landing.

In each instance the fundamental phenomenon and potential applications of the methodology will be discussed.   Among the reactions is a surface assisted Birch reduction, Fischer indole synthesis, and Borsche-Drechsel cyclization and the Eberlin transacylation.

Dr Lekha Sleno

Assistant Professor, Faculty of Sciences, Chemistry Department, Université du Québec à Montréal

Bioanalytical LC-MS applied to reactive drug metabolites and metabolomics

Our laboratory is involved in developing novel bioanalytical LC-MS methods applied to the analyses of endogenous metabolites (“metabolomics”), reactive drug metabolites and their covalent binding to proteins. The first part of the presentation will describe a strategy for detecting reactive metabolites in vitro using a novel glutathione trapping agent based on mass defect and isotope pattern filtering. Atrazine, a common pesticide, will be shown as an example in the discovery of new reactive metabolites using high-resolution LC-MS. Covalent binding can also be directly assessed by LC-MS following the complete digestion of proteins. In the area of metabolomics, it is generally impossible to analyse all endogenous metabolites in a complex sample. One option is to have a more “targeted metabolite profiling” strategy, focusing on a specific class of molecules that can be compared between biological samples. An example of this will be shown for a recent study on carotenoid profiling in algal samples. Also, an important challenge in metabolomics studies is the analysis of very polar metabolites. Normal aqueous phase chromatography is highly amenable to MS analysis and has been used successfully. An interesting application of phosphorylated compounds will be discussed, as well as a method for assessing perturbations in tryptophan metabolism.

Pierre Chaurand, Ph.D.

Département de chimie, université de Montréal

MALDI Imaging Mass Spectrometry: Principle, State of the Art and Future Challenges

MALDI-based imaging mass spectrometry is a new technology that allows to map different biocompounds and xenobiotics directly from thin tissue sections. Numerous classes of biomolecules including metabolites, phospholipids, peptides and proteins can be detected and mapped in direct correlation with the underlying histology. Molecular profiles and images depend on the types of tissues or cells studied and certain signals can be directly correlated with the health status of the tissue specimen. Indeed, the technology is sufficiently sensitive to detect variations in the molecular composition induced by the presence of disease or by drug uptake. Numerous technical advances such as automated matrix deposition and the development of in situ chemistries now allow us to study the proteomic content of fresh frozen and formalin fixed paraffin embedded tissue specimens.

After an introduction of the technology and a description of current progresses the different fields of research of imaging mass spectrometry will be presented. In particular its enormous potential in clinical settings in complement to traditional histopathology and its important role in the study of drug distribution and effects in various biological tissues will be described. Finally, a critical outlook will be made towards the developments to be made for the technology to become a mainstream analytical tool.

Daniel Figeys

CRC in Proteomics and Systems Biology, Director Institute of systems Biology, University of Ottawa

Proteomics and lipidomics of human diseases 

Proteomics and lipidomics to better understand biological processes associated with diseases, such as neurodegenerative and cardiovascular diseases that pose some interesting technical challenges inview of the limited amount of sample available when studying specific regions of the brain.  Although, proteomics can routinely identify and quantify 1000’s of proteins, this is generally done using  a large amount of material.  Here, we will present technologies to study minute amounts of specific regions of the brain using proteomics and lipidomics.  We are also studying the role of the regulation of LDL and VLDL in liver cells.  This requires studying biological processes from the ER, Golgi, endosome, and plasma membrane.  We will present some of our approach to study the proteome and the lipidome at the sub-cellular level.