CRMS Seminars 2015

SEMINAR
YORK UNIVERSITY
CENTRE FOR RESEARCH IN MASS SPECTROMETRY

DR. ANDREW JAMES, PhD | Sr. Scientist |ANALYTICAL RESEARCH & DEVELOPMENT NA | SANOFI PASTEUR LTD | CONNAUGHT CAMPUS | TORONTO | ONTARIO

Derek WilsonANDREW JAMES obtained his Ph.D. from the University of Toronto in the field of Biological Chemistry in 2003.  After a post-doctoral fellowship in clinical chemistry at the Toronto General Hospital within the University Health Network, Andrew shifted into biotechnology.  He started his industrial career as an application chemist at AB Sciex in the field of proteomic mass spectrometry.  At Sciex, he developed his foundational knowledge of mass spectrometry covering applications in protein ID, small molecule and peptide quantitation, MALDI based tissue imaging, and bioinformatics analysis of MS datasets.  Andrew then joined the Tony Pawson lab as an MS expert at the Samuel Lunenfeld Research Institute of Mount Sinai Hospital.  Within the Pawson lab, he worked on MS applications in cell signaling and systems/network biology.  He then transitioned to Sanofi Pasteur as a Senior Scientist in Biochemistry within the Analytical R&D department.  His current areas of research include: (i) development of novel MS based assays for characterization and release of new vaccine candidates, (ii) assay qualification/validation, and (iii) implementation of novel MS instrumentation and workflows designed to address unmet needs in development and project investigations (including peptide quantitation,  epitope mapping using HDX-MS, ion mobility characterization of intact proteins/peptides, and immuno-modulatory proteomics).  Andrew was recently appointed to the position of Adjunct Professor at York University with the department of Chemistry and is associated with the Center for Research in Mass Spectrometry.

“NOVEL APPROACHES TO TARGETED PROTEIN ANALYSIS USING LC-MRM IN AN HSV2 VACCINE CANDIDATE”
Date: THURSDAY NOVEMBER 5, 2015
Location: 306 Lumbers Building
Time: 3:00 PM


Click here to download pdf.

Abstract:
The capabilities of mass spectrometry are continuously increasing within the bio-pharmaceutical industry.  Until recently, the primary focus of mass spectrometry in vaccine research and development has been as a characterization and identity tool, particularly for recombinant protein candidates.  However, newer mass spectrometry workflows are being evaluated for both targeted analysis and quantitation of proteins in more complex vaccine candidates.  This work focuses on the development of a targeted assay for two HSV-1 proteins: HELI and DNBI (UL5 and UL29 gene products), detected in an HSV-2 replication deficient, live-attenuated vaccine candidate.  The HELI and DNBI proteins are expressed by a complementary cell line, AV529, used to produce the HSV529 candidate.  After detection and characterization of the vaccine candidate for HELI and DNBI components using nanoLC techniques; the development of a novel MRM assay using a high flow UPLC and a simplified chromatographic method was designed.  Sensitivity of the MRM assay for HELI and DNBI was established at high flow, and linearity of response was shown in the range of the LLOQ.  Our results demonstrate the unique sensitivity of MRM methods for detection of targeted proteins, and establish specificity for the HSV-1 proteins HELI and DNBI, in the candidate vaccine lots.

MS-ESE Seminars 2015-2016

  Date Title Speaker University

  Jan 22

Understanding Intrinsic Biomolecule
Conformation and Solvation Effects using Mass Spectrometry and Laser-Induced Fluorescence

Rita Straus

University of Toron

  Jan 22

"Automated Immuno-Pulldown Upstream of HPLC-MS/MS for Quantitation of Biologic Drugs"

Brendon Seale

University of Toronto

  Feb 5

 "Moving in on the Action: An Experimental Comparison of Fluorescence Excitation with Photodissociation Action Spectroscopy"

Sydney Wellman

University of Toronto

  Feb 5

"New Methods for Ionizing Peptides"

Yating Wang

York University

  Feb 19

"Development of Continuous Small Scale Chemical Manufacturing Technologies With Mass Spectrometric Readout"

Stas Beloborodov

York University

  Feb 19

" From CRMS to Catalytic Converters!"

Voislav Blagojovic

York University

  Mar 5

"Bioaccumulation of Ionic liquids in Zebra Fish (Danio Renio) by Desorption Electrospray Ionization Mass Spectrometry Imaging (DESI-MSI)"

Consuelo Perez

York University

  Mar 5

" Development of Time-resolved Electrospray Mass
Spectrometry for Epitope Mapping in Drug Discovery"

Kerene Brown

York University

  Apr 29

"Chemical Synthesis and Reaction Monitoring Using Mass Spectrometry"

Dr. R. Graham Cooks

 Purdue University

       

 

CRMS Seminars 2012-2013

Dr. Lucia Sepiashvilli - Phd Student at the Kislinger Group; University of Toronto; (Former York Student)
“Cell Line-based Discovery of Candidate Biomarkers for Head and Neck Squamous Cell Carcinoma”
Date: WEDNESDAY, SEPTEMBER 26, 2012
Location: Lumbers Building 306

Time: 1:00 PM

Abstract: Head and neck squamous cell carcinomas (HNSCC) can arise from the oral cavity, oropharynx, larynx or hypopharynx, and is the sixth leading cancer by incidence worldwide. The 5-year survival rate of HNSCC patients remains static at 40-60%. Hence, biomarkers which can improve detection of HNSCC or early recurrences should improve clinical outcome. Mass spectrometry-based proteomics methods have emerged as promising approaches for biomarker discovery. As one approach, mass-spectrometric identification of proteins shed or secreted from cancer cells can contribute to the identification of potential biomarkers for HNSCC and our understanding of tumor behavior. In the current study, mass spectrometry-based proteomic profiling was performed on the conditioned media (i.e. secretome) of head and neck cancer (HNC) cell lines in addition to gene expression microarrays to identify over-expressed transcripts in the HNSCC cells in comparison to a normal control cell line. This integrated dataset was systematically mined using publicly available resources (Human Protein Atlas and published proteomic/transcriptomic data) to prioritize putative candidates for validation. Subsequently, quantitative real-time PCR (qRT-PCR), Western Blotting, immunohistochemistry (IHC), and ELISAs were performed to verify selected markers. Our integrated analyses identified 90 putative protein biomarkers that were secreted or shed to the extracellular space and overexpressed in HNSCC cell lines, relative to controls. Subsequently, the over-expression of 5 markers was verified in vitro at the transcriptional and translational levels using qRT-PCR and Western Blotting, respectively. IHC-based validation conducted in two independent cohorts comprising of 40 and 39 HNSCC biopsies revealed that high tumor expression of PLAU, IGFBP7, MMP14 and THBS1 were associated with inferior disease-free survival, and increased risk of disease progression or relapse. Furthermore, as demonstrated using ELISAs, circulating levels of PLAU and IGFBP7 were significantly higher in the plasma of HNSCC patients compared to healthy individuals.


Terry D. Cyr, Ph.D. Research Scientist, Centre for Vaccine Evaluation; Biologics and Genetic Therapies Directorate
“Use of mass spectrometry for the analysis of influenza vaccine”.
Date: October 9th, 2012
Location: Lumbers Building 306

Time: 1:00 PM

Abstract: The presentation will focus on the application of proteomics methodology for the analysis of influenza vaccines.  The limitations of methods currently used for analyzing the key antigens in annual vaccines and in potential pandemic scenarios will be discussed along with advantages of this alternate methodology.  The standard proteomics methods have been optimized for simultaneous, rapid identification of both multiple influenza strains and egg protein contaminants.  The proteins, identified in the first stage, are then quantified by MSE using the top three hits from each protein and an internal standard.  The combination of new methods developed within the group provide alternative means of assessing products in emergency situations as well as the means of addressing inconsistencies between current international standards.


Dr. Ryan Julian. Department of Chemistry University of California Riverside, CA
"Taming Radicals for Novel Peptide and Protein Fragmentation"
Date: Tuesday, November 13, 2012
Location: 103 Life Science Building (LSB)

Time: 1:30 PM

Abstract: Radical reactions play a very important role in the biochemistry of proteins in both productive and harmful ways. Radicals are critical in cell signaling, catalysis, and energy production. Unchecked radicals lead to oxidative stress, which is likely the reason that radical damage is strongly correlated with numerous diseases. Protein radicals are therefore inherently interesting and are involved in several projects that are ongoing within the Julian lab. These projects aim to reveal both practical and fundamental biochemical information. We use photodissociation to site- specifically generate radicals for a variety of experiments including: 1) directing fragmentation of proteins or peptides in gas phase experiments, 2) elucidation of gas phase protein structure, 3) identification of post-translational modifications including racemization of amino acids, and 4) elucidation of fundamental peptide and protein radical chemistry. To carry out these experiments, we have developed a suite of chemical functionalities that undergo direct, homolytic dissociation upon activation by a UV photon. Typically, this involves cleavage of a carbon-iodine or carbon-sulfur bond with a 266nm photon to generate a radical, followed by examination of the outcome via various mass spectrometry based methods.


Jan Hendrikse, Ph.D., Principal Scientist & Vladimir Romanov, Ph.D. (Former York Student).
Smiths Detection Science & Technology Group
“Use of IMS/MS to clarify the origin of ion signals in fieldable IMS instruments”
Date: Wednesday, December 5, 2012
Location: Lumbers Building 306

Time: 1:00 PM

Abstract: Ion mobility spectrometry (IMS) is one of the key technologies used by Smiths Detection for detection, identification and monitoring of explosives and illegal drugs. Vapors of these compounds are ionized by radio-active beta emission (63Ni is the most popular such source) and then the ions are separated on the basis of their mobility in an electric field. A brief overview of the principle of operation of the commercial ion mobility spectrometers and applications in the war against terror will be presented during the talk.

While IMS systems are able to detect very small amounts of known analytes by any user without sample preparation, the method is not meant for the identification of unknowns. In order to correlate unknown peaks in our IMS spectra, a Smiths Detection Ionscan® 400B ion mobility spectrometry (IMS) has been interfaced to a QTRAP 2000 mass spectrometer (MS) by our Canadian team. The IMS-MS system enables identification of false positive responses in the field, and a variety of quality and contamination issues, by providing information about mass to charge ratio of the ions separated by size in the IMS. An example of capabilities of such system will be shown in the presentation.

 


Special Seminar
Dr. Richard Oleschuk; Professor Department of Chemistry Queen's University
Much ado about nothing”: Holes; how we make them, modify them and use them in analytical measurements.
Date: Tuesday, December 11, 2012
Location: Lumbers Building 306

Time: 11:00 PM

Abstract:  None


Jean-Philippe Lambert, PhD; Post-doctoral Fellowship Supervised by Dr. Anne-Claude Gingras and Dr. Tony Pawson Samuel Lunenfeld Research Institute, Toronto, ON
“Use of data dependent and independent mass spectrometry acquisition for the systematic study of the human acetylome components”.
Date: Wednesday, January 23, 2013
Location: 317 PETRIE SCIENCE BUILDING

Time: 1:00 PM

 

Abstract: Lysine acetylation is one of the key post-translational modifications regulating chromatin structure and function. Recent proteomics studies have detected lysine acetylation on a wide array of proteins beyond chromatin-containing histones, implying a broader role in cellular func-tions. However, how specificity is acquired within the acetylation system remains largely unknown. Multiple components of the human acety-lome are misregulated in diverse cancers or directly involved in numerous diseases such as schizophrenia, and thus constitute attractive tar-gets for therapeutic modulation. In order to characterize the acetylome machinery in an unbiased manner, we embarked on the systematic characterization of all human acetyltransferases, deacetylases and bromodomain-containing proteins (the only known acetylation mark “reader”). Using an array of mass spectrometry techniques, we are exploring the protein complexes surrounding acetylome components, and are identifying novel substrates for the acetylation machinery. Firstly, protein complexes encompassing all acetylome components have been defined by an affinity purification approach optimized for chromatin-associated proteins. This revealed intricate interaction networks involv-ing acetylome components, which are being validated by reciprocal purifications and targeted in vitro binding assays, and that implicate spe-cific acetylome components in a wide array of biological pathways. Additionally, to gain further biological insight in these novel interaction networks, we are defining their response to chemical and genetic perturbations, with an emphasis on bromodomain inhibitors, including the recently described inhibitor JQ1. JQ1 displaces BET-family bromodomain containing proteins from their acetyllysine targets (in that case, his-tones), resulting in strong anticancer properties: here we are using JQ1 as a tool compound to analyze network rewiring. Finally, we are eluci-dating the specificity of human bromodomains for mono- and poly-acetylated substrates by combining recombinant bromodomain pulldowns with data independent mass spectrometry acquisition (SWATH). This unbiased workflow enabled the systematic quantitation of acetylated substrates and the definition of specificity in the acetylation system.


Dr. Ann M. English, Department of Chemistry and Biochemistry, and Centre for Biological Applications of Mass Spectrometry, Concordia University, Montreal, QC, Canada;
“LC-MS profiling of age-related oxidative modifications in copper-zinc superoxide dismutase linked to
neurodegenerative diseases and asthma”
Date: Thursday, February 21, 2013
Location: 317 Petrie Science Building

Time: 1:00 PM

Abstract: Aggregation of the antioxidant enzyme, copper-zinc superoxide dismutase (CuZnSOD), has been reported in patients with age-related neurodegenerative dis-eases such as Amyotrophic Lateral Sclerosis (ALS) and Alzheimer’s. Over 100 mutations in CuZnSOD have been described in familial ALS but 90% of cases are of unknown etiology and are classified as sporadic ALS. Aggregation, and hence the development of ALS and Alzheimer’s, are attributed to age-related post-translational modifications (PTMs) in CuZnSOD. Thus, we are exploiting yeast as a model eukaryotic cell that rapidly ages to monitor PTMs in CuZnSOD. On gel filtration of cell lysates we isolated a high-mass inactive form of CuZnSOD, and found by FT-MS that cysteine 146 (C146) is oxidized to the sulfonic acid in this form. Importantly, C146 was also converted to the sulfonic acid in CuZnSOD isolated from the brains of Alzheimer’s patients(1) and the C146R mutation has been reported in familial ALS.(2) Notably, C146 forms a C146-C55 disulfide bridge that is critical in stabilizing the native CuZnSOD homodimer. In high-mass CuZnSOD, we further observe oxidation of histidine 71 (H71) and H120, which ligate the active-site zinc and catalytic copper, respectively. We hypothe-size that age-induced oxidation of C146, H71, and H120 contribute to the development of ALS as well as Alzheimer’s by releasing the redox-active catalytic copper, which would likely increase oxidative stress and CuZnSOD aggregation. By monitoring GFP (green fluorescent protein) fluorescence of CuZnSOD-GFP from an isogenic yeast strain expressing this fusion protein, we note that the cellular content of high-mass CuZnSOD-GFP increases ~10-fold from day 3 to day 7. We are currently characterizing CuZnSOD and CuZnSOD-GFP from 15- and 20-day yeast to establish if aggregated CuZnSOD continues to accumulate in old cells. CuZnSOD activity also is known to be decreased by inflammatory processes in the airway epithelium and C146 oxidation was reported in CuZ-nSOD purified from erythrocytes of asthmatic patients.(3) To mimic the inflammatory state of asthma we are examining modification of CuZnSOD after chal-lenging yeast cells with H2O2. Our LC-MS profiling will shed new light at the molecular level on how oxidative stress promotes the development of age-related neurodegenerative diseases and asthma.


Dr. Lekha Sleno; Associate Professor in Department of Chemistry; Université du Québec à Montréal
Montreal, QC, Canada.
 “Bioanalytical mass spectrometry: applications in semi-targeted metabolomics and protein
modifications”.
Date: Tuesday, March 19, 2013
Location: 317 PETRIE SCIENCE BUILDING
Time: 1:00 PM

Abstract:  I n the area of metabolomics, it is impossible to analyse all endogenous metabolites from a complex sample in one analysis. Therefore, a more targeted metabolite profiling strategy is useful when certain classes of molecules are of interest. For a comparison be-tween biological samples, differential isotope-labeling methods have been developed for the relative quantitation of acid and amine-containing metabolites. Using a high-resolution high-speed quadrupole time-of-flight mass spectrometer, we are able to profile labeled compounds and automatically trigger MS/MS with very fast scanning capabilities. Post-acquisition processing is also able to filter out peaks with specified isotope patterns unique to be labeled compounds as well as based on characteristic fragment ions from the derivati-zation reagent. This approach has been applied to oxidative stress in human cultured cells and an ALS disease model in C. elegans, in the hopes of identifying potential metabolite biomarkers.

The analysis of covalent modification of proteins is also a field of interest in our research group. Two examples will be presented. First, the covalent binding of a novel inhibitor of the RNase activity of Ire1, a transmembrane protein-kinase, was investigated. This work proved crucial for understanding the mechanism of action of this novel drug, and will therefore be useful in the optimization of non-covalent inhib-itors. We have also examined modifications on a human matrix metalloproteinase (MMP-13) thought to be involved in osteoarthritis. The lipid peroxidation product 4-hydroxy-2-(E)-nonenal (HNE), generated under oxidative stress, is known to play a crucial role in cartilage deg-radation, however, the mechanism is not yet fully understood. Results from LC-MS/MS analysis suggest that HNE can covalently bind to MMP-13 at several sites. A concentration-dependent study was performed to pinpoint the most active sites of modification, since previous data has shown that lower concentrations of HNE lead to the activation of MMP-13 and higher concentrations can subsequently deacti-vate the protease.


Dr. Kristina Håkansson; Department of Chemistry, University of Michigan
“Gas-Phase Ion-Electron and Ion-Photon Reactions for Biomolecular Structural Determination”
Date: Wednesday, April 17, 2013
Location: 317 PETRIE SCIENCE BUILDING
Time: 1:00 PM

Abstract:  Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) provides unprecedented mass accuracy and resolution compared to other types of mass analyzers. In addition, this MS technique offers gas-phase dissociation approaches (i.e., tandem mass spectrometry or MS/MS) that are complementary to the traditionally used collision-activated dissociation (CAD). For example, vibrational activation can be accomplished via IR photon absorption in infrared multiphoton dissociation (IRMPD), which eliminates the need to introduce collision gas. Furthermore, gas-phase ion-electron reactions, including electron capture dissociation (ECD) and electron detachment dissociation (EDD), trigger radical ion chemistry that results in unique fragmentation pathways compared to CAD and IRMPD. Fragment ion spectra are interpreted to deduce molecular structure.

However, the powerful method of ECD requires at least two positive charges (because capture of one electron reduces the total charge by one and mass spectrometers cannot detect neutrals) and, thus, analysis of acidic biomolecules, which show improved ionization in negative ion mode, is challenging. We have explored the utility of divalent metals as charge carriers in ECD and found that positive ion mode ionization efficiency improves for sulfonated peptides and acidic glycans from glycoproteins.

In addition, ECD of metal-adducted sulfonated species proceeds with partial or complete retention of the highly gas-phase labile sulfonate, thereby allowing determination of its location. ECD of metal-adducted glycans provides intriguing fragmentation patterns with highly sought after sugar cross-ring fragmentation (which can provide linkage information) being dominant in several cases. EDD, which operates in negative ion mode, has lower fragmentation efficiency than ECD but also appears valuable for glycan analysis: We have shown that EDD provides structural information that is complementary to that obtained from ECD, CAD, and IRMPD, including additional cross-ring cleavages.

In more recent efforts, we have utilized chloride adducts to extend the utility of EDD (which requires at least two negative charges) and begun to explore electroninduced dissociation (EID) of singly charged analytes. Similar to metal-assisted ECD, chloride-assisted EDD results in complementary fragmentation pathways for carbohydrates, thereby generating more extensive glycan structural information. EID of manganese-adducted fatty acids allows double bond locations to be determined.

Finally, we have discovered that negatively charged biomolecular ions can capture electrons (rather than lose electrons such as in EDD, or undergo electronic excitation such as in EID). This phenomenon, which we termed negative ion electron capture dissociation (niECD), results in charged-increased species that undergo dissociation analogous to that in ECD of cationic species. niECD is exciting for several reasons, e.g.: 1) negative ion mode analysis yields improved ionization efficiency for many important acidic molecules, including phosphorylated and sulfonated peptides. 2) FT-ICR MS detection efficiency is proportional to charge and thus niECD results in improved detection limits. 3) niECD is compatible with singly charged ions and thus allows coupling with matrix-assisted laser desorption/ionization (MALDI).