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Nuclear Magnetic Resonance Page 8
JANUARY 2011 Volume 15, Number 1
Multiplexed iTRAQ labeling Challenges and Opportunities
Page 12
Dr. Anne-Claude Gingras
Lunenfeld researchers develop innovative, freely available software to purify mass spectrometry data
Researchers at the Samuel Lunenfeld Research Institute of Mount Sinai Hospital and the University of Toronto, as well as colleagues in Michigan and Scotland, have developed an innovative computa-
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tional approach—the first of its kind worldwide—designed to analyze mass spectrometry data. The software, called SAINT (Significance Analysis of INTeractome), will allow researchers globally to quickly as-
e n v i ronm e nt
sess the reliability and accuracy of protein binding data helping to further their studies of cancer and other illnesses. The tool was developed by Lunenfeld Principal Investigator Dr. AnneClaude Gingras (Lea Reichmann Research chair in Cancer Proteomics and assistant professor in the Department of Molecular Genetics at the University of Toronto), and Dr. Alexey Nesvizhskii (assistant professor in the Department of Pathology and in the Center for Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor) to meet a key challenge in the field of protein mass spectrometry (a tech-
R&D News.......................... 1 Appointments..................... 6 Pharma Notes..................... 6 New Products................... 15 Calendar........................... 17 Career Spotlight............... 18
nology that helps researchers separate, identify, and quantify specific proteins): namely, to identify and quantify ‘true’ protein interactions gleaned from mass spectrometry data, and filter them from proteinbased contaminants in the sample data. Previously, other approaches to analyze mass spectrometry data have not allowed for a probabilitybased model to measure and account for errors in a data set. “SAINT allows researchers to identify the real protein interactions in their sample, and to exclude the false positives generated through contaminants,” said Dr. Gingras. “In effect, the software applies a much needed filter to purify the data and remove ‘noise.’” SAINT was introduced earlier this year by Drs. Gingras, Nesvizhskii and Mike Tyers, when their teams generated a comprehensive road map of the signaling proteins that control many aspects of cellular behaviour in yeast cells—a discovery reported in a May issue of Science. However, the updated approach can be applied to a wider variety of datasets of various sizes and levels of protein network density. “The first version of SAINT was intended to help us analyze very large-scale datasets, which is something that only a few laboratories worldwide are generating,” said Dr. Nesvizhskii. “We then realized that, with some modifications, the same approach could be extended to researchers specifically interested in knowing what a few proteins interact with inside the cell. This makes our approach very useful to most cancer biologists using mass spectrometry, as it enables them to quantify their interaction data.” The development of SAINT was supported by several agencies, including the Canadian Institutes of Health Research and the National Institutes of Health, as well as the Mount Sinai Hospital Foundation.
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January 2011 Laboratory Focus www.bioscienceworld.ca
news
SFU co-op student shines at TRIUMF How does a student with no science background wind up doing a stellar job as a communications assistant for TRIUMF, Canada’s top particle and nuclear physics laboratory? Dec.2010Nunc.BD.LabFocus.pdf
Given drive, imagination and opportunity, anything is possible in Simon Fraser University’s Work Integrated Learning (WIL) program, where students gain on-thejob experience applying their 12/22/10 10:09:38 AM
higher education in a practical setting. Just ask Jessica Coccimiglio, a fourth year cooperative (co-op) education student pursuing a joint major in communication and interactive arts and technol-
ogy at SFU. The 21-year-old Vancouverite has finished her third co-op term at the facility and Tim Meyer, head of TRIUMF’s Strategic PlanContinued on page 3
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January 2011
News
Continued from page 2
Jessica Coccimiglio
ning and Communications Office is having trouble saying goodbye. “I found TRIUMF’s communications assistant job through WIL’s Simplicity on-
line postings,” says Coccimiglio, who produced more than eight articles publicizing TRIUMF, since starting her last co-op term in September. They are posted online along
with external news releases sought out by Coccimiglio from TRIUMF’s many research partners, including SFU. Writing about and photo-
graphing discoveries, international conferences and distinguished visitors at TRIUMF has kept Coccimiglio on her cerebral toes. Genuinely curious about research, Coccimiglio has progressed from being a science neophyte to writing news releases about complex topics such as testing diamonds for use as particle detectors and the evolution of dark matter and anti-matter. Among her most memorable photography assignments was capturing SFU president Andrew Petter touring TRIUMF. “That was slightly intimidating,” says Coccimiglio, who feels her TRIUMF co-op terms have prepared her to work effectively in communications and marketing in any field. “Many of the articles I’ve written for TRIUMF have required a significant amount of effort to research the basics of the science on which I am reporting. Fortunately, scientists here are incredibly helpful and
patient, many of them taking time to meet with me so that they can answer my questions.” As for Meyer, who regularly hires undergrad co-op students nationally, he feels TRIUMF struck gold with Coccimiglio. “Jessica has been a great asset because of her eagerness to learn the science and look for the story behind the story,” explains Meyer. “It’s been a bonus this term to have someone with such school spirit; we had a blast with the visit of president Petter and even did a small student artist-inresidence project with an SFU arts professor thanks to Jessica’s contacts.” Meyer says he typically receives up to 70 applications every time TRIUMF’s communications assistant job is posted. TRIUMF seeks to fill the position with a student who has the right combination of grades, experience and originality, not necessarily science training.
wATeRlOO ReseARCh TeAM develOPs bReAKThROugh TeChNiQue TO sTudY biRTh defeCTs A University of Waterloo-led engineering research team has discovered a breakthrough technique that allows experts to see how human cells and tissues evolve and grow in the embryo. Video force microscopy (VFM), as the technique is known, will allow medical researchers to visualize and quantify the forces that are at work in cells and tissues, including those that ultimately form eyes, ears, nose, limbs and other features crucial to embryos and newborns. These forces are produced by the movements of microscopic-sized tissues early in development. “These movements involve what can be thought of as a tug-of-war between various cells and tissues,” said Wayne Brodland, a professor of civil and environmental engineering who led the international research team. “In a tug-ofwar analogy, VFM technique allows us to ‘see’ how hard each member of each team is pulling at any given moment.”
The new method is described in a research paper, entitled Mapping the Dynamic Forces Driving Ventral Furrow Formation in Drosophila Using a Novel Technique: Video Force Microscopy (VFM), published in the Proceedings of the National Academy of Sciences. Brodland said that during early embryo develop-
ment, sheets of tissue move in precise ways in order to form organs and other critical structures. “Although much is known about the genes expressed in many of these tissues, the mechanical forces that drive motions has remained largely a mystery.” The new VFM technique is key to medical research because it is widely held that
aberrations in these driving forces are the reason that tissues sometimes do not move in a normal way and a malformation - in short, a birth defect - arises. Common examples of such irregularities in humans include spina bifida, cleft lip and palate and cardiac septum defects. VFM makes it possible to determine these forces from time-lapse movies. “VFM allows us to learn the degree to which the driving forces are in error in cases
where defects arise,” Brodland said. “Our studies have shown that certain defects can be produced by irregularities in which the magnitudes of the driving forces are changed by as little as 20 per cent and for periods of time as short as a few hours. This knowledge is important for designing clinical strategies to prevent these defects.” The work was funded by the Human Frontiers Research Program (HFSP), Natural Sciences and Engineering Research Council Canada (NSERC) and international agencies.
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news NSERC funding creates $3 million Senior Industrial Research Chair in Biomaterials at Trent University vice-chancellor of Trent University. “The biomaterials research led by Dr. Narine and his team of researchers has the potential of adding substantial value to the University. The research being conducted in Peterborough at Trent University will have an impact at a local, regional, national and a global level. We are extremely grateful for NSERC’s support.” “Our research offers an alternative solution – to employ agriculturallysourced renewable feed stocks (fats and oils) to design functional materials more environmentally benign and more in sync with the natural carbon cycle so as to abate and combat climate change,” said Dr. Narine,
A research funding announcement will result in the creation of a new $3 million Senior Industrial Research chair at Trent University focusing on Biomaterials. Over $1.5 million in new NSERC funding was provided to create the “NSERC/GFO/ERS Senior Industrial Research chair in Lipid Derived Biomaterials,” awarded to Dr. Suresh Narine, director, Trent Biomaterials Research Program, and professor, physics and astronomy and chemistry. “The new NSERC Senior Industrial Research chair represents a unique addition to Trent University’s research and development strengths,” said Dr. Steven E. Franklin, president and
Dr. Suresh Narine, director, Trent Biomaterials Research Program, and professor, Physics & Astronomy, and Chemistry. who was recruited to Trent from the University of Alberta in September, 2009. “It is research that is both timely and necessary. On behalf of my team of researchers and Trent University, I would like to offer our deep appre-
ciation to NSERC and our generous founding industrial partners whose match investments are strategically placing Trent’s Biomaterials Research Program among one of the top facilities of its kind in the world.”
Breakthrough Treatment For Blocked Arteries Dr. Bradley Strauss, chief of Sunnybrook’s Schulich Heart Centre and Reichmann chair of Cardiovascular Sciences is conducting the world’s first clinical trial of a groundbreaking
Patients who have run out of treatment options for a common heart condition have found new reason for hope in the world’s first trial of a plaque-softening enzyme.
Chemical Institute of Canada
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treatment for patients with coronary arteries that are completely blocked by plaque. The promising new therapy is the first biological solution ever developed to treat chronic total occlusions (CTOs). It involves injecting the enzyme collagenase into a blocked artery to soften the plaque so a cardiologist can perform traditional angioplasty by sliding a small guide-wire through the otherwise impenetrable blockage and inserting a stent to re-open the artery and restore blood flow. About 25 to 30 per cent of people tested with an angiogram at the Schulich Heart Centre are found to have at least one total blockage. Although not all patients with CTOs need to have angioplasty to open up the blocked artery, those who experience common symptoms like chest pain and difficulty breathing can benefit greatly from it. In fact, successful angioplasty can relieve their symptoms and significantly improve their quality of life almost immediately. “Although many patients with this condition experience painful symptoms that would be relieved with angioplasty, the blockages are like cement and it is often impossible to get a guide-wire through them,” says Dr. Strauss, who is also a professor at the University of Toronto. Until now, efforts to treat patients with CTOs have been focused on designing stronger, more deliberate guide-wires to forge through the blockage. The success rate of this method is currently around 50 per cent, compared to 95 per cent for arteries that are only partially blocked. But this new approach to treatment could change that.
Together with his team of clinicianscientists at Sunnybrook’s Schulich Heart Centre, Dr. Strauss is conducting a phase one, first-in-human clinical trial to test the safety and effectiveness of this new treatment. With a careful eye on adverse effects, the procedure has been preformed on four groups of patients - using increasing amounts of collagenase in each group. At each dose, results have been reviewed by an independent data safety monitoring board. Participants were selected from a group of Sunnybrook patients who have undergone at least one failed angioplasty attempt. Results of the trial will be used to determine the best dose for a large-scale clinical trial. Todate, Dr. Strauss has performed the procedure on 19 of the 20 patients involved in the trial with a nearly 85 per cent success rate. The clinical trial is funded by the Canadian Institutes of Health Research (CIHR).
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Laboratory Focus January 2011
news Thunder Bay DNA Lab receives ASCLD/LAB-international accreditation for forensic science testing Warnex Inc. announces that its Warnex PRO-DNA services division has received accreditation from the American Society of Crime Lab Directors/Laboratory Accreditation Board - International® (ASCLD/LAB-International®) for forensic science testing. Warnex was specifically accredited in the categories of nuclear and mitochondrial DNA testing, relationship testing and body fluid identification. The lab expects a big boost in business after earning the international accreditation for forensic science testing. The thumbs up from the American Society of Crime Lab Directors/Laboratory Accreditation Board–International for Warnex’s PRO-DNA lab means it’ll be able to bid on U.S. government-funded contracts, Warnex president and CEO Mark Busgang said. “Most of the DNA testing
world in the United States is funded through (the Department of) Homeland Security,” he said. “So, you were basically shut out of that kind of stuff. We’ve started marketing . . . our services, and in fact we’ve had interest from 30odd states,” Busgang said. “It’s huge, it’s really huge.” The local lab — one of three Canadian facilities owned by Warnex — specializes in mitochondrial, or ancient, DNA, Busgang said. It is the only private laboratory in Canada providing STR, Y-STR, miniSTR, and mitochondrial DNA testing services for forensic and parentage testing purposes. Mitochondrial DNA testing is crucial in cases where nuclear DNA testing is not possible such as for shed hairs at crime scenes, identification of missing persons (skeletal remains with degraded DNA), and when there is insufficient nuclear DNA.
Warnex PRO-DNA Services has state-of-the-art facilities and is also accredited by the Standards Council of Canada (SCC) as a DNA testing laboratory for forensics, parentage and other familial relationships under ISO 17025/CAN-
P-4E and CAN-P-1578. The lab offers its forensic services to law enforcement, lawyers, investigators and private clients worldwide. The company bought the Thunder Bay facility last year. Warnex also announced
that it has signed a funding agreement with the National Research Council of Canada’s industrial research assistance program to develop its capabilities to analyze touch DNA, or skin cells, left on an object.
Allon Therapeutics Inc. announces that professor Illana Gozes, Allon’s founding scientist and the discoverer of Allon’s clinical-stage neuroprotective drug candidate davunetide, has been awarded the Olson Prize for her research insights into the behavioural effects of peptides by the journal PEPTIDES. Awarded annually for the most meritorious original research article on the behavioural effects of peptides published during the previous year, the Olson Prize honours the outstanding contributions of Drs. Gayle A. and Richard D. Olson to the field of peptide research. Prof. Gozes’ article, entitled “NAP (davunetide) enhances cognitive behavior in the STOP heterozygous mouse—A microtubule-deficient model of schizophrenia”, was published in PEPTIDES in April 2010, and involved studies in mice that are bred to be deficient in a protein called stable tubule-only polypeptide (STOP) protein. Associated with proper
function of microtubular network, the STOP protein is important for the survival of the nerve cell. The deficient mice are a reliable model for schizophrenia and, in Prof. Gozes’ study, the mice showed schizophrenia-like symptoms (hyperactivity) that
were ameliorated by chronic treatment with the antipsychotic drug, clozapine. Prof. Gozes’ study showed that daily intranasal treatment with NAP (davunetide) significantly decreased hyperactivity and also protected the visual memory of the
STOP-deficient mice. This result is consistent with previous research by Prof. Gozes in mouse models where brain pathology is characterized by the hyperphosphorylation of the microtubule-associated protein tau, often referred to as tauopathies.
Illana Gozes
Allon’s founding scientist Illana Gozes awarded Olson Prize from PEPTIDES journal
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January 2011 Laboratory Focus www.bioscienceworld.ca
APPOiNTMeNTs
Dr. Mark J. Poznansky
He also serves as chair of Let’s Talk Science, and until recently, served as chief science advisor to the CEO of the Thunder Bay Regional Research Institute. He is a board member of the Innovation Institute of Ontario, founding member and past chair of the Council for Health Research in Canada, and has also chaired the Scientific Advisory Boards of the Canadian Medical Discoveries Fund and MDS Capital Corp. Dr. Poznansky was made a member of the Order of Ontario in 2004 and the Order of Canada in 2005. Dr. Poznansky replaces Dr. Christian Burks who has served as President and CEO since 2004.
as president and CEO. For the past two years, Dr. Poznansky has been running his own consultancy group offering a range of services including program reviews, strategic planning, change management and leadership training to clients in government, hospitals, universities and the private sector. Before this, he was the president, CEO and Scientific director of the Robarts Research Institute in London, ON. Dr. Poznansky has also served as president and CEO of London, ON based Viron Therapeutics Inc. from 2004 to 2006 and founded the London Biotechnology Incubator Inc., Ontario’s first life sciences incubator, in 2002. Since 2004, Dr. Poznansky has served on the OGI Board of directors and from 2008 as chair.
OncoGenex Pharmaceuticals, Inc. has appointed Michelle Burris to the position of executive vice president, operations and chief financial officer. Ms. Burris, a seasoned public company executive, brings over 20 years of financial and operations management experience to OncoGenex. In addition to her new role, she is currently a member of the board of directors of OncoGenex, which she joined following the merger with Sonus Pharmaceuticals. Prior to the merger, Burris had served on the Sonus board of directors since 2004. Burris most recently served as senior vice president and chief operating officer for Trubion Pharmaceuticals, Inc. until its recent acquisition by Emergent BioSolutions, Inc. She joined Trubion in 2006 as senior vice president and chief financial officer after holding similar positions at
The Ontario Genomics Institute (OGI), announces the appointment of Dr. Mark J. Poznansky
Medicago Inc. (Québec, QC), a biotechnology company focused on developing highly effective and affordable vaccines based on proprietary manufacturing technologies and Virus-Like Particles (VLPs), announces that the last dose of vaccine has been administered completing the first part of the company’s Phase II human clinical trial with its H5N1 Avian Influenza vaccine. The company expects to present interim Phase II clinical trial results with its H5N1 pandemic influenza vaccine in January, 2011. The Phase II randomized, placebo controlled clinical trial is designed to evaluate the safety and immunogenicity of different doses of the vaccine. Specifically, the vaccine will be studied in approximately 255 healthy adults between the ages of 18 to 60 years. In the first part of the study conducted in 2010, 135 healthy adults were administered twice with either
a placebo or the H5N1 vaccine at varying doses to determine the optimal dose. In the second part of the study to be conducted in 2011, healthy adults will receive an injection of either a placebo or the H5N1 vaccine at the optimal dose. To date, all results have shown the H5N1 vaccine is safe and well tolerated. Atrium Innovations Inc. (Québec, QC) announces the purchase of Belgium-based Minami Nutrition, a niche brand of superior quality scientifically proven Omega-3 products primarily sold in several European countries. Minami’s product portfolio consists of 16 Omega-3 supplements with revenues derived largely from Belgium, Holland, Ireland and the UK. Minami’s products distinguish themselves through an exclusive sourcing of oil with a high level of concentration and purity and a small ecological
Dendreon Corporation and Corixa Corporation. Prior to entering the biotechnology field, she served in numerous management positions at The Boeing Company and, prior to that, as a research analyst at Cypress International Inc., a Washington, D.C.-based high-technology consulting firm. Medicago Inc. has engaged Paul Kirkconnell, a seasoned advisor to the pharmaceutical and vaccine industry, as a consultant on business development matters. Paul Kirkconnell, founder of P-A-K Limited, provides biopharmaceutical companies with strategic investment and corporate development advice. Prior to founding P-A-K Limited, he was managing director of DRI Capital, a private investment company specialized in biopharmaceutical royalty acquisition, with over $1 billion under management. Prior to this, Mr. Kirkconnell was president of Aventis Capital, the equity investment arm of Aventis (now Sanofi Aventis) with about $750 million under management. He also served as corporate vice president of Business Development of Aventis Pasteur where he led their worldwide vaccine business development programs, evaluating corporate acquisitions, and was responsible for deal structures and terms with respect to licensing opportunities. Nelson J. Chai has been elected to Thermo Fisher Scientific Inc.’s board of directors, bringing the
footprint. Some competing brands may require a larger number of softgel capsules to deliver the full benefit of Omega-3 on a daily basis. In contrast, with up to 90 to 95 per cent of pure Omega-3 per softgel, Minami’s products require considerably fewer capsules. Based on a unique, exclusive and patented manufacturing process, the purity of Minami’s Omega-3 supplements surpasses industry standards for PCBs, dioxins and heavy metals. Finally, no chemical products are used in the manufacturing process and the oil is derived from nonendangered species of fish or algae. Minami’s fish oil has Eco-Management and Audit Scheme (EMAS) certification which is approved by the European Parliament. iCo Therapeutics Inc. (Vancouver, BC) has granted Immune Pharmaceuticals (IMPH) based
Nelson J. Chai
company’s total number of directors to 11. In addition, Chai has also been appointed to the board’s Audit Committee. In addition, Chai is the executive vice president, chief administrative officer and head of strategy for CIT Group, a bank holding company that provides lending, advisory and leasing services to small and mid-sized companies and operates in over 50 countries. Previously, he served as president, Asia-Pacific for Bank of America Corporation and executive vice president and chief financial officer of Merrill Lynch & Co. Mr. Chai also served in senior positions at Allied Signal and Philip Morris before joining medical diagnostics provider Dade-Behring in 1997. He entered the financial services field in 2000 when he joined Archipelago Holdings, the first all-electronic stock exchange in the United States. When the firm was acquired by the New York Stock Exchange (NYSE) in 2006, Mr. Chai became the chief financial officer of NYSE Euronext.
PhARMA NOTes in Israel and the United States, an option to an exclusive license for the development and commercialization rights to the systemic uses of iCo-008, iCo’s human monoclonal antibody targeting eotaxin-1. The license option is for systemic uses including: inflammatory bowel disease and severe asthma, while iCo has retained worldwide exclusive rights to all ocular applications. IMPH will pay iCo a non-refundable option fee creditable upon conversion against an upfront license fee payment of US $1 million. iCo may receive up to an additional US $32 million in milestone payments as well as royalties on net sales of licensed products.
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Feature
Using Nuclear Magnetic Resonance
to Test Fat Content
in Foods The quickening pace of business caused one of Europe’s leading independent testing laboratories to seek an alternative to the standard solvent extraction/acid hydrolysis (Soxhlet) method for testing the amount of fat in a variety of foods. This contract laboratory, with numerous sites throughout the UK and Ireland, provides quality control analytical services to the food industry. By converting from the wet chemistry method to an MQC benchtop nuclear magnetic resonance (NMR) analyzer for
How one Contract Laboratory Dramatically Increased Sample Throughput
measuring fat content of foods, the lab reaped significant economic and environmental benefits. Figure 1 is a brief overview of NMR’s advantages over other secondary methods.
Standard wet chemistry methods result in bottlenecks Customers send foodstuff samples to this contract quality control laboratory, which has a specialty in fast turnaround service. A typical request includes five or six measurements, including fat (oil) content. The Soxhlet
method used for the oil measurement is slow, with measurements taking as long as six hours. This situation led to serious bottlenecks that were reducing throughput and affecting the lab’s ability to deliver its promised rapid analysis service. The process is also rather cumbersome, can be inaccurate, and requires highly skilled personnel. In addition, many of the hazardous chemicals used are becoming increasingly unacceptable according to international environmental standards.
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Laboratory Focus January 2011
Feature Figure 1
Table 1
Key features of NMR
Comparison of wet chemistry and NMR-MQC instrument
Alternatives investigated to speed testing throughput The lab began seeking a rapid technique that would improve turnaround time without increasing operating costs, but would also be comparable to the industry standard Soxhlet technique. There are a number of analytical methods that can be used to conduct the testing. Such methods are often referred to as secondary techniques, since they are usually set-up to match the results produced by solvent extraction. To provide a result equivalent to the traditional extraction techniques, secondary techniques must be correlated against the reference technique used. Although they may be fast in use, many secondary techniques need to be calibrated and maintained regularly. Maintenance and consumables add significantly to the cost of ownership. For example, Supercritical Fluid Extraction (SFE) is reasonably fast, but it requires high maintenance and the cost of compressed CO2 that is used to extract oil is also significant. Near Infra-Red (NIR) is sometimes used, but it is generally sensitive to the surface rather than the bulk of the sample, and has substantial calibration and calibration maintenance issues. NIR calibration is complex because measurements are sensitive to product granularity and other physical characteristics and can be affected by additives such as seasoning, making it difficult to maintain accurate calibrations on a large variety of product types. This gives NIR limited applicability for the quality control of fat content in foodstuffs. In contrast to the standard wet chemistry methods and various secondary techniques, low field nuclear magnetic resonance (NMR) provides a fast, direct and user friendly method for determination of the fat and oil content in foodstuffs. The technique is based on measurement of the NMR response obtained from fat in the product, and quantification of the fat content by simple and direct calibration without the use of chemometrics. The instrument is extremely easy to operate and does not require the use of skilled chemists or NMR specialists. NMR can be calibrated to cover a concentration range from 0.5 to 100 per cent fat. The user can produce a primary calibration using a single sample of fat. NMR is very stable over the long-term, and therefore requires infrequent recalibration. Sample measurement time is short, typically about 20 seconds, allowing a high throughput of samples and efficient laboratory operation. Minimal sample preparation is required because the entire sample is normally loaded into a tube and measured directly, and there are several different size tubes available.
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Feature Figure 2
Currently, about 90-95 per cent of the laboratory’s samples are run on the MQC instrument and the lab has increased its efficiency because of the rapid measurement capability.
Fat (Oil) Content in Foodstuffs
Calibration obtained for fat content in foodstuffs; standard deviation of the linear fit is 0.20 % by mass, correlation coefficient R2 = 1.00. Measurements were made using an Oxford Instruments MQC-23 benchtop NMR analyzer equipped with a 26 mm diameter probe.
With NMR, no solvents are required since the sample is analyzed in its natural state. The instrument facilitates bulk measurement; the signal is generated from the whole sample, ensuring that the result embodies everything inside the sample, not just on the surface. NMR is virtually insensitive to sample granularity and such additives as spices, flavors, colors and salt. Finally, unlike Soxhlet,
the NMR technique is nondestructive, so any required repeatability measurements can be made easily.
NMR selected to solve throughput challenges After reviewing the potential alternative solutions to bottlenecks associated with fat measurements, the laboratory contacted Oxford Instruments, which offers a benchtop NMR instrument widely
used in industry to measure the oil content in foodstuffs and oil seeds. Oxford Instruments recommended its powerful but compact MQC benchtop NMR analyzer for this application because it offers the analyst the benefits of accurate quantitative results, combined with sampling ease and convenience. To verify whether the MQC NMR instrument would meet the laboratory’s needs, appli-
Table 2
Results of instrument repeatability test
Figure 3
Measurement comparison
cations specialists from Oxford Instruments collected and tested samples of some of the foods the lab typically analyzes and compared NMR measurements with fat measurement values obtained with the Soxhlet method. The goals of the testing process were to analyze 80 per cent of samples using the MQC NMR instrument, achieve correlation to within 5 per cent of the wet chemistry method, and achieve a repeatability of within 5 per cent. Applications specialists sampled a range of foods, with fat contents ranging from 2.1 to 40.2 per cent by mass, including baked cheese, muesli, milk powder, chicken pow-
der, trifle, garlic bread, macaroni and cheese, meat, and chicken sandwich filler. Sampling was conducted using an Oxford Instruments MQC23 benchtop NMR analyzer equipped with a 26 millimeter (mm) diameter probe. As shown on Table 1, the NMR results compared very closely to wet chemistry. Figure 2 shows a calibration for the samples, indicating an excellent linear correlation between the NMR response and the concentration of fats in the products. Instrument repeatability was tested by measuring one sample 10 times. Prior to each measurement the sample was placed in a heating block for 20 minutes. Since the magnet is temperature stabilized at 40°C, precision is enhanced by conditioning the sample at 40°C prior to inserting it into the NMR magnet. Conditioning samples prior to measurements aids repeatability, since the NMR signal is temperature sensitive. In addition, some samples must be heated to melt the fat so it becomes visible to the NMR. The sample only needs to be returned to the block for a further 20 minutes if the measurement is going to be repeated on that sample. Table 2 illustrates the repeatability test results, which were well within the desired range. Laboratory managers reported that the testing results exceeded their expectations, and the group purchased an MQC low-field, benchtop NMR analyzer. Currently, about 90 to 95 per cent of the laboratory’s samples are
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run on the MQC instrument and the lab has increased its efficiency because of the rapid measurement capability. Figure 3 compares the number of analyses per hour and the skills required for the NMR technique versus that of Soxhlet. According to Kulsum Jassat, sales manager for the UK and Europe, “Throughput has increased from 80 to 200 samples per day. With solvent extraction, each sample took approximately six hours from set-up to completion, or a rate of about one sample per hour. With NMR, it is usually less than one minute per sample after sample setup.” She adds, “The company achieved a return on investment (ROI) within five months of purchasing the instrument.” The purchase of the NMR MQC instrument allowed the laboratory to replace four of its Soxhlet units. This significantly reduced the cost of purchase and disposal of solvents, increased available laboratory space, reduced running costs, achieved environmental benefits associated with reduced solvent usage, and facilitated better deployment of laboratory staff. Since there is a slight calibration drift over about six months to a year, the laboratory will still need to recalibrate periodically against the primary technique or with set-up samples supplied by Oxford Instruments. During installation, set-up samples can be made to physically capture and maintain the original calibration, saving time during instrument recalibration. They also save time by allowing calibrations to be transferred to other similarly configured Oxford MQC NMR instruments. While not required by most potential users of NMR instruments, this particular contract lab chose to get its new fat measurement method accredited by the United Kingdom Accreditation Service (UKAS); the national accreditation body that uses a set of internationally agreed standards to assess organizations that provide certification, testing, inspection, and calibration services. The lab developed a universal method using a sample blending process, sample conditioning to ensure the optimal temperature for reading by the temperature-sen-
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Laboratory Focus January 2011
sitive NMR signal, followed by analysis using the MQC instrument. They used hundreds of data samples from Soxhlet and NMR-MQC to prove they were getting similar results within a degree of tolerance. This method has been UKAS-validated and fully accredited since 2009.
NMR can eliminate quality control bottlenecks NMR provides a fast and reliable method to increase throughput for quality control testing of fat content in foodstuffs. Although past history may have placed the venerable Soxhlet solvent extrac-
feATuRe tion technique in the role of industry standard, for those seeking to eliminate quality
control bottlenecks, the MQC NMR analyzer is poised to take over the lead.
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Feature
Multiplexed iTRAQ Labeling in Quantitative Proteomics:
Challenges
and Opportunities By By J i e ran L i , P e i h on g Z h o u , J i an C h e n an d K e nn e t h R . E v an s
Quantitative, mass spectrometry-based methodologies are proving to be a powerful tool in protein biomarker research. One such approach is the application of multiplexed isobaric tags for relative and absolute quantification (iTRAQ) technology, in which Isobarically-labeled peptides from different biological conditions can be mixed and analyzed simultaneously using tandem mass spectrometry (MS) technology. This allows up to eight samples to be analyzed simultaneously, thereby reducing the run-to-run variability issues and increasing reliability and precision of MS data. However, optimization of the method is warranted as variations in sample preparation can significantly impact the outcome of MS analysis, and moving from 4-plexed iTRAQ to the 8-plexed iTRAQ results in fewer proteins being identified. The objective of this report is to provide our perspectives on how to improve sample preparation and to establish a minimal modified standard procedure for quantitative analysis of proteins with multiplex iTRAQ labeling methodology. Our optimized method, which includes removal of detergents and optimization of labeling conditions, has enabled us to achieve excellent performance using multiplexed iTRAQ labeling techniques. This report also discusses issues related to increasing peptide charges that can cause complexity in protein identification after iTRAQ labeling, an issue that has not been reported by the manufacturer of the iTRAQ reagent kit.
Sample cleanup process The quality of the protein sample is the most important factor in apply-
ing complexed iTRAQ reagents labeling since interfering substances may have accumulated from the initial sample preparation steps (e.g. cell lysis). Samples often contain substances such as Thiols, denaturants or high amounts of detergents, which, together with any primary amines that are present may interfere with the iTRAQ Reagents. In the present experiment, cell lysate from RIPA buffer (PIERCE, USA) containing high amounts of detergent as well as urine containing high amounts of urea were used. A sample clean-up step is an essential process prior to application of iTRAQ reagents. We have selected Norgen’s ProteoSpin total protein detergent clean-up kit and Norgen’s urine protein concentration kit for urine samples as our method of choice to remove detergent/denaturant before using iTRAQ
labeling kit. This spin column format clean-up kit is ion-exchange based and binds particularly well to multiply-charged large molecules like protein. With these methods, a 10mM sodium phosphate elution buffer is fully compatible with the downstream iTRAQ labeling process and LC-MS/ MS analysis. Since trace amounts of detergents could strongly interact with proteins identification, we found that washing twice instead of once seems to eliminate final concentration of detergent in the multiplexed sample. This showed that the lower concentration of detergent in the eluant, there would be less background interference with MS analysis.
Denaturation of proteins According to the iTRAQ labeling protocol provided by the manufacturer, 1ul of denaturant (2 per cent SDS) is
needed to add into each sample prior to digestion. Following this protocol, we found that the number of identified proteins was lower than we expected. In order to address this problem, we have set up an experiment in which SDS was either added prior to digestion or not. These data are shown in Table 1. The sample from Norgen’s clean-up column with SDS added before digestion had less protein identification (IDs). Comparing to 4plex (four iTRAQ labeled samples were combined), the number of protein IDs in 7plex (seven iTRAQ labeled samples were combined) was significantly decreased. Total spectra percentage in Table 1 indicates the purity of sample. The sample with SDS had much lower percentage than that without SDS. The presence of ionic or non-ionic detergents is well known to destroy chromatographic resolution
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13
Laboratory Focus January 2011
feature even in very low concentrations (Swiderick et al, 1995), and 0.02 per cent SDS interferes with LC/MS/MS (manufacture’s instruction). If SDS is used during electrospray ionization (ESI) mass spectrometry, a shift of the charge envelope may take place. The samples undergoing Norgen’s detergent clean-up column may retain residual detergents. Adding denaturant reagent before digestion likely increases final concentration of SDS in each sample. In combining all iTRAQ labeled samples in the final step of the multiplexed labeling procedure there is an accumulation in the concentration of detergent. The outcome of this is a reduction in the number of proteins identified.
Reduction of proteins Tri-(2-carboxyethyl-phosphine (TCEP) is more stable and effective in basic condition than DTT in the reduction of proteins (Getz et al., 1999) and has therefore been selected as the reducing reagent in the iTRAQ labeling procedure. As the manufacturer’s protocol, 2ul of 25mM TCEP were added and incubated at 60ºC for 1hr. Wang et al (2010) reported that the reduction by TCEP should be effective at room temperature, heating the peptide with TCEP led to the formation of a new modification corresponding to the loss of 32 Da, suggesting conversion of cysteine into alanine. Considering this observation, an experiment was carried out to determine the optimal incubation condition of TCEP under non-additional SDS. The samples were incubated at 60ºC for 1hr and at room temperature for 1hr or 2hrs with gentle shaking. The results showed that the number of protein identifications was similar following incubation with TCEP at room temperature for 1hr (>95% protein ID=206) and 2hrs (>95% protein ID=197), and decreased as the sample was incubated at high temperature (>95% protein ID=93). Of note, special care should be taken to avoid exposure of disulfide bond containing samples to heat at basic pH since the disulfide bond containing peptides are prone to
b-elimination and thus the formation of dehydroalanine (Wang et al., 2010).
Trypsin digestion Since trypsin autohydrolyzes overtime and loses activity during incubation time, we initially use half the amount
of trypsin and incubate at room temperature for 1-3hrs, followed by the addition of the second half into the sample and then incubate at 37ºC overnight. Finehout et al (2005) reported trypsin digestion at pH 7.8-8.1 is in the optimal range for TPCK-treat-
ed trypsin in terms of kinetic constants calculation. We used HPLC-water to adjust pH 8.5 to 8.1 before trypsin digestion and raised pH by adding up to 5ul of TEAB (Triethylammonium bicarbonate) during the labeling step.
Strong cation exchange (SCX) chromatography for sample fractionation The dynamic range of protein concentration in complex biological samples poses another serious challenge toward successful quantitative
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feATuRe Table 2
effect of iTRAQ labeling on protein identification Peptide Charge
Treatment
Protein ID (95% confidence)
2+
% in Total
3+
% in Total
4+
% in Total
5+
% in Total
6+
% in Total
248
63.75
116
29.82
25
6.43
0
0
0
0
52
1hr Labeling
26
14.53
79
44.13
59
32.96
11
6.15
4
2.25
29
2hrs Labeling
24
13.64
85
48.3
56
31.82
11
6.25
0
0
31
4hrs Labeling
13
5.14
192
75.89
40
15.82
7
2.77
1
0.4
25
unlabeled
Table 1
effect of sds on protein identification SDS added before digestion
Non-SDS before digestion
4plex iTRAQ labeling
4plex iTRAQ labeling
7plex iTRAQ labeling
Protein detected (>95%)
143
638
284
% Total spectra
33.5
48.2
50.7
*The data are from fractionated peptides of cell lysates. 2ug of peptides were used for LC-MS/MS analysis (125min gradient).
proteomic analysis. For multiplexed iTRAQ experiments, the bias toward identification of high abundance proteins is particularly challenging. Sample fractionation is therefore required in order to achieve a satisfactory analytical outcome. Ion exchange chromatography based peptide fractionation methodology is widely used for the purpose of reducing sample complexity and enhancing the chance of detecting low abundant proteins. In our process, a narrow bore strong cation exchange column (2 x 100 mm, Biobasic SCX, Thermo, USA) is used because of its adequate loading capacity and lower elution volume. Up to 2mL, 300 µg of iTRAQ labeled protein sample was injected and washed with solvent A for 10 minutes to eliminate unbind iTRAQ reagent and reaction by-products. A salt gradient from 0 per cent solvent B to 40 per cent solvent B in 30 minutes was employed to elute peptides for collection in the eppendorf tubes every 2 minutes. Solvent A contains 10mM ammonia acetate in water with 25 per cent of acetonitrile (pH 3.0). Solvent B is solvent A with addition of 0.35M KCl. The flow rate of SCX fractionation is 0.2mL/ min. The collected fractions had been dried in a speedvac. This SCX methodology delivers optimal fractionation for downstream MS analysis.
Increasing peptide charges in applying isobaric-tags labeling The data in Table 2 showed that most of the isobarically-labeled peptides possess 3+ and 4+ ions, while no-labeling peptides possess 2+ and 3+ ions. This may be one of reasons why the number of protein ID decreased after iTRAQ labeling. Further study is required to determine how to identify more isobarically-labeled peptides.
Conclusion: Sample preparation is the one of the most critical processes for achieving optimal MS analysis results. Protein identification has been greatly reduced by using isobaric-tags labeling when working with manufacturer’s protocol. With our modified sample preparation and fractionation protocol, we are able to identify more than 600 proteins using 7-plexed iTRAQ labeled samples, this is same as 4-plexed iTRAQ labeled samples.
Materials and Methods Materials: All test cell line and urine samples are provided by our academic collaborators with appropriate Research Ethical Board approval as required. iTRAQ Reagents-8plex
kit, buffer kit and Trypsin with CaCl2 were purchased from AB SCIEX (Foster City, USA). ProteoSpin Detergent Clean-up kit were purchased from Norgen Biotek Corp. (Thorold, ON, Canada). Mass-spectrometry analysis was conducted using a hybrid quadrupole/Time-of-flight mass spectrometer (QStar Elite®, AB Sciex, USA), with an Easy-nLC® nanoflow HPLC (Proxeon, Denmark) as the front end. Protein Extraction: 5x107 cells were first lysed by RIPA lysis buffer (25 mM Tris•HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) containing Halt Protease and Phosphatase Inhibitor Cocktail (PIERCE, USA), and detergent-free lysate was obtained by using Norgen’s ProteoSpin Detergent Cleanup kit. 1.5ml of human urine sample was used to extract proteins by Norgen’s urine protein concentration kit. The purified proteins were measured by Bio-Rad’s DC Protein Assay kit, (Bio-Rad, Canada). A total 50 µg of sample from clean-up cell lysate were used for tryptic digestion. LC-MS/MS analysis: The separation system consisted of a trap column (300µm ID, Agilent, USA) and an analytical column (75 µm ID) packed in-house with 5µm, 300Å Zorbax SB-C18 beads. Separation was performed using a linear binary gradient where solvent A is 2% acetonitrile in water with 0.1% (v/v) formic acid, and solvent B is 2% water in acetonitrile with 0.1% (v/v) formic acid. The separation was done at a flow rate of 300 nL/ min using 60min and 125min gradient profile. 60mins gradient is 6 to 35% B in 30 min, 35% to 65%B in 5min, 65%-95%B in 4min, 95%B for 3min, 95%-4% B in 3min and 4%B in 3min. 125min gradient is 6% to 35% B in 99min, 35% to 65% B in 4min, 65% to 95%B in 3min, 95% B 3min, 95% to 4% in 3 min and 4% in 3min. Mass spec data were acquired
with Analyst QS 2.0 software from ABSciex. Each data acquisition cycle consisted of one MS survey scan and 3 information dependent (IDA) MS/ MS production ion scans. MS/MS accumulation time was set automatically. Q2 transmission window was set manually to balance the need for enhancing the iTRAQ reporter ion region and production ion region. Collision energy was set automatically to enhance the iTRAQ reporter ion region. Ion spray voltage was set at 2100 volts, ion source gas 1 was set at 17, curtain gas set at 20, and interface heater temperature set at 150oC. MS/MS Mass spec data were searched against human protein database from Swissprot with ProteinPilot® software from ABSciex.
References: 1. Getz EB, Xiao M, Chakrabarty T, Cooke R, Selvin PR. A. 1999. Comparison between the sulfhydryl reductants tris(2carboxyethyl)phosphine and dithiothreitol for use in protein biochemistry. Anal Biochem 273: 73-80 2. Erin J. Finehout, Jason R. Cantor and Kelvin H. Lee. 2005. Kinetic characterization of sequencing grade modified trypsin. Proteomics 5: 2319–2321 3. Zhouxi Wang, Tomas Rejtar, Zhaohui Sunny Zhou and Barry L. Karger. 2010. Desulfurization of cysteine-containing peptides resulting from sample preparation for protein characterization by mass spectrometry. Rapid Commun. Mass Spectrom. 24: 267–275
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New Products
MICROPLATE WASHER DISPENSER BioTek has released a new vacuum filtration module for it’s EL406™ Microplate Washer Dispenser. The module can be used for automated vacuum filtration. Now, the entire line of BioTek micoplate washers can be automated for washing of polystyrene and magnetic bead-based assays such as the Luminex xMAP® assay platforms. Each washer comes with software through which users can define protocols for unattended operation. The ELx50™ offers 96-well precise strip or full plate washing for ELISAs, biomagnetic separations and vacuum filtration processes. The ELx405™ is for washing ELISAs and cell based assays; offering 96 and 384-well microplates. The EL406™ offers quick 96, 384 and 1536-well microplate washing. Both ELx405 and EL406 models are robotic compatible.
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BIOO SCIENTIFIC QPCR ANALYSIS OF MICRORNA AND SMALL RNAS The MiraMas kit for SYBR Green qPCR-based profiling of microRNAs and other small RNAs incorporates a one tube protocol to convert all of the micro RNAs in a sample into cDNA. The kit offers flexibility in terms of sample size and number of reactions performed. The kit leverages a proprietary 3’ ligation strategy, allowing researchers to produce cDNA libraries from up to 150 total or small RNA enriched samples per kit. Using higher input amounts of RNA, hundreds of microRNA targets can be screened in up to 30 samples per kit. The Micro RNA targets are amplified using microRNAspecific Forward primers and the Universal Reverse primer included in the kit. THe microRNA-specific Forward primers are designed using an algorithm that does not require modified nucleotides.
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MINIFLASH FLASHPOINT DETECTOR The MINIFLASH Touch, flashpoint tester used to determine the flashpoint of liquids and solids, users a large colour screen and runs on Microsoft Windows software which features a menu navigation system that requires no training for flashpoint testing. Incorporating a touchscreen design, the MINIFLASH Touch offers extended temperature range from 0 to 400°C. The unit includes a few different connection outputs including USB, Ethernet and LAN. The MINIFLASH series uses the ASTM D7094 standard of flashpoint determination which offers the highest repeatability and reproducibility. The unit’s detection method measures the instantaneous pressure increase inside a continuously closed test chamber resulting from a highenergy electric arc that ignites vapour from a 2mL sample. Maximum safety is ensured by the MINIFLASH’s continuously closed cup design and small sample volume. The unit’s design makes it ideal for mobile labs, military, and other applications demanding a rugged design.
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SUPERCRITICAL FLUID EXTRACTOR In its latest model of supercritical fluid extractors, the SFT-110, Supercritical Fluid Technologies (SFT) has completely redesigned the restrictor valve with an integrated micrometer to allow for precise flow control. Also, a completely removable oven lid and large side panel allow the user access to the high pressure vessel. An indicator light on the SFT-10 pump module alerts the user to proper operation of the Peltier pre-cooler, ensuring CO2 is maintained in the liquid state. An outlet from the restrictor ensures that users will not accidentally damage the outlet tube when inserting it into the collection container. An alternative to traditional extraction methods that use organic solvents, supercritical fluid extractors use carbon dioxide for extraction of a variety of materials.
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INJECTION PORT LINERS VICI’s new Injection Port Liner reduces the breakdown of Endrin and DDT during injections while increasing the interval between linter changes. With non-deactivated liners and those filled with non-deactivated glass woll, DDT and Endrin are easily degraded in the injection port. VICI has discovered that when they apply a highly cross-linked siloxane over a conventionally deactivated liner, the result is an injection port liner that contributes less to breakdown than any other component of the injection system. Sold in a package of five be used in VICI injectors or other model injector brands.
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HIGH-SPEED, HIGH-VOLUME CENTRIFUGE The Thermo Scientific RC BIOS centrifuge system for high-speed, high-volume, batch bioprocessing applications integrates a six-litre Thermo Scientific Fiberlite carbon fibre rotor and true fit one-litre Thermo Scientific Nalgene bottles to achieve superior yields with faster processing time. The system can spin up to 15860 xg, yet offers the smallest footprint available in its class and quiet performance. The system has a high-capacity (maximum 12 litres), and a power-saving design that reduces overall processing and development costs. It also includes a refrigeration system to ensure sample quality which, in turn, reduces lot-to-lot variability. Optional software included with the centrifuge is the new Thermo Scientific Centri-Log data collection software.
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NEW PRODUCTS 520Di dispensing pump
psi. The 520Di has full dispensing capability, enabling accurate batch dispensing and automatic or singleshot operation. While the product is being pumped, a ramp and drip feature allows soft start and graduated dosing to reduce splashing or foaming. Users are also able to carry out periodic dose recalibration ‘on-the-fly’ without disturbing the flow of the batch. Up to 50 individual dispensing programs, including all dose parameters and calibration,
Watson-Marlow offers its 520Di dispensing pump for accurate metering, dosing and transferring of sensitive fluids in biopharmaceutical environments. Ideal for filtration, fermentation and dispensing applications, the 520Di is well suited for filling lines in pharmaceutical and biotech production. The pump accepts eight different tubing materials and sizes up to 9.6mm for flow rates ranging from 4 microlitres/min up to 3.5 liters/min and pressures up to 100
can be stored in the memory with full, 8-character file names. The 520Di’s new brushless DC motor has a speed range of 3500:1 and over a million to 1 flow range, offering total flexibility of speed control combined with precision and repeatability for the most demanding dispensing applications. A number of different pumpheads can be fitted to suit the user’s needs.
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January 2011 Laboratory Focus
CAReeR sPOTlighT Bio-economy Career Profile Compiled by BioTalent Canada Position: Manufacturing Chemist Salary Range: $60,000 to $80,000 per year
2011 dubbed iNTeRNATiONAl YeAR Of CheMisTRY Thanks to an initiative between IUPAC (International Union of Pure and Applied Chemistry) and UNESCO (United Nations Educational, Scientific, and Cultural Organization), 2011 has been dubbed the International Year of Chemistry. The year of celebrations falls on the 100th anniversary of Madame Marie Curie’s Nobel Prize win, as well as the founding on the International Association of Chemical Societies. Canada’s celebrations will include emphasis on important discoveries made by Canadian chemists, which include how to produce insulin for treating diabetes, how to extract canola oil from an inedible planet, and how to detect fingerprints at crime scenes. There are also a series of local and national events being hosted to celebrate the International Year of Chemistry such as a YouTube contest; a cross-country speaking tour by Joe Schwarcz, author, radio host and director of McGill University’s Office for Science and Society; the Science Rendezvous, a day-long program where scientists will interact with members of the public in malls, coffee shops and libraries; and National Chemistry Week, where businesses, elementary schools, colleges and universities will hold lectures, lab tours and competitions to spark interest in chemistry. “The International Year of Chemistry is a global celebration, so we wanted to highlight the role that Canadian chemistry has played in improving the well-being of people around the world,” said David Dolphin, chair of the Chemical Institute of Canada’s IYC organizing committee. The global festivities will kick off at the UNESCO headquarters in Paris, France, with a two-day ceremony, January 27th to 28th which will feature prominent scientists from around the world, plus debates on global trends and perspectives in chemistry.
What I do:
My day-to-day work includes overseeing all duties connected to the chemical process. I ensure that the quality of the product is in compliance with the customer specifications’ requirements. My position is part analysis and development in the laboratory, and part management of technicians and operators related to the production processes. I spend about 60 per cent of my day in the laboratory and 40 per cent on the manufacturing floor.
What education and skills do candidates need for this position?
I have a Masters of Chemistry Degree, majoring in Chemical Science. I have also upgraded my skills in Canada in the pharmaceuticals industry as an Analytical Chemist. My position requires a strong background in chemistry and good communication skills to explain basic chemistry to the technicians. The safe use of chemicals in the manufacturing process is an ongoing issue for my company. As a Manufacturing Chemist, you also need good computer skills, as there is a lot of paperwork and document writing. To get a job in the industry, I would encourage you to get as much background in chemistry as you can. When entering the workforce, look around for opportunities to gain knowledge of industries with which you are unfamiliar and work hard at lifelong learning.
What are the best parts of your job?
I find my work very interesting. I have gained great understanding about an industry of which I had no practical prior knowledge. Being a recent immigrant, I was unsure of how my skills and abilities would translate in Canada. Since working for Anod-Tech, I realize that my background and knowledge was acceptable, and my skills are in demand in Canada.
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