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9th Montreal Post-ASMS Symposium

Free to attend Mass Spec Users’ Meeting

Sep. 17, 2014, Montreal (Holiday Inn Pointe Claire)

Many Thanks to our Sponsors of the 9th Montreal Post-ASMS Symposium

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Acknowledgement

to the following for CFABS Mass Spec Meetings & Symposiums

Analytical Steering Committee Board (ASCB)

• Prof. Christoph Borchers, Uvic - Genome BC Protein Centre

• Prof. Lekha Sleno, UQAM (Universite du Quebec a Montreal)

• Dr. Eric Reiner, Ontario Ministry of Environment

• Prof. Lars Konermann, University of Western Ontario

Technology Advisory Committee (TAC):

• Mr. Maroun El Khoury, Thermo Scientific

• Mr. Patrick Savory, Waters

• Mr. Tom Moy, AB Sciex

• Dr. Laurie Allan, Bruker Daltonics

• Dr. Marcus Kim, Agilent Technologies•

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PROGRAM AGENDA

03:30pm-04:00pm – Welcome Coffee/Tea and Refreshments

Plenary Oral Session - Chair: Prof. Lekha Sleno

04:00pm-04:20pm – "Development of a Novel 2D LC/MRM-MS Approach for Deeper and Broader Quantitation of Putative Protein Biomarkers in Human Plasma" - Presented by Prof. Christoph Borchers (University of Victoria-Genome BC Proteomics Centre, Victoria, BC)

04:20pm-04:40pm – "Parameters Affecting the Formation of Perfluoroalkyl Acids in 20 Canadian Wastewater Treatment Plants" - Presented by Dr. Mehran Alaee (Environment Canada, Burlington, ON)

04:40pm-05:00pm – "Integrating mass spectrometry with structural biology in the Mass Spec Studio" - Presented by Prof. David Schriemer (University of Calgary, Calgary, AB)

05:00pm-06:00pm – Networking Buffet Dinner

Parallel Oral Session 1 - Chair: Dr. Eric Reiner

06:00pm-06:20pm – "Consequences of tumor invasion of the skeleton" - Presented by Prof. Richard Kremer (McGill University, Montreal, QC)

06:20pm-06:40pm – "Resolution of Sildenafil-d8 Ionization Dependence on Sildenafil Concentrations " - Presented by Pierre-Yves Caron (inVentiv Health Clinique, Quebec City, QC)

06:40pm-07:00pm – "Strategies and Techniques for Identifying Unknown Compounds in Environmental Samples " - Presented by Dr. Eric Reiner (Ministry of Environment, Toronto, ON)

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07:00pm-07:20pm – "Conformational Dynamics of DNA G-Quadruplex in Solution Studied by Kinetic Capillary Electrophoresis Coupled On-line with Mass Spectrometry" - Presented by Prof. Maxim Berezovski (University of Ottawa, Ottawa, ON)

07:20pm-07:45pm – Parallel Poster Presentations Session 1 Chaired by Dr. Alexandra Furtos (University of Montreal, Montreal, QC)

Parallel Oral Session 2 - Chair: Christoph Borchers

06:00pm-06:20pm – "Nanodiscs and CaR-ESI-MS: A novel method for the discovery of protein-glycosphingolipid interactions" - Presented by Prof. John Klassen (University of Alberta, Edmonton, AB)

06:20pm-06:40pm – "Improved detection of SUMOylated peptides in large scale proteomic analyses using High Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) " - Presented by Dr. Eric Bonneil (University of Montreal, Montreal, QC)

06:40pm-07:00pm – "Proteomics profiling of pediatric serum and discovery of biomarkers for differentiation of the cause of febrile illnesses in Madagascar " - Presented by Laetitia Cortes (Caprion, Montreal, QC)

07:00pm-07:20pm – "Absolute quantitation of NAPQI-modified serum albumin from rat plasma samples by LC-MS/MS: monitoring acetaminophen toxicity" - Presented by Prof. Lekha Sleno (UQAM, Montreal, QC)

07:20pm-07:45pm – Parallel Poster Presentation Session 2 Chaired by Dr. Mark Watson (Caprion, Montreal, QC)

07:45pm-08:15pm – Dessert with Coffee/Tea

Plenary Oral Session - Chair: Prof. Lekha Sleno 08:15pm-08:40pm – "Environmental analysis of poly- and perfluoroalkyl compounds using a Q-

Exactive Orbitrap: optimization for a laser diode thermal desorption method " - Presented by Prof. Sebastien Sauve (University of Montreal, Montreal, QC)

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08:40pm-09:00pm – "Multi-modality 3-D imaging of a human carotid atherosclerotic plaque: Correlating in vivo ultrasound and imaging mass spectrometry " - Presented by Prof. Pierre Chaurand (University of Montreal, Montreal, QC)

09:00pm-09:15pm – "Quantification of Growth Hormone Receptor Antagonist Pegvisomant by LC-MS/MS in Rat Plasma: Method Development Considerations for PEGylated Proteins" - Presented by Dr. Fabio Garofolo (Algorithme Pharma, Laval, QC)

List of Poster Presentations

Poster Parallel Session 1 Chaired by Dr. Alexandra Furtos

1. "Rapid Screening of Adulterated & Counterfeit Products using Bench-Top High Resolution Mass Spectrometer and mzCloud Database Search" - Presented by Philippe Lebel1 or Alexandra Furtos1 Philippe Lebel1; Alexandra Furtos1; Karen Waldron1; Kate Comstock2; Tim Stratton2; Maroun EI Khoury2 1Université de Montréal, Montréal, Qc, Canada; 2Thermo Fisher Scientific, San Jose, CA, USA

2. "Improvement of Sensitivity and Robustness of an LCMSMS Quantitation Method for Digoxin,

Controlling the Reactivity of the Deuterated Internal Standard" - Presented by Luc Bouchard Luc Bouchard; Carine Levesque; Nathalie Pelletier; Nadine Boudreau; Ann Levesque inVentiv Health Clinical, Quebec, Canada

3. "Quantitative performance of the Q-Exactive high-resolution accurate-mass (HR/AM) spectrometer for the analysis of tetracyclines in a complex environmental matrix" - Presented by Morgan Solliec Morgan Solliec; Audrey Roy-Lachapelle Université de Montréal, Montréal, CANADA

4. "Assessment of relative efficiency and selectivity of extraction methods for global metabolomics by LC-MS" - Presented by Dmitri Sitnikov Dmitri Sitnikov; Dajana Vuckovic Concordia University, Montreal, CA

5. "Ultra-fast LDTD-APCI-MS/MS analysis of gamma-hydroxybutyric acid and its precursors in

beverages and biological samples" - Presented by Paul Fayad1 Paul Fayad1; Sung Vo Duy1; André Lajeunesse2; Sébastien Sauvé1 1Université de Montréal, Montreal, QC, Canada; 2Université du Québec à Trois-Rivière, Trois-Rivières, QC, Canada

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Poster Parallel Session 2 Chaired by Dr. Mark Watson

1. "Large-scale analysis of lysine SUMOylation by SUMO remnant immunoaffinity profiling" - Presented by Frederic Lamoliatte1 Frederic Lamoliatte1; Danielle Caron1; Chantal Durette1; Louiza Mahrouche1; Mohamed Ali Maroui2; Olivier Caron-Lizotte1; Eric Bonneil1; Mounira Chelbi-alix2; Pierre Thibault1 1Institute for Research in Immunology and Cancer, Montréal, Qc, CANADA; 2CNRS FRE3235, Université Paris Descartes, Paris, France

2. "Development of imaging MS methods to monitor the molecular composition of latent fingermarks"

- Presented by Nidia Lauzon Nidia Lauzon1; Matthew Howland2; Martin Dufresne1; Vinita Chauhan2; Pierre Chaurand1 1Université de Montréal, Montreal, CANADA; 2Health Canada, Ottawa, Canada

3. "Proteomic analysis of wastewater cultured Chlamydomonas reinhardtii: Comparing protein

expression for enhanced phycoremediation and biomass production" - Presented by Anil Patel1 Anil Patel1; Eric Huang1; Mark Lefsrud1; Etienne Low-De'carie2 1Université de Montréal, Montréal, Qc, Canada; 2Thermo Fisher Scientific, San Jose, CA, USA

4. "Identifying reactive metabolite protein targets in liver microsomes using click chemistry and

affinity purification" - Presented by Andre Leblanc Andre Leblanc; Tze Chieh Shiao; René Roy; Lekha Sleno UQAM, Montreal, CANADA

5. "Site-specific quantitation of lysine acetylation in isomeric peptides of histones H3 and H4" -

Presented by Nebiyu Abshiru1, 2 Nebiyu Abshiru1, 2; Olivier Caron-Lizotte1, 2; Roshan Elizabeth1, 2; Alain Verreault1, 2; Pierre Thibault1, 2 1University de Montreal, Montreal, CANADA; 2Institute for Research in Immunolgy and Cancer, Montreal, QC

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Oral Sessions Abstracts & Speaker Biographies

 Oral 01: “Development of a Novel 2D LC/MRM-MS Approach for Deeper and Broader Quantitation of Putative Protein Biomarkers in Human Plasma”

Speaker: Prof. Christoph Borchers (University of Victoria-Genome BC Proteomics Centre, Victoria, BC)

Introduction Rapid, sensitive, and reproducible protein quantitation methods are required to verify and validate the growing list of disease biomarker candidates in human biofluids such as blood plasma. Although immunoassays are ideal for use in clinical validation and employment, a bottom-up proteomic approach utilizing stable isotope-labeled standards (SIS) and LC/MRM-MS has emerged as a powerful and preferred technique for biomarker verification. The multiplexing ability and detection sensitivity of this method, however, remains rather limited, especially at levels below 5 ng/mL. We have therefore developed a novel 2D RPLC/MRM-MS method, utilizing high and low pH separations with internal SIS peptides for the highly multiplexed and sensitive quantitation of >250 high-to-low abundance disease biomarker candidates in undepleted human plasma.

Methods Tryptic digestion of a pooled plasma control sample was conducted following a standard, sample pretreatment workflow. A complex mixture of 1035 synthetic SIS peptides (corresponding to 423 proteins) was spiked in post-digestion, for normalization. Samples were then extracted and fractionated by rapid 2D RPLC, configured in an off-line alkaline (ammonium hydroxide, pH 10) separation, followed by an on-line acidic (formic acid, pH 3) separation. The on-line LC step was interfaced to a triple quadrupole mass spectrometer for dynamic MRM measurements via positive ESI on an Agilent 6490 triple quadrupole mass spectrometer. Development involved optimization and interference testing, with quantitation conducted on 13 LC fractions. Application of this method to patient samples is currently being explored.

Preliminary Results/Abstract We have previously demonstrated the ability to quantitate the top 6 order-of-magnitude concentration range of plasma proteins with only a simple sample pre-treatment and without the need for antibody depletion or enrichment, or additional chromatographic or electrophoretic fractionation (Percy et al. BBA, 2013). There, standard-flow RPLC/MRM-MS enabled the quantitation of proteins with concentrations as low as 44 ng/mL (myeloblastin). To enhance the depth and breadth of quantitation beyond that level, a novel 2D-LC fractionation method was developed. This method utilized alkaline and acid separations, with the former using an ammonium hydroxide-based mobile phase, in contrast to the ammonium formate or ammonium acetate mobile phases conventionally used in proteomics. Preliminary optimization involved tuning the transition-specific parameters, adjusting the high-pH LC gradient, and evaluating several fraction-pooling strategies. The final method involved the analysis of 13 LC fractions (consisting of ca. 80 peptides/fraction) which were obtained by selectively pooling some of the 47 fractions which were obtained over a 31 min run. Subsequent analysis of technical replicates (n = 3) showed a high level of consistency in peptide elution and signal, and good quantitative performance. Concentrations of 256 disease-related plasma proteins spanning an 8 order-of-magnitude range (from 15 mg/mL to 450 pg/mL) could be determined. This represented a 2 order of magnitude enhancement in the quantitative depth over that attainable without fractionation, as revealed by parallel 1D LC/MRM-MS experiments with matched proteomic samples. From this comparison, 83 proteins were quantified only by 2D, with 18 proteins quantified below 5 ng/mL and 55 presenting concentrations below the 44 ng/mL limit found in our 1D results. The improvements provide a springboard for application to our complete panel (634 interference-free peptides

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corresponding to 256 disease-linked proteins), or a subset thereof. We are currently exploring method applications to patient samples for protein biomarker verification.

Novel Aspect Simple and fast LC fractionation toward more comprehensive and multiplexed MRM quantitation of high-to-low abundance proteins in undepleted human plasma.

Speaker Biography:

Christoph H. Borchers, Ph.D., Full Professor, University of Victoria

Dr. Borchers received his B.S., M.S. and Ph.D. from the University of Konstanz, Germany. After his post-doctoral training and employment as a staff scientist at NIEHS/NIH/RTP, in North Carolina, he became the director of the UNC-Duke Proteomics Facility and held a faculty position at the UNC Medical School in Chapel Hill, NC (2001-2006). Since then, Dr. Borchers has been employed at the University of Victoria (UVic), Canada and holds the current positions of Professor in the Department of Biochemistry and Microbiology and the Don and Eleanor Rix BC Leadership Chair in Biomedical and Environmental Proteomics. He is also the Director of the UVic – Genome BC Proteomics Centre, which is one out of five Genome Canada funded Science & Technology Innovation Centres and the only one devoted to proteomics. Dr. Borchers is also appointed as Professor at McGill University in the Department of Oncology, Montreal, QC.

His research is centred around the improvement, development and application of proteomics technologies with a major focus on techniques for quantitative targeted proteomics for clinical diagnostics. Multiplexed LC-MRM-MS approaches and the immuno-MALDI (iMALDI) technique are of particular interest. Another focus of his research is on technology development and application of the combined approach of protein chemistry and mass spectrometry for structural proteomics. Dr. Borchers has published over 190 peer-reviewed papers in scientific journals and is the founder and CSO of two companies, Creative Molecules. Inc. and MRM Proteomics Inc. He is also involved in promoting proteomic research and education through his function as HUPO International Council Member, Scientific Director of the BC Proteomics Network and Vice-President, External of the Canadian National Proteomics Network.

Oral 02: “Parameters Affecting the Formation of Perfluoroalkyl Acids in 20 Canadian Wastewater Treatment Plants”

Speaker: Dr. Mehran Alaee (Environment Canada, Burlington, ON)

Introduction Perfluoroalkyl acids (PFAAs) contain dual hydrophobic and hydrophilic moieties that make materials both oil and water resistant. This makes them useful in the production of apparel, carpets, and packaging products, as processing additives during fluoropolymer production, and as surfactants in consumer applications. However, PFAAs are potentially persistent in the environment, bioaccumulative, and toxic.

The objectives of this investigation were a) to determine PFAA concentrations in the liquid and solid streams of 5 different wastewater treatment types (facultative lagoon (FL), aerated lagoon (AL), chemically-assisted primary treatment (PT), secondary aerobic biological treatment (ST), and advanced biological nutrient removal treatment (AT)); b) study parameters affecting PFAA removal and fate, and c) conduct mass balances to delineate the fate of PFAAs.

Materials and methods Raw influent (RI), primary effluent (PE), and final effluent (FE) samples were 24-h equal volume composite collected. Primary sludge (PS) was sampled from the underflow of the primary clarification tank and waste biological sludge (WBS) was collected from the underflow of the secondary clarification tank. Treated

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biosolid was sampled after the final treatment step. PS, WBS, and biosolids were collected as grab samples. Each WWTP was sampled for 3 consecutive days during the summer (June to September) and winter (January to April) seasons, in 2009 or 2010. PFAAs were extracted using solid phase extraction and then analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS).

Results and discussion PFAAs were analyzed in 386 liquid samples: RI (n=149), PE (n=90), and FE (n=147). In order to evaluate the performance of different WWTPs and seasonal differences, PFAA formation percentage was calculated by %formation= ((effluent concentration – influent concentration)/influent concentration) x100. The formation of PFAAs is likely due to the breakdown of PFAA precursors during wastewater treatment. Previous investigations showed that fluorotelomer alcohols (FTOHs) were transformed to PFOA, PFNA, and PFHxA [1] while N-ethyl perfluorooctane sulfonamidoethanol (N-etFOSE) was transformed to PFOS [2]. Precursors have been detected in the atmosphere above the primary clarifier of a Canadian WWTP and their levels decreased at the aeration tank (~50%), probably due to their conversion to PFAAs [3].

PFAA levels were evaluated in 110 PS, 76 WBS, and 108 treated biosolid samples. Higher concentrations of PFAAs were obtained in WBS compared to PS maybe due to the longer contact time and recirculation of WBS in the aeration tank and secondary clarifier, giving more opportunity for PFAAs sorption onto sludge. PS and WBS may also have different sorption tendencies. In biosolids, PFAAs were detected in all samples where PFOS was the most prevalent compound (median 13 ng/g). The next highest concentrated compounds were PFOA>PFDA>PFNA, and even-chain length perfluorinated carboxylic acids (PFCAs) like PFOA, PFDA, and PFDoA that accounted for 79±12% of total PFAAs.

Finally, mass balances were conducted to evaluate PFAA mass loadings in FE and biosolids as well as their formation. Total PFAAs mass loadings in FE were significantly higher (median 5400 mg/d) compared to biosolids (median 160 mg/d). Estimation of the FE:biosolids ratio in mass loadings ranged from 0.8 to 38 (median: 11) and from 0.2 to 170 (median: 5.8) in plants with PT and ST/AT, respectively, suggesting a higher burden of PFAAs to the environment through effluent..

Conclusions Investigation of parameters affecting PFAAs formation showed that PT processes formed the least PFAAs compared to biological treatment processes, possibly due to minimum biological activity and short HRT. Higher temperature and longer HRT increased the formation of PFAAs. Mass balances confirmed the formation of PFAAs, the low sorption of PFAAs onto biosolids and their consequent release to the aquatic environment at mass loadings up to 140 g/day. Additional studies on levels of PFAA precursors and their transformation patterns would be beneficial to better understand how PFAA are formed in WWTPs.

References:

[1] N. Wang, B. Szostek, R.C. Buck, P.W. Folsom, L.M. Sulecki, J.T. Gannon, 8-2 Fluorotelomer alcohol aerobic soil biodegradation: Pathways, metabolites, and metabolite yields, Chemosphere 75 (2009) 1089.

[2] K.R. Rhoads, E. M.-L. Janssen, R. Luthy, C. S. Criddle, Aerobic biotransformation and fate of N-ethyl perfluorooctane sulfonamidoethanol (N-EtFOSE) in activated sludge, Environ. Sci. Technol. 42 (2008) 2873.

[3] L. Ahrens, M. Shoeib, T. Harner, S.C. Lee, R. Guo, E.J. Reiner, Wastewater treatment plant and landfills as sources of polyfluoroalkyl compounds to the atmosphere, Environ. Sci. Technol. 45 (2011) 8098.

Speaker Biography

Dr. Alaee is a research scientist at the Water Science and Technology Directorate of Environment Canada, and an adjunct professor at the Department of Chemistry, Queen’s University. He received his B.Sc. in Biochemistry from Concordia University, and his Ph.D. in analytical chemistry from the University of Guelph.

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Mehran’s research interest is in the determination of sources, and fate of emerging persistent organic pollutants (POPs) such as brominated flame-retardants (BFRs), siloxanes and phthalates to the Great Lakes and the Arctic. Last year, he completed a 3-year study to evaluate the impact of siloxanes on the Canadian Environment and Health of Canadians. He is currently focusing on the sources and fate of phthalates in the Canadian environment.

Dr. Alaee has authored and co-authored numerous papers on the occurrence and fate of POPS including BFRs and siloxanes in the environment. He has also edited and co-edited several special issues of Chemosphere on BFRs, siloxanes and other POPs.

Mehran is member of the editorial boards of Chemosphere. He is also a member of the International Advisory Board for the International Symposium on Halogenated Environmental Organic Pollutants and POPs, co-chaired Dioxin 2005, and is co-chair of Dioxin 2017 to be held in Vancouver. He is a member of the International Advisory Board for International Symposium on Brominated Flame Retardants in the Environment symposium and chaired BFR 2004.

Oral 03: “Integrating mass spectrometry with structural biology in the Mass Spec Studio”

Speaker: Prof. David Schriemer (University of Calgary, Calgary, AB)

Combining biophysical data from multiple sources is critical for developing accurate structural and functional models of dynamic multi-protein systems. Mass spectrometry can be used to measure the insertion location for a wide range of chemical probes, and such insertion data provide a rich but disparate set of topographical modeling restraints. We have developed routines that integrate mass spec data with protein modeling activities. These routines are built on a novel architecture for app development (the Mass Spec Studio). The presentation will briefly highlight the concepts and technologies behind this new approach to software development in mass spectrometry, and then describe an app package useful for MS-driven integrative structural biology.

The structure biology app package mines any labeling data from any mass spectrometer in a proteomics-grade manner, and converts label data into molecular docking restraints. These restraints are implemented within the HADDOCK framework to drive complex protein modeling problems. Specifically, support is provided for covalent labeling chemistries and hydrogen/deuterium exchange (HX). It includes novel acquisition strategies such as targeted HX-MS2 and data-independent HX-MS2 (CID and ETD-based). The MS2 modes extend measurements to highly complex protein systems, which we demonstrate through the analysis and modeling of microtubule-ligand interactions.

Speaker Biography:

Dr. David Schriemer studied chemistry and mathematics at the University of Manitoba and the University of Winnipeg and obtained his Ph.D. in chemistry at the University of Alberta (1997). As a postdoctoral fellow and in research leadership for two biotechnology companies, he developed MS-based technologies for drug discovery and protein interaction analysis. He returned to academia in 2001, where he is an Associate Professor in the Department of Biochemistry & Molecular Biology, and an adjunct in the Department of Chemistry. He is also the Director of the SAMS Centre for Proteomics at the University of Calgary.

His research group fuses a fundamental interest in measurement science with a fascination for the inner workings of cell division. One goal is to understand the role of microtubules in orchestrating the separation of replicated chromosomes, and to turn this enhanced understanding into novel therapies. The group has been driving mass spectrometry as an approach for modeling complex protein systems – in both structural and conformational terms.

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It has allowed them to generate new knowledge about tubulin self-assembly and most recently, it has supported a program in rational drug design centred on a novel strategy for antimitotic drug therapy. Structural mass spectrometry is data-intensive, requiring a strong focus on computational methods and informatics. His group has produced software adopted by researchers and instrument vendors alike.

Dr. Schriemer holds a Canada Research Chair in Chemical Biology and is an AIHS Heritage Senior Scholar.

Oral 04: “Consequences of tumor invasion of the skeleton”

Speaker: Prof. Richard Kremer (McGill University, Montreal, QC)

Skeletal metastases have devastating consequences on patient’s quality of life leading to intractable pain ,pathological fractures and frequently hypercalcemia.In 1889 Stephen Paget proposed the “seed and soil” hypothesis in a landmark article published in the Lancet entitiled”The distribution of secondary growths in cancer of the breast”. Paget observed that patients with breast cancer often developed secondary tumors in the liver. He postulated that tumor cells (the seed) evading from the primary tumor can only successfully establish at distant sites (the soil) that have a fertile growth environment.Consequently,successful seeding of these cancer cells depends largely on their interaction with the stroma, or tumor microenvironment in target organs.

Over the past 30 years this hypothesis has been carefully tested in breast cancer models spreading to the skeleton. When cancer cells seed in the bone microenvironment, they trigger the activation of receptor activator of nuclear factor-kB ligand (RANKL), a critical mediator of bone turnover expressed by the osteoblast and the osteocyte which then binds to RANK at the surface of the osteoclast to activate bone resorption.Osteoclasts activation in the bone microenvironment(“soil”) also triggers the production of transforming growth factor-β (TGF-β), and insulin-like growth factor 1 IGF-1, TGF-β, IGF-1 and RANKL which then promote tumor cell proliferation within bone contributing to a vicious cycle of mutual cooperation. This vicious cycle is thought to play a critical role in the establishment and progression of skeletal metastases.

Therapies aimed at reducing the skeletal complications of cancer have primarily targeted the “soil”. Bisphosphonates are small molecules analogs of inorganic pyrophosphate that have a high affinity for bone and suppress bone resorption by causing osteoclast apoptosis .Aminobisphosphonates such as pamidronate and zoledronate administered at high dose intravenously in women with breast cancer with established bone metastases can significantly reduce pain and other skeletal complications. In contrast none of the studies to date evaluating bisphosphonates in a secondary prevention framework showed any effect on bone metastases. A recent study using a monoclonal antibody against RANKL (Denosumab) showed a protective effect against the development of bone metastases in patients with prostate cancer. Our group, in a prospective cohort study, recently reported that use of bisphosphonates for osteoporosis prevention or treatment was associated with a reduced risk of developing bone metastases in women diagnosed with breast cancer and improved overall and cancer-related survival. Clinical trials are currently underway to evaluate the efficacy of bisphosphonates and Denosumab in a variety of cancer as a preventative strategy in bone metastases.

In contrast to the many studies aimed at suppressing the activity of the bone microenvironment (‘soil”), very little attention has been paid to directly targeting the metastatic cells in bone. We will review potential strategies aimed at targeting both the “soil” and the “seed”..

Speaker Biography:

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Richard Kremer, MD, Ph.D. FRCPC

Dr Richard Kremer is currently Professor and Director of the Bone and Mineral Unit in the Department of Medicine of McGill University. He is also the co-leader of the Musculoskeletal Axis of the McGill University Health Centre. Born in Paris, Dr Kremer received his MD (1977) and Ph.D. (1979) degrees at the Pierre et Marie Curie University of Paris, and completed his internal medicine and endocrinology residency at La Pitié-Salpêtrière Hospital in Paris. He then moved to Canada and completed his residency training at the Montreal General Hospital, Royal Victoria and Ottawa Civic Hospitals. He pursued his research training at the Royal Victoria Hospital and joined the Division of Endocrinology as a full-time physician-scientist in 1989.

His research focuses on the role of calcium regulating hormones in health and disease. Over the past twenty years his laboratory has explored the role of vitamin D and parathyroid hormone related peptide (PTHrP) in cancer and musculoskeletal disorders. His team was the first to demonstrate the efficacy of vitamin D on bone aging and prevention of fat accumulation in the bone marrow. Recently, he co-authored the first evidence linking vitamin D insufficiency, muscle fat accumulation and muscle strength. His laboratory established the role of the hormone PTHrP as a biomarker of survival in cancer and more recently as a critical mediator of tumor initiation and progression in breast cancer. His clinical research unit located at the McGill University health Center also participates in major multicenter trials on osteoporosis and bone–related cancer trials.

Dr Kremer is the recipient of several prizes and awards including the Chercheur National award from the Fonds de Recherches en Santé du Québec. He has made significant contributions to the Canadian Institutes of Health Research (CIHR), the US National Institutes of Health (NIH) and the US National Cancer Research Institute (NCI) on grant and award committees. He is a reviewer and sits on the Editorial Board for several international journals and is a consultant to industry, academic and lay organizations in his capacity as a clinician scientist and expert in metabolic bone diseases.

Oral 5: “Resolution of Sildenafil-d8 Ionization Dependence on Sildenafil Concentrations”

Speaker: Pierre-Yves Caron (inVentiv Health Clinique, Quebec City, QC)

Introduction Sildenafil is a selective phosphodiesterase type 5 (PDE5) inhibitor. PDE5 is responsible for breaking down cyclic guanyl monophosphate (cGMP), a vasodilator produced in response to nitric oxide. Internal standard ionic suppression was observed when Sildenafil and Desmethyl Sildenafil upper limit of quantitation was raised from 250 ng/mL up to 1500 ng/mL and 125 ng/mL up to 750 ng/mL, respectively. This ionic suppression gave a difference of approximately 20% (up to 30%) in internal standard area response between a LLOQ and a ULOQ sample. Because of this important variation, a modification in chromatography and ionization mode was performed to solve this issue.

Methods Original chromatography used a Zorbax SB-C18 50x4.6 analytical column and ionization was performed with a turbo ion spray source operated in positive mode. Ion spray voltage was set at 4500 under a temperature of 400°C. Nebulizer and auxiliary gases as well as mass spectrometer parameters were set to achieve the best analyte and internal standard response. Transitions monitored were 475.3 ? 283.1, 461.4 ? 283.2 and 483.6 ? 283.1 for Sildenafil, Desmethyl Sildenafil and internal standard respectively. The new chromatography uses an ACE 3 C18 30x4.6 analytical column and ionization procedure uses an APCI source, still operated in positive mode and with a needle corona potential of 3. Most of the MS parameters are slightly modified to increase signal response.

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Preliminary Results/Abstract Internal standard ionic suppression was not observed when the assay used a range of 1-250 ng/mL and 0.5-125 ng/mL for Sildenafil and Desmethyl Sildenafil, respectively. This problem became apparent when ULOQ was incresed. Internal standard area response varied based on the concentration of Sildenafil in the sample. A difference of approximately 25% could be observed between a LLOQ and a ULOQ sample, ranging from 6300000 to 7800000. Although that the accuracy for sildenafil was not affected since the IS is Sildenafil-d8, the high end of the curve for Desmethyl Sildenafil was positively biased up to 24%. An improvement in chromatography was made in order to solve the problem and the analytical column modification gave a shorter retention time, narrower peaks and an increase in peak height, sharpness and signal to noise ratio. These improvements did not solve the ionic suppression problems, but the increase in effectiveness made the use of APCI ionization source possible. The new source parameters were optimized and internal standard ionization became stable and reproducible. The new difference of internal standard response was approximately less than 5%. The loss of signal response caused by the ionization mode modification was mitigated by the increase in chromatography efficiency. For Desmethyl Sildenafil, the accuracy at the high end of the calibration curve was 106%. Selectivity was also evaluated with the new conditions. The new method was also tested successfully for the matrix effect including hemolyzed and lipemic plasma. The assay was used for study sample analysis and was shown to be reproducible with reassay confirmation rate near 100%.

Novel Aspect Sildenafil-d8 ionization dependence on Sildenafil concentrations was solved by modifying chromatography and ionization mode.

Speaker Biography:

Pierre-Yves Caron, M. Sc., Research Scientist, inVentiv Health clinique

Pierre-Yves Caron received his B. Sc. in Chemistry from Université de Sherbrooke, Québec, in 2008. He then pursued his M. Sc. studies in synthetic organic chemistry under the supervision of professor Pierre Deslongchamps at Université de Sherbrooke, which he completed in 2010. He also published scientific articles in peer-reviewed journals and presented posters in scientific conventions such as ASMS and AAPS. He cumulates experience in pharmaceutical chemistry, drug discovery, synthetic organic chemistry, analytical chemistry and bioanalytical method development. He joined inVentiv Health clinique in 2011 and has since then worked as a Research Scientist in bioanalytical method development.

Oral 06: “Strategies and Techniques for Identifying Unknown Compounds in Environmental Samples”

Speaker: Dr. Eric Reiner (Ministry of Environment, Toronto, ON)

Introduction There are approximately 100,000 industrial chemicals or chemicals of commerce used currently, but only a small fraction are monitored routinely. The Stockholm Convention on persistent organic pollutants (POPs) targets 24 halogenated organic compounds or compound classes. Environmental samples contain hundreds or thousands of non-targeted compounds, some of which may pose a risk to the environment or human health. Their identification can be very challenging because most routine analytical techniques are transparent to non-targeted compounds. (Ultra)High resolution mass spectrometry (MS) and multidimensional gas chromatography (GCxGC) are complementary analytical techniques for non-targeted analysis of environmental samples. This contribution deals with the strategies implemented by our group to identify halogenated compounds, which are often POPs, in a range of challenging environmental samples.

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Methods GCxGC-TOF experiments were performed using a LECO modified Agilent 6890 system with secondary oven/quad jet modulator connected to a LECO Pegasus 4D TOF or electron capture detector. High resolution TOF experiments (8000 FWHM) were obtained using a Waters GCT connected to an Agilent 6890 GC that has been converted to a GCxGC instrument with the addition of a Zoex single jet loop modulator system. Ultrahigh resolution mass spectrometry experiments were performed using a Varian TQ (triple quadrupole)-FTMS consisting of a Varian CP-3800 GC, 920-MS and 9.4 T magnet. For all GC or GCxGC experiments, the instruments were operated in the electron ionization (EI) mode (70eV). Polar sample extracts were analyzed by direct infusion electrospray ionization (ESI) using a Varian 901-MS.

Preliminary Results/Abstract Multidimensional chromatography offers unparalleled separating power. As a consequence, GCxGC-TOF chromatograms often display thousands of peaks and the task of reviewing and interpreting such enormous data sets can be extremely laborious and time-consuming. One strategy to deal with this is to use automated software (scripts) to rapidly and automatically identify halogenated compounds in complex GCxGC-TOF chromatograms. This approach exploits the signature isotope patterns of Cl and Br, which have long been used for manual spectral interpretation. A single efficient script that can recognize a common range of isotope ratios exhibited by all Cl, Br and mixed Cl/Br compounds will be discussed. Another approach for the identification of halogenated compounds exploits the negative mass defects of F, Cl and Br. (Ultra)high resolution mass spectra of environmental samples often display thousands of peaks. Distinguishing halogenated and non-halogenated peaks can be a significant challenge, but is made straightforward by constructing a mass defect plot, i.e., the nominal mass of each mass spectral peak is graphed vs. the corresponding mass defect. One observes that halogenated and non-halogenated ions occupy different regions of the plot. The interpretation of mass defect plots may also be aided by converting the experimentally measured masses (IUPAC) to other mass scales defined by F, Cl and Br substitutions, and this greatly simplifies the identification of homologues and related compounds. Examples of a number of newly identified compounds in complex environmental samples that have been characterized using one or a combination of the approaches described above will be presented. These include sediment and fish samples as well as samples from an electronics recycling plant fire, a dust sample from an electronics recycling facility and the controlled burn of halogenated polymers, furniture and electronic equipment.

Novel Aspect Advanced analytical instrumentation and techniques can be used to systematically identify unknown contaminants in environmental samples.

Speaker Biography:

Eric Reiner is a senior mass spectrometry research scientist at the Laboratory Services Branch of the Ontario Ministry of the Environment. He also holds adjunct professor status at the Department of Chemistry at the University of Toronto, University of Waterloo and Pennsylvania State University. Eric obtained his Ph.D. from the University of Toronto in 1986 in Analytical Chemistry / Mass Spectrometry under the direction of Prof. Alex G. Harrison. He has been working in the field of ultra-trace environmental analysis for more than 25 years and has authored / coauthored over 150 scientific publications. His research interests include: the analysis of dioxin-like and other persistent toxic organics including halogenated flame retardants, perfluorinated compounds and industrial chemicals using advanced analytical techniques such as Fast GC, 2 Dimensional GC, time of flight mass spectrometry (TOFMS), tandem mass spectrometry (MS/MS), high resolution mass spectrometry (HRMS) and analytical laboratory automation; The determination of the fate, transport and spatial and temporal trends of persistent toxic organics in a variety of environmental matrices; The identification and detection of unknown halogenated and persistent organics in the environment; Development of analytical methods for new and emerging

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organics using a variety of instrumentation including automation and prescreening procedures to increase analytical throughput and reduce costs; Development of materials and procedures for performance evaluation and quality control testing.

Oral 7: “Conformational Dynamics of DNA G-Quadruplex in Solution Studied by Kinetic Capillary Electrophoresis Coupled On-line with Mass Spectrometry”

Speaker: Maxim Berezovski (University of Ottawa, Ottawa, ON)

Introduction G-quadruplexes (GQs) are non-canonical secondary structures formed from G-rich sequences of nucleic acids, and play important roles in the regulation of gene transcription and translation. Formation of GQ in a telomere region causes inhibition of telomerase activity with subsequent obsolescence and cell death. GQ structures are found in some promoters of oncogenes, such as c-MYC, BCL-2, c-KIT, K-ras, VEGF. Therefore, GQs could be a key therapeutic target for anti-cancer drugs. While the idea of GQ stabilizing/destabilizing compounds looks promising for switching genes on and off, it is critical to measure kinetics of GQ folding in solution for efficient drug design and high-throughput screening of drug candidates.

Methods In this article, we demonstrate the power of kinetic capillary electrophoresis coupled on-line with mass spectrometry (KCE-MS) to monitor individual DNA conformers and revealing rate and equilibrium constants of GQ DNA folding upon the binding to potassium ions. We connect KCE with MS on-line by electrospray ionization (ESI), a soft ionization technique, which keeps non-covalent complexes intact. The advantages of KCE-MS are that (i) DNA interacts with a ligand and folds at near physiological conditions, and all kinetic and thermodynamic parameters are measured in solution but not a gas phase; (ii) DNA and ligands don't need special labeling for the MS detection; and (iii) interactions/foldings of several DNAs and ligands can be studied simultaneously in one capillary microreactor.

Preliminary Results/Abstract G-quadruplex forming DNA/RNA sequences play an important role in the regulation of biological functions and development of new anticancer and anti-aging drugs. In this work, we coupled on-line Kinetic Capillary Electrophoresis with Mass Spectrometry (KCE-MS) to study conformational dynamics of DNA G-quadruplexes. We showed that peak's shift and its widening in KCE can be used for measuring rate and equilibrium constants for DNA-metal affinity interactions and G-quadruplex formation in solution; and ion mobility mass spectrometry provided information about relative sizes, absolute molecular masses and stoichiometry of DNA complexes in gas phase. Together, KCE and MS separate efficiently a thrombin binding aptamer d[GGTTGGTGTGGTTGG] from mutated sequences based on affinity to potassium, and reveal its apparent equilibrium folding constant (KF ~ 150 µM), folding rate constant (kon ~ 1.70×103 s-1M-1), unfolding rate constant (koff ~ 0.25 s-1), half-life time of the G-quadruplex (t1/2 ~ 2.8 s), and its relaxation time (? ~ 3.9 ms at physiological 150 mM [K+]). In addition, KCE-MS allows screening for a GQ-stabilizing/destabilizing effect of DNA binding dyes and an ant-cancer drug - cisplatin.

Novel Aspect KCE-MS establishes a new paradigm that separation methods with MS detection can be used as comprehensive kinetic tools of DNA.

Speaker Biography

Maxim V. Berezovski received M.Sc. in Biochemistry in1994 from Novosibirsk State University (Siberia, Russia).

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After serving as a CEO of a pharmaceutical company for 6 years in Russia, he moved to Canada and earned his Ph.D. in Bioanalytical Chemistry from York University at the Sergey Krylov lab in 2005, and then was trained as an NSERC postdoctoral fellow in Tak Mak’s lab at the University of Toronto from 2006 to 2008. Since 2009 Dr. Berezovski is an Assistant Professor and leads a Bioanalytical and Molecular Interaction laboratory in the Department of Chemistry at the University of Ottawa. He is also cross-appointed with Department of Biology, and an active member of Centre for Catalysis Research and Innovation. Dr. Berezovski is the author of 3 book chapters, 2 patents and 55 articles with >1070 citations and h index 22. His research focuses on: (1) bioseparation - the study of affinity interactions and conformational dynamics of biomolecules by kinetic capillary electrophoresis and mass spectrometry; (2) biopharmaceuticals - the selection of DNA aptamers to proteins, live cells, bacteria and viruses, and the application of the aptamers as imaging probes and therapeutic agents; (3) biosensors - the development of sensors for microRNAs, cancer cells and pathogens.

Oral 08: “Nanodiscs and CaR-ESI-MS: A novel method for the discovery of protein-glycosphingolipid interactions”

Speaker: Prof. John Klassen (University of Alberta, Edmonton, AB)

Introduction Catch and release electrospray ionization mass spectrometry (CaR-ESI-MS) has tremendous potential for rapidly screening carbohydrate libraries against target proteins to identify and quantify new protein-carbohydrate interactions. However, the method is limited to the analysis of carbohydrates that are relatively soluble in water. Here, we describe a new method, employing nanodiscs and CaR-ESI-MS, for the discovery of protein interactions with insoluble glycosphingolipids. Glycosphingolipids are readily incorporated into nanodiscs, which are discoidal phospholipid bilayers surrounded by a membrane scaffold protein, allowing them to mimic their natural cellular environment while being soluble in aqueous solution. The results of control experiments reveal that the assay allows for simultaneous detection of both low and high affinity glycosphinglolipid ligands and has the ability to rank their relative affinities.

Methods Nanodiscs were prepared by dissolving lipid films made up of a mixture of DMPC and glycosphingolipid with membrane scaffold protein (MSP1E1) and sodium cholate (25 mM) to give a final molar ratio of 100:1 lipid:MSP1E1. Nanodiscs were then formed by the slow removal of sodium cholate at 25 °C by the addition of biobeads. Finally, nanodiscs were purified using size exclusion chromatography and concentrated into 200 mM ammonium acetate pH 6.8 prior to ESI-MS analysis. A Synapt G2S HDMS mass spectrometer (Waters) was used to detect protein-glycosphingolipid complexes present in solution. Collision-induced dissociation was performed on the protein-glycosphingolipid complexes in the Trap region to release the ligands. Accurate mass measurement allowed for ligand identification.

Preliminary Results/Abstract

Two proteins were selected as model glycosphingolipid-binding proteins for this study: cholera toxin B subunit homopentamer (CTB5) and the heat labile toxin B subunit homopentamer (HLTB5), which both bind preferentially to the ganglioside GM1. Application of the CaR-ESI-MS assay to CTB5 and a library of nanodiscs that each contains one of three gangliosides (GM1, GM2 or GM3) or nanodiscs that contain a mixture of all three glycosphingolipids demonstrated that the assay allows for the simultaneous detection of both high and low affinity glycosphingolipid ligands.To determine whether the specificity of binding can also be determined using this method, the CaR-ESI-MS assay was applied to CTB5 and a nanodisc containing seven gangliosides (GM1, GM2, GM3, GD1a, GD1b, GD2 and GT1b) . Using collision-induced dissociation to release bound ligands, it was found that CTB5 binds to GM1, GM2, GM3 and GD1b in aqueous solution at pH 7. Moreover, the relative abundances of the ligands

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released from the CTB5-glycosphingolipid complexes were found to qualitatively reflect the affinities of the oligosaccharide ligands for CTB5 in solution. Finally, to demonstrate the utility of the CaR-ESI-MS assay for discovering new glycosphingolipid ligands, it was applied to solutions of CTB5 or HLTB5 with nanodiscs containing glycolipids extracted from porcine brain. Notably, these measurements led to the discovery of a neolacto glycosphingolipid as a new cholera toxin ligand.

Novel Aspect Novel method based on nanodiscs and electrospray ionization mass spectrometry for screening glycosphingolipid libraries for protein interactions is described.

Speaker Biography:

John S. Klassen received a B.Sc (Honours) in chemistry from Queen’s University in 1991. He pursued his doctoral research in the area of gas-phase ion chemistry under the supervision of Prof. Paul Kebarle at the University of Alberta and received his PhD in 1996. He spent the following year as a NSERC postdoctoral fellow at the University of California at Berkeley in the lab of Prof. Evan Williams. In 1998 he returned to University of Alberta as an Assistant Professor in the Department of Chemistry. He was promoted to Associate Professor in 2004 and to Full Professor in 2008. He is a Principal Investigator in the Alberta Glycomics Centre, director of the Alberta Glycan Screening Facility and serves on the editorial board of the International Journal of Mass Spectrometry. His research focuses on the development and application of mass spectrometry methods to characterize non-covalent protein complexes, with an emphasis on protein-carbohydrate interactions. His contributions to the fields of mass spectrometry and bioanalytical chemistry have been recognized with an American Society for Mass Spectrometry Research Award (2000), the Canadian Society for Mass Spectrometry Award (2004), a Petro-Canada Young Innovator Award (2004) and the F.P. Lossing Award for distinguished contributions to mass spectrometry in Canada (2011). In 2012, he was a co-recipient of NSERC’s Brockhouse Canada Prize for Interdisciplinary Research in Science and Engineering.

Oral 09: “Improved detection of SUMOylated peptides in large scale proteomic analyses using High Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS)”

Speaker: Dr. Eric Bonneil (University of Montreal, Montreal, QC)

Introduction Small Ubiquitin-related MOdifier (SUMO) are proteins involved in intracellular trafficking, genomic integrity and signaling. Sumoylation sites are challenging to identify due to the limited number of substrates, the dynamic nature of the modification, and the complicated MS/MS resulting from lysine residues modified with a SUMO chain extending up to 32 amino acids. To address this last issue we previously generated HEK293 cells stably expressing functional His6-SUMO mutants leaving a 5 amino-acid SUMO remnant after tryptic digestion. SUMOylated peptides form abundant triply-protonated precursor ions due in part to the free amino group on the remnant SUMO chain. We exploited this property using high Field Asymetric waveform Ion Mobility spectrometry (FAIMS) to enhance the detection of SUMOylated peptides in large-scale nano-LCMSMS experiments.

Methods Synthetic SUMO peptides were spiked into a yeast tryptic digest. His6-SUMO mutants were expressed in HEK293 cells. Cells were lysed and centrifuged to isolate nuclear extracts. SUMOylated proteins were enriched on a Ni-NTA column and digested with trypsin. Two separate FAIMS devices with different inner electrodes (6.5mm-radius and 7.75 mm-radius) were evaluated in this study. The 7.75 mm electrode leaves a 1.25mm analytical gap enabling the use of 100% N2 as a carrier gas. Optimal transmission of SUMOylated peptides with nano-LC-FAIMS-MSMS was performed at different compensation voltages (CVs) using yeast digests spiked with a mixture of synthetic SUMO peptides. MS/MS spectra were analyzed with Mascot and Peaks using a customized script to retrieve characteristic fragment ion features of SUMOylated peptides

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Preliminary Results/Abstract Because of the expected change in collisional cross sections and charge of branched SUMO peptides, we reasoned that these modified peptides could be transmitted by FAIMS at higher CV values than their non-SUMOylated linear counterparts. Preliminary results obtained on tryptic digest of HEK293 cells with a conventional FAIMS device showed modest enhancement of sensitivity since only 25 sumoylated peptides were detected compare to 19 without FAIMS. This is partly explained by the low resolution and ion transmission loss occurring within the FAIMS interface. Consequently, we used a new FAIMS device with a reduced gap between electrodes to improve peak capacity and ion transmission. Infusion experiments performed using 5 synthetic SUMO peptides revealed an enhancement of 3-fold in peak capacity with an average peak width at half height of 3-4 V. Interestingly, triply charged SUMO peptides were transmitted at CV values strikingly lower than those observed for typical tryptic peptide ions. Nano-LC-MS/MS analysis of a SUMO peptide library spiked in a yeast tryptic digest enabled the identification of 8716 unique peptides (1368 proteins) with FAIMS compared to 4192 peptides (662 proteins) without FAIMS. We also identified 209 and 134 unique SUMOylated peptides with and without FAIMS respectively. Interestingly, we noted that 50% of identified SUMOylated peptides were transmitted between CV values of -89V and -53V. We exploited the relatively narrow CV domain where SUMOylated peptides were transmitted to enhance the identification of these modified peptides in large-scale SUMO proteome analysis of HEK293 cells.

Novel Aspect LC-FAIMS-MS/MS on an Orbitrap mass spectrometer enhance the selectivity and comprehensiveness of SUMO proteome analysis in human cells.

Speaker Biography

Eric Bonneil received his Master degree from the Universite de Rouen in France in 1995 then his PhD from the Université de Montréal in 2000. He did his post-doc at the NRC in Ottawa from 2000 to 2001, then worked for Caprion proteomics as a senior Scientist from 2001 to 2004. Since 2004 he is the proteomic platform manager at the IRIC in University of Montreal.

Oral 10: “Proteomics profiling of pediatric serum and discovery of biomarkers for differentiation of the cause of febrile illnesses in Madagascar”

Speaker: Laetitia Cortes (Caprion, Montreal, QC)

Introduction Proteomics profiling of serum of children under 5 years old is uncommon, even more so for children affected by febrile illnesses in malaria endemic regions. In these children, unexplained fever is often caused by acute lower respiratory infections (ALRI) which may be of viral, bacterial, fungal or parasitic origin. In malaria-endemic regions, diagnosis is further complicated since malaria is commonly assumed in children that present with fever. The overall objective of this project was therefore to discover candidate biomarkers in sera of children with unexplained acute febrile illness in a malaria endemic region. The impact of demographic and environmental factors such as age and nutritional status was also evaluated.

Methods One hundred sixty-eight (168) serum samples from a malaria endemic area in Madagascar, representing malarial, viral and/or bacterial infections were profiled by mass spectrometry (MS). Samples were depleted of abundant serum proteins using tandem depletion chromatography that coupled in series IgG-HSA and IgY14/Supermix columns. The depleted samples were trypsin-digested, desalted and stored at -20°C. No fractionation step was performed prior LC-MS/MS analysis, where ~1 µg per sample was injected onto a NanoAcquity UPLC coupled to a Q-Exactive mass spectrometer. Peptide separation was achieved using a C18 column. MS analysis identified 1150 non-redundant proteins (approximately 18,000 unique peptides). A multifactor

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ANOVA analysis was applied to identify proteins that were differentially expressed between the various disease groups.

Preliminary Results/Abstract The number of proteins identified in the pediatric samples was on average 32% higher compared to adult sera. In addition to proteins representing the host response to infection, other proteins representing age-related processes such as cartilage and central nervous system development were identified in the children. These proteins may represent new pools of candidate biomarkers specific to pediatric samples. A subset of proteins also appeared to be associated with the nutritional status of the children, and thus likely affected the host response to infection. Most of the differentially expressed proteins identified had known associations with the host response, such as proteins associated with the immune response, lesion formation, oxidative stress and cell death. These functions were generally enriched in all disease types, although particular subsets of proteins belonging to these functions appeared associated with specific pathogen infections. A preliminary step-wise algorithm using the differentially expressed proteins able to distinguish disease and disease combinations that require different medical interventions was developed and is currently being evaluated.

Novel Aspect Biomarkers associated with specific febrile illnesses in children were identified, and used as novel candidate classifiers differentiating clinically relevant diseases.

Speaker Biography

Laetitia is a Group Leader in the Bioinformatics department of Caprion Proteomics, where she is involved in data analysis, visualization and interpretation of client studies.

Laetitia's trainings include a B.Sc. in Biology from Université de Montréal, a M.Sc. in Applied Microbiology from INRS-Institut Armand-Frappier and a DESS in Bioinformatics from UQAM.

Oral 11: “Absolute quantitation of NAPQI-modified serum albumin from rat plasma samples by LC-MS/MS: monitoring acetaminophen toxicity”

Speaker: Prof. Lekha Sleno (UQAM, Montreal, QC)

Introduction Acetaminophen (APAP) toxicity is the most common cause of acute liver failure in the United States. The hepatotoxicity of acetaminophen is caused by an electrophilic reactive metabolite of acetaminophen, N-acetyl p-benzoquinone imine (NAPQI), that forms covalent adducts with nucleophilic thiol groups in liver proteins, causing hepatic necrosis. Serum albumin (SA) is known to be covalently modified by NAPQI and is present at high concentrations in the bloodstream. SA is therefore a potential biomarker to assess the levels of protein modification by NAPQI. To date, no quantitative method exists for NAPQI modified serum albumin. Here, we present a highly sensitive method for the absolute quantitation of modified serum albumin from in vivo rat plasma samples by LC-MS/MS.

Methods Plasma samples were digested using pepsin and the albumin active site peptide containing Cys34, modified by NAPQI, was targeted for quantitation. For accurate absolute quantitation, a standard was designed using an iodo-analog of acetaminophen to quantitatively modify RSA cysteines. This protein standard yields a target peptide that is a positional isomer to the one modified by NAPQI. A deuterated version was used to produce an internal standard added prior to digestion to all standards and unknown samples, correcting for all steps during sample preparation. The digested plasma samples undergo solid phase extraction, and ion exchange fractionation prior to injection. LC-MS/MS analysis was performed on an AB Sciex 5500 QTRAP in positive MRM mode.

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Preliminary Results/Abstract An LC-MS/MS method was developed to quantify NAPQI-modified serum albumin from rat plasma with a limit of quantitation of 31.25 ng modified RSA present in 100 µl plasma, representing approximately 0.0006 % of the total RSA. The linear dynamic range spans two and a half orders of magnitude. The precision and accuracy of the method were assessed using a 10 point standard curve prepared in triplicate with excellent accuracies and reproducibility. The method was applied to quantify modified RSA in plasma samples from rats (n=4) dosed at low (non-toxic) levels of acetaminophen (75 mg/kg). While no peaks of interest were detected in blank plasma samples, we measured concentrations between 380 and 480 ng/100 µl plasma in dosed samples, illustrating the potential of this approach to directly monitor acetaminophen covalent binding from in vivo samples. The developed quantitation method relies on a novel protein standard design based on iodoacetamide chemistry for modifying cysteine residues in standard albumin. This modified RSA, spiked into blank rat plasma, is used for an external calibration curve while a deuterated (d4) version of the analog is used as internal standard. Several clean-up steps were shown to be necessary in order to remove complex background from digested plasma. Mixed-mode anion exchange solid phase extraction as well as strong cation exchange HPLC fractionation is crucial for achieving the high sensitivity of the final method. After digestion, the resulting peptides are positional isomers of NAPQI modified RSA peptides. Using synthesized standards of our targeted peptic peptide (LQKC*PYEE) containing both the NAPQI and the iodo-APAP modifications, we show that the standard and target peptides' chromatographic, ionization and fragmentation behaviors are extremely similar. We also show that this standard is essential to the accuracy of the method by comparing to other methods commonly used for protein quantitation.

Novel Aspect Absolute quantitation of NAPQI-modified albumin for monitoring acetaminophen toxicity able to monitor non-toxic levels of adducts in plasma

Speaker Biography:

Lekha Sleno received her PhD in chemistry from Dalhousie University in 2006, where her work involved small molecule mass spectrometry. She then completed two postdoctoral fellowships at the University of Geneva in Switzerland (in pharmaceutical analytical chemistry) and the University of Toronto (at the Donnelly CCBR in proteomics research). She is currently associate professor in the chemistry/biochemistry department at UQAM (Université du Québec à Montréal). Her research interests include bioanalytical mass spectrometry applied to metabolomics and covalent binding of reactive drug metabolites.

Oral 12: “Environmental analysis of poly- and perfluoroalkyl compounds using a Q-Exactive Orbitrap: optimization for a laser diode thermal desorption method”

Speaker: Prof. Sebastien Sauve (University of Montreal, Montreal,QC)

Introduction Contaminants of emerging concern (CECs) encompass a wide array of molecules, which include pharmaceutical and personal care products, UV filters, brominated flame retardants, or poly- and perfluoroalkyl substances (PFASs). PFASs are composed of two moieties, a fluorinated carbonaceous tail (both lipophobic and hydrophobic) and generally a polar head (hydrophilic), and display excellent tensioactive properties. They are thus strategic chemicals for electroplating, leather treatment, aqueous film-forming foams (AFFFs), lubricants and coatings, and fluoropolymer chemistry. However, PFASs are extremely persistent in the environment and long-chain ones have been shown to be bioaccumulative.

Methods A Laser Diode Thermal Desorption Atmospheric Pressure Chemical Ionization (LDTD/APCI) was coupled with a Q-Exactive Orbitrap mass spectrometer to develop a high-throughput, ultra-fast analysis technique

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for the quantification of 19 selected poly- and perfluoroalkyl compounds (PFASs) in water and sediment. Analysis time between each sample was reduced to less than 20s, all target molecules being analyzed in a single run with the use of optimal LDTD settings were investigated using either one-factor-at-a-time or experimental design methodologies, while Orbitrap parameters were optimized simultaneously by means of a Box Behnken design.

Preliminary Results/Abstract The optimization scheme, which combined both one-factor-at-a-time methods and experimental designs, led to the choice of optimal parameters for the determination of 19 PFASs in a 20-s single run. LDTD optimal settings were achieved by combining a high laser power (70%) and low carrier gas flow (2 L min-1), and mass spectrometry parameters were simultaneously investigated in a three-factor, three-level Box Behnken design, prompting us to choose a maximum AGC target (5?106), a resolution of 70 000 and a 50 ms injection time. Environmentally relevant method detection limits were reported, falling in the range 0.3 - 4 ng L-1 in the waste water matrix, and 0.03 - 2 ng g-1 dw in the sediments. Target compounds were unequivocally detected in several wastewater influents (< LOD - 23 ng L-1 in the dissolved phase and < LOD - 74 ng g-1 dw in the suspended particulate matter, SPM), as well as in surface waters from the Montreal area, albeit at lower concentrations (< LOD - 0.8 ng L-1). The LDTD/APCI-Orbitrap method proved reliable, with values close to those obtained with LC/HESI-Orbitrap, yet could not discriminate between different PFAS isomers and exhibited more variability. However, eliminating the chromatographic step prior to MS analysis reduced solvent consumption and potential contamination risk from Teflon tubings, noise level, and analysis times, making it an economic alternative for high-throughput sample screening. In waste water samples, 10 out of 19 PFAS were quantified either in the dissolved phase or the SPM. The main species found in the dissolved phase was PFOA which was systematically quantified with concentrations in the range 4.5 - 23 ng L-1. PFOS and PFOA were found in tap water (0.59-0.69 ng L-1), levels lower than USEPA provisional drinking water guidelines (200 ng L-1 for PFOS and 400 ng L-1 for PFOA).

Novel Aspect Coupling an LDTD/APCI interface to a Q-Exactive Orbitrap instrument is innovative, especially to quantify emerging contaminants such as PFASs.

Speaker Biography

Sébastien Sauvé is full professor in Environmental Analytical Chemistry at the Université de Montréal which he joined in 2001. He has a B.Sc. and M.Sc. from McGill University, and a Ph.D. from Cornell University. He directs a team of about fifteen students and researchers who focus their work partly on ‘traditional’ contaminants such as lead, copper, arsenic, and partly on emerging contaminants, such as pharmaceuticals, cyanotoxins and nanoparticles. He has published over 125 scientific articles and book chapters.

Oral 13: “Multi-modality 3-D imaging of a human carotid atherosclerotic plaque: Correlating in vivo ultrasound and imaging mass spectrometry”

Speaker: Prof. Pierre Chaurand (University of Montreal, Montreal, QC)

Introduction Within this work, we report a 3-D imaging mass spectrometry (IMS) method to morphologically determine the molecular composition of vascular tissues afflicted with cardiovascular disease, the leading cause of death worldwide. Here we expand beyond the simple correlation of IMS results to histological stainings with an additional level of correlation to 3-D in vivo ultrasound data. Using a human carotid with significant atherosclerotic plaque build-up, we were able to perform multiple label-free chemical 3-D reconstructions of the plaque structure by IMS. IMS measurements were performed on a surgically excised atherosclerotic plaque using two approaches: MALDI- and Silver Assisted LDI-IMS. IMS results were then aligned to 3-D in vivo ultrasound imagery allowing an enhanced comprehension of plaque formation and instability.

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Methods A surgically excised 1.4 cm atherosclerotic plaque from a human carotid was cut in 10 µm sections throughout its length. Using serial sections, MALDI-IMS, silver assisted LDI IMS (AgLDI), and histological stainings were performed every 100 µm. All IMS experiments were performed at 100 µm of spatial resolution, therefore defining 100 µm voxels for 3-D reconstruction. IMS data was acquired using a Bruker UltraFleXtreme TOF-TOF system with 1,5-diaminonapthelene (DAN) as matrix for phospholipid analysis and sputtered metallic silver for cholesterol and olefin analysis. Data processing and analysis was done in the R environment. 2-D data visualization was done with Bruker FlexImaging 3.0. 3-D visualization was done with ImageJ. Ultrasound data was analyzed using Plaque Texture Analysis software (LifeQMedical Ltd).

Preliminary Results/Abstract The 3-D IMS reconstruction for the full length of the plaque required the analysis of 140 sections for each IMS modality (MALDI and AgLDI). This translates to over 700,000 mass spectra and approximately 131 hours of data acquisition. Positive and negative mode MALDI IMS data using DAN matrix allowed visualization of the distribution of phospholipids within the plaque whereas AgLDI IMS allowed monitoring of the localization of free fatty acids and cholesterol. Analysis of 3-D imaging data revealed several important morphological structures within the plaque defined by different molecular compositions. The identified structures include plaque adventitia, media, intima as well as a lipid core within the plaque and the surrounding connective and smooth muscle tissue of the arterial wall. All of these structures are clearly visualized in both 2-D and 3-D and compared with corresponding histological and immunohistochemistry staining. The 3-D IMS data visualizations include both individual ion volumes and 'soft segmentation' maps of the 3-D dataset obtained by data clustering algorithms that automatically reveal morphological structures. Lysophospholipids originating from both phosphotidylcholine and phosphoethanolamine were identified as most abundant within the lipid core and are strongly associated with plaque development. In preparation for surgery, ultrasound technology is a common imaging technique for the precise location of plaques within the carotids. Post-operational correlation of the 3-D IMS data to the 3-D in vivo ultrasound is performed using landmark features in the plaque to associate a molecular composition to observed density volumes. Such a powerful approach will allow a better comprehension of plaque formation and the identification of molecular events leading to plaque instability.stract

Novel Aspect Multimodal 3-D IMS approaches applied to large dimension tissue specimens. Correlation to 3-D in vivo ultrasound imaging.

Speaker Biography:

For the past 20 years, Prof. Pierre Chaurand has contributed to the development and characterization of various aspects of matrix-assisted laser desorption ionization (MALDI) mass spectrometry. During his Ph.D. years (1991-1994, Université de Paris Sud, Orsay, France), his contributions ranged from the fundamental understanding of ion production via the MALDI process and subsequent detection by impact on various surfaces including microchannel plate detectors. During his post-doctoral training (1994-1998, University of Düsseldorf, Germany), Prof. Chaurand’s efforts were focused on the design and construction of MALDI time-of flight mass spectrometers optimized for peptide sequencing by post-source decay. Prof. Chaurand has then spent 11 years of his professional career (1998-2009) has research faculty at Vanderbilt University (Nashville TN, USA) contributing to the development of a technology named 'imaging mass spectrometry', which through the direct analysis of thin tissue sections by MALDI mass spectrometry, allows the profiling and mapping of biomolecules including proteins, lipids and other metabolites, as well as administered pharmaceuticals. In 2009, Prof. Chaurand has joined the Dept of Chemistry at the University of Montreal, where his research efforts for the development of the imaging mass spectrometry technology are continued.

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Oral 14: “Quantification of Growth Hormone Receptor Antagonist Pegvisomant by LC-MS/MS in Rat Plasma: Method Development Considerations for PEGylated Proteins”

Speaker: Dr. Fabio Garofolo (Algorithme Pharma, Laval, QC)

Introduction PEGylation is a widely used approach to improve the pharmacokinetic properties of therapeutic protein drugs. However, quantification of PEGylated proteins in plasma by LC-MS/MS encompasses challenges that may not be encountered in non-PEGylated proteins and therefore requires careful consideration. Depending on the method of conjugation, PEGylation at the targeted amino acids may not be stoichiometric or consistent, therefore requiring a surrogate peptide to be void of potential PEGylation sites and thus reducing the pool of candidate signature peptides amenable for quantification. Herein, we present an LC-MS/MS approach for the quantification of Pegvisomant, a recombinant human growth hormone (hGH) structural analog PEGylated on several lysine residues and used as a therapeutic hGH receptor antagonist indicated for the treatment of acromegaly.

Methods Rat plasma was spiked with a Somavert formulation containing 5 mg/mL of Pegvisomant protein. As internal standard, the hGH was also spiked in the plasma samples. Samples were diluted 5-fold with NH4HCO3 to allow protein reduction with DTT (5 mM) at 60ºC for 20 minutes, followed by alkylation with iodoacetamide (10 mM) for 15 minutes at 20ºC. Reduced and alkylated plasma proteins were then subjected to tryptic digest at 60ºC for 60 minutes (400 mg/mL final trypsin concentration). A 0.1% HCOOH and acetonitrile gradient was applied to a Waters Acquity CSH column (50 x 2.1 mm, 1.7µm) using a UHPLC pump (Waters) and the Pegvisomant peptide was monitored in a QTRAP triple-quadrupole instrument (AB Sciex) operated in ESI(+) MRM mode.

Preliminary Results/Abstract Pegvisomant is a PEGylated version of the hGH with several amino acid substitutions that allow the molecule to bind the growth hormone receptor while preventing its dimerization and thus, activation. The protein portion of Pegvisomant has a molecular weight of 22 kDa, to which 4 to 6 molecules of polyethylene glycol (PEG) are covalently attached to lysine residues, resulting in a heterogeneous population of molecules with molecular weights of 42, 47 and 52 kDa. To avoid any possible inconsistencies and in order to ensure specificity, the arginine-flanked, Pegvisomant-specific tryptic peptide (R)VSTFLR (corresponding to the tryptic peptide (K)VETFLR in the hGH) was chosen as the signature peptide for quantification. During the early phase of tryptic digestion optimization, it was found that urea-mediated sample denaturing was not only unnecessary but also resulted in lower detection levels. However, reduction with DTT and subsequent alkylation with iodoacetamide was essential to the detection of the signature peptide VSTFLR, most likely due to its proximity to an adjacent cysteine involved in a disulfide bond. Direct plasma tryptic digest was performed without enrichment or purification steps, thus rendering the method attractive for high throughput purposes. The major product ions generated from fragmentation of the VSTFLR doubly charged parent ion (m/z 361.7) corresponded to the y4 (m/z 536.3) and y5 (m/z 623.4) fragments which confirmed the identitiy of the selected signature peptide. For quantification purposes, the y5 fragment was used due to lower associated interfering ions. Under these conditions, the assay was linear and quantitative between 50 ng/mL - 50 mg/mL. This research highlights how the judicious choice of a surrogate peptide, optimization of sample preparation steps, as well as optimal tuning of UHPLC and MS parameters can circumvent the challenges of PEGylated protein quantification and result in the development of industry-tailored, cost-efficient bioanalytical methods.

Novel Aspect Quantification by UHPLC-MS/MS of the PEGylated protein Pegvisomant in the ng/mL range without prior protein purification or enrichment.

Speaker Biography

Dr. Fabio Garofolo has been working in the pharmaceutical bioanalytical and LC-MS analytical fields for more than

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20 years. He has also been heavily involved and committed to working as a volunteer for pharmaceutical and scientific non-profit organizations with the mission to promote the interactions among industrial, academic and regulatory bodies to provide education and forums for discussion in the pharmaceutical practices.

Dr. Garofolo has progressed throughout in his career in both the pharma/biotech and CRO industries and benefited from successfully overcoming the prevalent challenges in drug development. Since 2005 he has held the position of Vice-President Bioanalytical Services at Algorithme Pharma. Dr. Garofolo has over 130 publications & presentations in international conferences. He developed around 300 innovative analytical methods. He designed and invented 3 innovative bioanalytical approaches and is the recipient of the following Lilly awards: Achievement (2001); Global (2002); Emmerson (2003). His current interests include analysis of large molecules by LCMS, HRMS, DBS, and Emerging Technologies.