Dr

Dr. Keith Ashman has extensive experience in the field of protein chemistry and automation. His achievements include: the first successful online HPLC separation for analysing the PTH amino acids produced by an Edman protein sequencer; the design and implementation of the first automated system for the in-gel proteolytic cleavage of gel separated proteins. He has held positions at the Max-Planck Institute for Molecular Genetics in Berlin, Germany, the European Molecular Biology Laboratory in Heidelberg, Germany, The Centre for Animal Biotechnology Melbourne, Australia, and at the Samuel Lunenfeld Institute in Toronto, Canada. He is currently a senior Scientist at MDS Sciex in Toronto.

Abstract

The key to any protein analysis is to prepare a “good sample”. However, since proteins exist in a wide range of concentrations and exhibit very diverse chemical properties, it is generally not easy to obtain the “good sample” or even a bad one. In this talk I would like to outline some of the methods that are in use and briefly discuss some new approaches to sample preparation such as LC-MALDI and the application of orthogonal Time of Flight mass spectrometry to protein analysis.

2^nd T.O.MS seminar (June 3, 2004)

Jeffery Smith

Jeff Smith completed his Honours Bachelor of Science in Biochemistry at Trent University in 2000, attaining the status of both Dean’s Honours Roll, as well as President’s Honours Roll. He began his Ph.D. at the Centre for Research in Mass Spectrometry with Professor K. W. Michael Siu in the Department of Chemistry, York University, in the fall of 2000. Since then he has published his work studying the non-covalent interactions within nitric oxide synthase using mass spectrometric methods, and is currently pursuing the identification of the protein compliment of the cilia of Tetrahymena thermophila. He is planning on defending his Ph.D. dissertation in the fall of this year.

Abstract

The ciliated protozoan Tetrahymena thermophila is a unicellular eukaryote characterized by nuclear dimorphism, with a degree of cellular structural and functional complexity comparable to that of human and other metazoan cells. It has proven extremely valuable as a model organism for many genetic and molecular biological studies. It contains an estimated 20-40,000 proteins, a rich resource for proteome analysis. Furthermore, its genome has recently been sequenced (http://www.tigr.org/tdb/e2k1/ttg/). Complete proteome databases have been made in-house by translating two forms of T. thermophila’s recently sequenced, raw genomic information in six reading frames. Two dimensional LC separations of a tryptic digest of the ciliary proteins have been conducted using SCX followed by RP LC using a LC Packings Nanobore LC system; the RP LC eluent was directly infused into a QSTAR Pulsar™ instrument for ESI MS/MS analysis. MS/MS data were searched in-house using Mascot™ software equipped with the aforementioned translated databases. Searching our preliminary data against the translated databases has resulted in high confidence identifications of 66 different axonemal proteins, as well as a further 34 proteins of comparatively lower confidence. “Scaffold” translations (large regions of translated genomic information) were treated as large proteins (2 – 75 MDa), and matching peptides localized in close proximity to each other indicated a coded region of the translated genome; these regions were BLASTed to find homologous proteins, and thus annotate the hit. The identification of proteins of lower confidence are currently being confirmed using new axonemal preparations. To the best of our knowledge, this is the first instance where a “scaffold” database has been used to successfully identify a proteome.

3rd T.O.MS seminar (September 10, 2004)

rick bagshaw

Rick Bagshaw is currently a graduate student in the department of Laboratory Medicine and Pathobiology and Collaborative Program in Neuroscience, conducting his work in Don Mahuran’s lab at the Hospital for Sick Children. He received his B.Sc. from Ryerson University in 1995 and then joined John Callahan’s lab at HSC where he studied lysosomal storage diseases prior to beginning his Ph.D. project on proteomics of the lysosomal membrane in January 2001. Rick is planning to defend a Ph.D. thesis at the end of 2004.

Abstract

When attempting comprehensive proteomic surveys of integral-membrane proteins, alternative techniques of protein sample preparation need to be to used because of the proteins’ hydrophobic nature. In my “sub-cellular”-proteomic study of the lysosomal membrane, I used a combination of denaturing Ion-exchange chromatography, SDS-PAGE, and LC-MS/MS as a strategy for protein separation and identification. In this talk I would like to present this protein identification strategy as applied to an integral-membrane protein fraction of the lysosome, some opinions on optimizing on-line LC:MS/MS for tryptic peptide separation and identification in proteomic surveys, and finally the identification of Alzheimer’s disease-associated γ-secretase complex proteins and a novel Arf-family protein in the lysosomal membrane.

T.O.MS guest seminar (October 7, 2004)

ANDREW ALPERT

In 1985 Dr. Alpert started PolyLC to develop HPLC materials and techniques. The company specializes in solutions to difficult protein and peptide separations and purifications, and manufactures chromatography columns and materials for such applications. Examples include the separation of closely related protein variants and the selective isolation of specific classes of peptides (disulfide-linked, phosphopeptides, etc.). Dr. Alpert introduced the technique of Hydrophilic Interaction Chromatography (HILIC) in 1990. This technique is suitable for analysis of polar solutes in general, just as reversed-phase HPLC is suitable for nonpolar solutes.

Abstract

Current interests to be covered in Dr. Alpert’s seminar include the development of techniques for analysis of particularly difficult proteins, including pathogenic prion proteins and proteomics analysis of both membrane and nonmembrane proteins.

4th T.O.MS seminar (November 18, 2004)

ERIC YANG

Eric studied the molecular interactions of the MEF2 transcription factor using the yeast two hybrid system during his graduate period at the University of Toronto. He then spent three years at York University working as a proteomics researcher under the supervision of Drs. Michael Siu and John McDermott. This work led to the identification of Chaperonin 10 and other markers for endometrial carcinoma using 2D-gel, SELDI, and ICAT approaches. Currently, Eric is directing the proteomics core facility at Sunnybrook and Women’s College Health Science Centre (http://swri.ca/services/proteomics).

Abstract

SELDI-TOF has been used to obtain protein profiles which are then used to differentiate/predict diseased from normal status of a sample. While it is relatively easy to obtain protein profiles, it is a different story to determine the identity of the potential markers. Two examples in identifying the potential cancer markers discovered by the SELDI-TOF approach will be discussed.
Typically, mass spectrometry-based protein identification requires complete genomic sequences. The second part of my talk will cover an application using MS-BLAST (http://dove.embl-heidelberg.de/Blast2/msblast.html) to identify proteins from distylous Turnera, in which little genomic information is available.
Finally, data will be presented on a seamless protocol including a silica carbide column (ProteoSpin^TM) to prepare tryptic digest of alkylated proteins for mass spectrometric analysis. Upon LC/MS-MS analysis of 10 fmol digested BSA, this strategy recovered 18 distinct peptides with 37% amino acid sequence coverage compared to 10 and 19% in the commercial sample. Interestingly, the peptides recovered by our protocol that were not present in the commercial supply contained at least 1 or 2 alkylated cysteines. The possible reasons for the alkylation-specific peptide gain in our protocol will be discussed.

5th T.O.MS seminar (January 20, 2005)

THOMAS KISLINGER

Thomas received his Master's Degree in Chemistry from the University of Munich, Germany (1998). During his Ph.D. he studied advanced glycation endproducts and their interaction with their cellular receptor RAGE at Columbia University and University of Erlangen, Germany. Since 2001 he has joined Andrew Emili as a postdoctoral fellow and has been concentrating his efforts on the development of LC-MS based methods for the analysis of mammalian proteomes.

Abstract

Development of a MudPIT based methodology for the high-throughput analysis of mouse tissues. The development of the methodology, some pitfalls and potential solutions will be discussed. Preliminary data obtained by the application to specific biological questions such as lung development, heart disease and tissue profiling will be presented.

6th T.O.MS seminar (March 18, 2005)

SERguei RASPoPOV

Dr. Raspopov has worked in research groups at the Univ. of Toronto (with Prof. J.C. Polanyi), the Univ. of Waterloo (Prof. T.B. McMahon) and York Univ. (Prof. K.W.M. Siu). His recent accomplishments include development of a new modification of High Pressure Mass Spectrometry, which allowed performing chemical equilibrium studies of biologically relevant compounds (amino acids) having low gas-phase volatility; discovery of a new most stable structure of the proton-bound dimer of glycine with unexpected binding motif; first study of the thermally-activated formation of peptide bond between amino acids in the gas phase and development of a novel technique (IRMPD in a QqTOF) for top-down characterization of proteins. He is currently involved in analytical method development using MS and separation techniques at Ontario Ministry of the Environment.

Abstract

Top-down sequencing is an alternative approach to protein analysis, by which the whole protein is subjected to fragmentation, as opposed to tryptic peptides characterized in the conventional bottom-up approach. This strategy provides more complete sequence data in shorter experiment time and offers a number of other significant advantages. However, it has a limited use at this time, since it is only amendable to FTICR instrumentation or by employing ion-ion reactions in customized ion traps (McLuckey group).

A novel experimental technique has been developed, allowing to perform efficient dissociation of peptides and intact proteins by IRMPD in a collision cell of a QqTOF instrument. Sequencing of selected proteins (equine myoglobin, bovine casein, human insulin and chaperonin 10) has been demonstrated using this approach. Fragmentation patterns and structural information obtained by this technique are similar to IRMPD performed in FTICR mass spectrometers. High fragmentation coverage was achieved, which permits confident identification of both proteins and species using database searching tools (Prosight PTM and MS Tag). Posttranslational modification were also detected and characterized, including phosphorylation of casein, disulphide bridges in insulin, removal of N-terminal methionine and acetylation of the second residue in chaperonin 10. Study of human chaperonin 10 (potential marker for endometrial cancer) also demonstrated applicability of this technique to analysis of biological samples, extracted from cell culture, purified by chromatographic techniques and ionized using nanospray.

7th T.O.MS seminar (May 25, 2005)

ROBIN HAW

Dr. Robin Haw received his Ph.D. in Genetics at the University of Nottingham in 1999 for his work in investigating the mechanisms of action of the yeast transcription factor Rap1p, under the supervision of Dr. Alistair Chambers. During his post-doctoral work at the National Institute of Bioscience and Human Technology in Japan, Robin continued his work on Yeast, studying regulation of glycolysis and pathogenesis in Candida. Prior to joining The Blueprint Initiative, Robin participated in the development and implementation novel methodologies for high-throughput synthetic lethality (SGA) screens and protein complex purifications as part of Genome Canada-funded yeast genomics and proteomics initiative, under Charlie Boone and Jack Greenblatt, at the Best Institute. Robin has been at Blueprint since 2003 as a molecular database curator, and now leads the curation effort on Blueprint’s contract with Science AAAS for re-development of the STKE resource.

Abstract

The Blueprint Initiative (http://www.blueprint.org/), is a not-for-profit, public good, data management research program affiliated with the University of Toronto. Blueprint has been established to develop and maintain the Biomolecular Interaction Network Database (BIND) as the world’s most comprehensive, free and publicly accessible interaction database. BIND’s success stems in part from high curation standards, which ensure consistent record quality and timely delivery to journal partners, including Science and Nature Publishing Group.

Biomolecular interaction data generated by large and expensive research efforts is valuable to on-going research. Simply publishing the data without archiving it results in a lost opportunity to maximise the impact of the interaction data to the research community. BIND is built on a robust data model capable of capturing biomolecular interaction data, from all organisms down to an atomic level of detail, which allows researchers to access interaction data that would otherwise only be accessible through time-consuming literature searches.

Access to information in a computable format, and in the context of other interaction data, will help scientists understand how complex molecules inside cells assemble to form living cells, both in healthy and disease states. For example, mass spectroscopy is a key technology to identify protein interactions within and between macromolecular complexes. However, this interaction data can be “noisy” or incomplete and there is a need for cross-validation, data integration and comparative analysis. BIND was conceived as a comprehensive archive of experimental molecular assembly data and provides the infrastructure to exchange and analyse interaction data from proteomic and other fast-evolving technologies.

8th T.O.MS seminar (September 23, 2005)

IAN STEWART

Dr. Stewart has been actively involved in mass spectrometry based research over the past fifteen years. In 1996, he received his PhD in Analytical Chemistry from the University of Alberta where he focused on Electrospray MS and instrument development for trace chemical measurements. He continued his research focusing on developing novel ion sources, fundamental research and application methods in support of trace element analysis and chemical speciation during his post-doctoral fellowships at the Ohio State University and at the NRC in Ottawa. Currently Dr. Stewart is the Director of Analytical Science at Protana Inc. (MDS Proteomics) where he has been heavily involved in the development of a robust MS based differential analysis platform for drug discovery and biomarker research. His current research focuses on developing quantitation strategies for the comparison and profiling of complex biological samples which includes labeling, post-translational modifications and data analysis challenges.

Abstract

The The ability to measure differences in protein abundance in order to address key biological and medical problems is rapidly becoming a requirement for mass spectrometry (MS) based proteomics. In the emerging field of biomarker discovery, the ability to achieve this goal is further challenged when working with plasma samples due to the presence of abundant proteins which often limit the lower level or dynamic range accessible by most MS methods. In addition, the complexity of such samples often requires MS instrumentation capable of high mass accuracy, resolution and dynamic range to accurately define, group and quantify peptides and proteins across sample data sets. The complexity of the samples also requires that the MS instrumentation has an effective duty cycle in order to properly reproduce and ‘MS/MS sample’ chromatographic peaks eluting into the MS. Finally, the need for some minimum statistical design places further requirements for the reproducible acquisition of large numbers of samples and the ability to process and sort large data sets.

In this study we describe the use of a multi-dimensional peptide fractionation approach followed by LTQ-FTMS (Thermo-Finnigan) for the label-free differential analysis and identification of protein biomarkers in mouse plasma samples derived from a db/db genetic mouse model of obesity and diabetes. The approach includes depletion of abundant proteins using the Agilent multiple removal affinity kit, off-line SCX fractionation of peptides followed by nano-LC/MS/MS analysis. For the purposes of this talk a description of the parameters used and experience with the LTQ-FTMS will be provided in conjunction with the a description of the results. Pooled mouse plasma from 9 control heterozygous and 9 disease homozygous db/db mice were used to generate 3 control and 3 disease sample sets. From each sample 20 SCX fractions were collected resulting in a total of 120 individual samples for LC/MS/MS analysis. Chromatographic peak detection, alignment and comparison across all 120 runs were performed using in-house software. The resulting set of over 5,000 unique sequenced peptides were grouped into ~2,000 unique protein clusters for both groups combined, using an algorithm that clusters proteins according to overlapping sets of peptides. Approximately 70 unique protein differential clusters encompassing ~400 peptides (ratios > 2, p-value < 0.05, false discovery rate of less than 0.1) were identified between db/db diabetic and control mice. Generally, biological annotation of these differential proteins showed functional roles consistent with changes in biological processes associated with diabetes. A preliminary validation study was conducted using immunoblot (Western) analysis of several of the biologically relevant differential proteins (normal plasma concentration 40-60 ng/ml), and the results indicate the same differential trend at the protein level as predicted by differential analysis of peptides by MS.

9th T.O.MS seminar (November 30, 2005)

LORNE TAYLOR

Lorne is currently a Staff Scientist at the Samuel Lunenfeld Research Institute responsible for assessing and implementing new mass spectral techniques for protein detection, mapping of post translational modifications and reagent based quantitation methods for SLRI biologists and Mt. Sinai research clinicians. Lorne has been involved with protein analyses for more than 20 years in both academic and industrial settings and has worked at MDS SCIEX in R&D and product development.

Abstract

Protein phosphorylation is a regulatory mechanism of signal transduction wherein it plays an important role in controlling a large number of cellular processes. MS based detection of phosphorylation sites on proteins is technically challenging. A new set of MS based tools for the detection and mapping of phosphorylation weill be discussed including a new LC-MALDI system from ABI-SCIEX which encorporates "direct drive" Exigent pumps, monolithic capillary LC and electrodeposition of samples onto MALDI targets. Phosphorylated protein standards, with and without titanium oxide enrichment of phosphorylated peptides were analyzed using a MALDI MS/MS system with a high rep rate laser. LC-MALDI advantages include increased sequence coverage, accurate mass MS and MS/MS and increased detection coverge of phosphorylated peptides.

10th T.O.MS seminar (January 27, 2006)

Rebecca Jockusch

Rebecca Jockusch is a new faculty member in the Chemistry Department at the University of Toronto where she will pursue combined mass spectrometry and optical spectroscopy studies. She is interested in the conformation, structure and interactions of biological molecules, with a particular focus on their intrinsic properties and how these are influenced by the environment, especially water. Rebecca received her PhD in Chemistry in 2001 from the University of California, Berkeley where she studied the gas-phase structure of non-covalent complexes using electrospray ionization Fourier transform mass spectrometry. Her post-doctoral studies, at the University of Oxford, focused on conformation and structure determination in isolated sugar and sugar-water clusters using UV and IR spectroscopy.

Abstract

Non-covalent interactions with molecules in the local environment, including water and cofactors, can have a dramatic effect of the structure and other properties of biological molecules. Better understanding these effects is important, not only from a fundamental perspective, but also to aid biological modelers and to answer an outstanding question of concern to people who use mass spectrometry as a tool to study biological systems: namely, how closely do gas-phase biomolecular properties (structure, stability, binding energies) resemble those of the solution?

We pursue a strategy of simplicity, first isolating biomolecules in the gas phase to study their intrinsic properties and then examining them in small clusters which have pieces of the local environment added back in. I will discuss results from my graduate studies examining the stabilization of gas-phase salt-bridges in hydrated amino acid-metal clusters and from my post-doctoral research examining the intrinsic conformational preferences of small saccharides and how they are affected by complexation with water. Finally, I will outline my plans to combine trapping mass spectrometry and fluorescence and fluorescence resonance energy transfer (FRET) as complementary techniques to characterize the structure and stability of biomolecules and their clusters in the gas phase.

11th T.O.MS seminar (March 30, 2006)

john marshall

John Marshall did his undergraduate degree at University if Toronto, his masters at the University of Waterloo and his Ph.D. with E. B. Dumbroff at Waterloo and Environment Canada. He has won University of Waterloo, the Ontario Provincial, NSERC National and Hospital for Sick Children Scholarship and Fellowship competitions. He worked as a Post-doc first with Eduardo Blumwald at UofT, then with Paul Walker at the Toronto General Hospital and subsequently with Sergio Grinstein at the Hospital for Sick Children. He spent two years in industry and reached the level of Vice President of Research and Development at SYNX Pharma. He then accepted an Assistant Professor position in the Department of Chemistry and Biology at Ryerson University in Sept 2003. He is a founding board member of the Ontario Cancer Biomarker Network and of YYZ Pharmatech Inc. and has authored some thirty published papers and patents to date.

Abstract

There is a need for openly available and explicitly detailed algorithms to non-redundantly enumerate and categorize the proteins detected in normal serum and to compare lists of proteins from different research groups or populations. The proteomic data published to date have been obtained by additive searches of MS/MS spectra against both human and non-human sequences including cDNAs and predicted hypothetical gene products. We mapped the results from 10 groups to the non-redundant reference sequence database by searches for MS/MS peptides and BLAST analysis of the full-length protein sequences to collapse the publicly available data into sets of representative non-redundant proteins. From the representative lists we estimated the common and unique proteins between publicly available lists of plasma and serum proteins. We observed that most groups identified proteins that have close homologues or exact sequences matches in the set of confirmed human expression products in the human Reference Sequence database. The number of potential sequences that found matches in RefSeq was larger for the exact peptide searching compared to full length BLAST matching. At least three groups agree on a set of 1671 types of proteins, while two groups agree on a set of 9258 proteins. Combining the data from all groups we found potentially 6669 proteins in RefSeq that had at least two different reported peptides. In general BLAST resulted in a lower number of non-redundant proteins.

12th T.O.MS seminar (July 16, 2006)

Scott D. Tanner

Associate Professor, Institute of Biomaterials and Biomedical Engineering, University of Toronto.

Last year, after 25 years of mass spectrometry development at MDS Sciex, Scott and his colleagues Vladimir Baranov and Dmitry Bandura, together with Olga Ornatsky of MDS Proteomics, spun into the University of Toronto. As an associate professor in the Institute of Biomaterials and Biomedical Engineering, his group is focused on the development of a mass spectrometer-based flow cytometer and reagents that enable massively multiplexed bioanalysis of single cells and particles. The work is already sufficiently successful that they are working on the spin out of their company DVS Sciences in January 2008.

'New Reagents and Instrument for Multiplexed Single Particle BioAssay with ICP-MS'

Abstract

Some 18 months ago, we had the honour to describe to you our ambitious plans for a multidisciplinary, multi-institute project to develop a mass spectrometer-based flow cytometer and to demonstrate its use for the subclassification of leukemia in patients’ samples. Since then, our group has moved to the University, renovated and moved into a new laboratory, established essential collaborations within and outside of the University, and made exceptional progress in the chemistry and technology that we described. We will show our current results for multiplexed analysis of single cells and beads using our now-functional research prototype instrument. These results have been further enabled with a new class of tagging reagents that provide 200 times better sensitivity than commercially available metal-labeled tags. We will also discuss a new opportunity and feasibility results for a bead-based equivalent of gene array analysis.

13th T.O.MS seminar (October 26, 2006)

Graham mcGibbon

Assistant Professor, Department of Biochemistry & Biomedical Sciences and Department of Chemistry McMaster University

'Functional proteomics: The long and winding road'

Abstract

At this juncture, it appears a germane question to ask whether or not, and if so how, small focused laboratory efforts to elucidate biological roles of individual proteins or protein classes by investigating their interactions using mass spectrometry can continue to provide insights that are both useful and complementary to larger scale systems biology efforts. There are a variety of experimental approaches of evolving suitability for probing interactions. Among these, mass spectrometry based technologies have advanced tremendously for effective protein identification. However, the complexity of samples remains a significant challenge to thorough analysis since protein quantities range from modest to minute and post translational modifications, which are crucial in cellular responses, can be sub-stoichiometric, heterogeneous and combinatorial. This presentation will address some aspects considered significant in the characterization of proteins of unknown or multiple functions. Selected bacterial proteins, and human cytoskeletal proteins and proteins involved in post translational modifications will be discussed along with aspects of the fairly standard instrumentation platforms and methods used in these analyses.

14th T.O.MS seminar (December 21, 2006)

anne-claude gingras

Assistant Professor, Department of Medical Genetics, University of Toronto.

1991-1994 BSc Biochimie, Laval University, Quebec

1994-2001 PhD Biochemistry, McGill University.

Supervisor: Nahum Sonenberg

Project: Signalling to translation initiation

2002-2005 Postdoctoral studies, Institute for Systems Biology, Seattle

Supervisor: Ruedi Aebersold

Project: Network of protein-protein interactions for PP2A-type phosphatases

2005- Investigator at Samuel Lunenfeld Research Institute at Mount Sinai Hospital

2006- Assistant Professor, Dept. of Medical Genetics, University of Toronto

‘Functional proteomics of serine/threonine phosphatases’

Abstract

The catalytic subunits of serine/threonine phosphatases assemble into multiple macromolecular complexes with other proteins (regulatory and adaptor proteins) that are thought to confer specificity to the dephosphorylation process. Identifying the interactions involving the catalytic subunits is therefore crucial to an understanding of the biological function of these phosphatases. Using an iterative affinity purification/mass spectrometry approach, we have defined stable protein-protein interaction networks centred around the catalytic subunits of human and yeast PP2A, PP4 and PP6. These networks now serve as a framework for functional analysis of phosphatase activity. A PP4-containing module involved in cisplatin sensitivity and checkpoint recovery after DNA damage will be presented. Building on our stable interaction networks, my laboratory is now using quantitative proteomic approaches to understand the dynamics of protein-protein interactions involving these phosphatases.

15th T.O.MS seminar (February 9, 2007)

eleftherios P. Diamandis

Eleftherios P. Diamandis, M. D., Ph. D., is currently Professor and Head, Division of Clinical Biochemistry, Department of Laboratory Medicine and Pathobiology, University of Toronto and Head, Section of Clinical Biochemistry, Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto.

Dr. Diamandis received his B.Sc. in Chemistry, Ph.D. in Analytical Chemistry and M.D. from the University of Athens, Greece and his Diploma in Clinical Biochemistry from the University of Toronto, Canada. He is a Certified Clinical Chemist by the Canadian Academy of Clinical Biochemistry and the American Board of Clinical Chemistry.

He published four books, 50 review papers and over 350 research papers. His interest is on tumor markers, genomic and proteomic technologies and their applications to diagnosis and monitoring of cancer.

‘STRATEGIES FOR DISCOVERING CANCER BIOMARKERS: A MASS SPECTROMETRIC APPROACH’

Abstract

Cancer biomarkers can be discovered by using various techniques, including microarrays, mass spectrometry, bioinformatics, molecular and other techniques. In this presentation, we will review briefly, the current strategies for discovering cancer biomarkers. We will also present one approach based on mass spectrometry and tissue culture systems with cancer cell lines. The approach will be exemplified by using breast cancer as an example. Potential difficulties and bottlenecks will be identified.

16th T.O.MS seminar (April 27, 2007)

RUNE LINDING

Rune Linding is an HFSP Research Fellow in the labs of Tony Pawson at the Samuel Lunenfeld Research Institute and Mike Yaffe at MIT where he is currently working on network medicine. Rune obtained his Ph. D. in 2004 on the subject of structural biocomputing, from the European Molecular Biology Laboratory (EMBL).

‘Systematic Discovery of In Vivo Phosphorylation Networks’

Abstract

Protein kinases transduce signals by phosphorylating specific substrates. Recent proteome-wide mapping of protein phosphorylation sites by mass spectrometry has discovered thousands of in vivo sites. However, in the majority of cases we do not know which of the ~518 human protein kinases are responsible for these events, resulting in fragmentary phosphorylation networks. This problem cannot be systematically resolved by current experimental or computational approaches. For example, individual kinases phosphorylate consensus sequence motifs, but these cannot accurately pair specific kinases with phosphorylation sites, potentially due to the importance of contextual factors, such as protein scaffolds, localization and expression in determining substrate specificity in the cell. We have therefore developed a method that augments consensus motifs with context for kinases and phosphoproteins. This increases the prediction accuracy to 81%, a 2.5 fold enhancement over the use of motifs alone, and improves the resolution from the kinase family-level to individual kinases. We demonstrate that context provides 60-80% of the predictive power for computationally assigning kinase-substrate specificity in the construction of in vivo phosphorylation networks. Building on the current human phospho-proteome we predict several novel phosphorylation networks of site-specific kinase-substrate interactions. We identified a subnetwork of DNA damage response (DDR) proteins and experimentally verify that human Rad50 is phosphorylated by ATM kinase in response to genotoxic stress. The DDR network suggests that ATM contributes significantly to the initiation and regulation of apoptosis. These results imply that molecular context is of great importance for in vivo kinase-substrate specificity and is needed in order to build phosphorylation networks from proteomics data. Our approach is completely general and can be applied to other types of post-translational modifications.

17th T.O.MS seminar (June 28, 2007)

Ken Yeung

Ken Yeung is an Associate Professor in the Department of Chemistry and Department of Biochemistry at the University of Western Ontario. He obtained his Ph.D. in Analytical Chemistry under the supervision of Charles Lucy (Calgary). He then worked with Richard Zare (Stanford) and Liang Li (Alberta) for his post-doctoral research before joining UWO. His research focuses on the development of new methods in the miniaturization of protein sample handling and separations prior to mass spectrometry.

‘The Past, Present (and Future) Role of Capillary Electrophoresis in Proteomics’

Abstract

The first commercial capillary electrophoresis (CE) instrument was introduced in the late 1980s. It was once believed to be a superior technology that could replace liquid chromatography. While the field has grown considerably in the past 20 years, and CE has become the predominant technique for a number of analyses (e.g., highly polar analytes, chiral pharmaceuticals), it is clear that liquid chromatography remains to be the method of choice in proteomics and many other applications.

In this seminar, the role of CE in proteomics research will be discussed. We will focus on the fundamental benefits of CE in proteomics as well as the technical challenges. Recent development in my group, in the area of microscale protein sample preparation for mass spectrometry, will be presented. They include the use of CE for protein enrichment, on-capillary proteolysis, and phosphopeptides fractionation.

18th T.O.MS seminar (October 12, 2007)

Panayiotis O. Vacratsis

Dr. Vacratsis is an assistant professor in the department of Chemistry and Biochemistry at the University of Windsor. His group specializes in applying biological mass spectrometry to study molecular mechanisms regulating protein kinases and phosphatases. In 2001, he received his PhD in Biochemistry and Molecular Biology from Michigan State University under the supervision of Dr. Kathleen Gallo. Before returning to his hometown of Windsor in 2003, Dr. Vacratsis was a NIH postdoctoral fellow in the lab of Dr. Jack Dixon at the University of Michigan.

'Desorbing Insights into Cell Signaling Pathways using MALDI-TOF Mass Spectrometry'

Abstract

Temporal and spatial regulation of signal transduction pathways is critical for a cell to properly respond to extracellular stimuli. These processes rely heavily on post-translational modifications and formation of multiprotein complexes to achieve transient changes in enzyme activity and substrate specificity. Mass spectrometry has become an essential discovery tool within this field due to its ability to map regulatory phosphorylation sites and elucidate novel signaling complexes. However, equally important to the mass spectrometry efforts are the interfacing techniques that are critical for capturing target molecules and reducing sample complexity. This presentation will highlight our twists to chromatography and mass spectrometry methods commonly used to study signal transduction systems. Also discussed will be how the application of these approaches has led to groundbreaking discoveries within poorly characterized signaling systems, creating the necessary framework to understand how these systems function in cellular regulation.

19th T.O.MS seminar (December 13, 2007)

Derek Wilson

Dr. Wilson is an Assistant Professor in the Department of Chemistry, Faculty of Science and Engineering at York University.
- BSc Trent (undergraduate thesis with Dr. Steven Rafferty) "Acid induced denaturation of the Core Oxygenase Domain of Inducible Nitric Oxide Synthase: A Study by CD, Fluorimetry and UV/visible Spectroscopy"
- PhD Western (with Dr. Lars Konermann, NSERC PGS-A and CGS-D) "Biochemical Kinetics Studied on the Millisecond Time-Scale by Electrospray Mass Spectrometry"
- Post-doc Cambridge (with Dr. Chris Dobson, NSERC PDF), NMR studies of amyloidosis in TTR105-115 and conformational dyanmics in Sso AcP

‘Time-resolved Electrospray Mass Spectrometry: A Powerful Tool for Monitoring Millisecond Time-scale Dynamics in Proteins’

Abstract

Proteins are dynamic molecules. Their biological function depends critically on processes that are inherently dynamic, i.e. ligand binding/release, conformational changes, bond vibrations. And yet the picture that emerges from the conventional tools used to study protein structure (predominantly X-ray crystallography) is overwhelmingly static.Crystal structures provide quantitative information only as to the ground state (or most readily crystalizable) conformation. This belies the emerging view that often biological activity arises not from the ground state, but rather from brief excursions to higher energy conformations. In any attempt to understand or predict protein function, it is critical that these transient conformations, and the dynamics linking them to the ground state, be well characterized. It should come as little surprise, then, that ‘structure based’ rational drug design and de novo protein engineering efforts have met with limited success. These efforts require highly accurate predictions of protein function that can only be made when the underlying dynamics are thoroughly understood.
My research is aimed at providing new insights into protein function by acquiring detailed, quantitative measurements of millisecond time-scale conformational dynamics. Our approach combines biophysical NMR, microfluidics and a powerful Electrospray Mass Spectrometry technique that I introduced in my PhD. This talk will focus on the Mass Spectrometry aspect, introducing the Time-resolved ESI-MS approach and it's applications in protein (mis)folding, dynamics and enzyme kinetics.

20^th T.O.MS seminar (February 22, 2008)

DARYL SMITH

Daryl obtained his Bachelor degree in biochemistry at Trent University in Peterborough, Ontario, and worked as a Quality Assurance Manager in a water testing laboratory before joining the Centre for Research in Mass Spectrometry in 2003 as a Laboratory Manager and doctorate trainee in chemistry.
His research centres around investigations of complex protein systems by liquid chromatography and tandem mass spectrometry including an investigation of the mitochondrial proteome of the ciliated protozoon Tetrahymena thermophila and the identification of protein biomarker in human ovarian cancer.

‘Exploring the Mitochondrial Proteome of the Ciliate Protozoon Tetrahymena thermophila: Direct Analysis by Tandem Mass Spectrometry’

Abstract

Using liquid chromatography (LC) and tandem mass spectrometry (MS/MS), we have undertaken to identify and annotate the proteins that comprise the mitochondria of Tetrahymena thermophila (TETTH), a ciliated protozoon. Here will be discussed the analytical methodologies used, the bioinformatics challenges that had to be overcome due to the fact that the TETTH proteome was previously unknown and therefore unavailable for database searching, and the biologically interesting results that were discovered.

BIOINFORMATICS: The identification of large numbers of proteins in complex solutions by LC and MS/MS is common practice for most proteomics-based research today and the handling of the large amount of data generated by this process is generally accomplished by submitting data to established protein databases using automated software. However, when exploring a frontier proteomic system, whose proteins are unknown, this approach is not possible and alternative methods must be developed. Here we will discuss the generation and interrogation of custom databases, created from translations of entire genomes or computationally predicted open reading frames.

BIOLOGY: 573 mitochondrial proteins were identified in total of which 545 were encoded by the nuclear genome and 28 by the mitochondrial genome. Of particular interest is one protein encoded by the mitochondrial genome that was not found by computational annotation protocols, but was discovered by our analysis. We have also identified a broad coverage of expected membrane bound and soluble proteins including members of the tricarboxilic acid cycle, electron transport chain and membrane translocases. Less expected, but not unprecedented, was the identification of several glycolytic enzymes, which appear to be operating inside the organelle. These results, as well as notes on organelle targeting, ORFan proteins and evolutionary insights will be discussed.

21^th T.O.MS seminar (October 31, 2008)

Parveen Sharma

PhD, University Of Birmingham, England Dept of Biochemistry with Dr. Barry Levine “Interactions of phospholamban with the Ca2+ -ATPase. (1997-2001)

Postdoctoral Fellowship (2001-2006). Dr. David MacLennan, Banting and Best Dept of Medical Research, “RyR mutations in malignant hyperthermia and central core disease”

Research Associate (2006-present), Dr Anthony Gramolini, Physiology, University Toronto. “Mass spectrometry analysis of cardiac and skeletal muscle”.

‘Identification and characterization of cell-surface associated proteins in the heart: Applications of a proteomic strategy’

Abstract

Cardiovascular disease is one of the leading causes of death in the developed world. With 50% of all drug targets being membrane proteins, insight into cardiomyocyte membranes and membrane associated proteins can provide a fundamental tool for innovative research into heart disease. However, the low abundance of membrane proteins together with technical issues of solubility has resulted in challenges to the large-scale proteomic studies of this class of proteins. In this study we have used cationic silica beads to enrich for cell-surface associated proteins from mouse neonatal primary cardiomyocytes. Through extensive MudPIT-based proteomics we have identified 3,420 proteins with high confidence. Through integrative data analysis and subtractive proteomics comparison to the cytoplasmic fraction, we arrived at a list of 379 high confidence cell surface associated proteins which included known membrane proteins. When compared to two previously published cardiac proteomes of over 6,000 proteins, we have identified 88 proteins not previously reported in cardiac tissue. Enrichment of cell surface associated proteins was confirmed by western blotting analysis of the silica bead bound fraction in comparison to levels found in the cytoplasmic fraction. Confocal microscopy of GFP labelled fusion proteins confirmed cell surface association in primary fibroblasts and cardiomyocytes. RT-PCR was used to validate cardiac expression of previously unannotated proteins. In this study, we have provided the first comprehensive study of cell surface-associated proteins in the heart.

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