short course summary - ASMS

The course will emphasize the key steps in protein identification: peptide-spectrum matching, error rate ... phases of utilizing mass spectrometry in the clinical diagnostics setting will be discussed with emphasis on the ... along with updated industry practices for experimental design, data interpretation, and data reporting.
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TWO-DAY SHORT COURSES – Saturday and Sunday, June 4 and 5 Go to www.asms.org for more detailed descriptions, information, and online registration. 01 Bioinformatics for Protein Identification Bioinformatics tools are routinely used to identify proteins from “shotgun” LC-MS/MS data sets. These tools, however, are often poorly understood by their users. This course seeks to familiarize proteomics researchers with the inner workings of the software that enables this field. The course will emphasize the key steps in protein identification: peptide-spectrum matching, error rate estimation, and protein assembly. It will build on these topics by discussing advanced techniques for MS/MS identification through spectral libraries and algorithms that leverage de novo sequence inference. Live demonstrations of the TransProteomic Pipeline, ProteoSAFe, and BumberDash / IDPicker tools will familiarize participants with the decisions that produce reliable results from these systems. Participants will emerge from the course with a solid understanding of the underlying algorithms that enable protein identification as well as the software available to accomplish this task. Instructors: Nuno Bandeira (University of California San Diego); Alexey Nesvizhskii (University of Michigan Health System); David Tabb (Stellenbosch University) 02 Clinical Diagnostics: Innovation, Validation, Implementation and Operation by Mass Spectrometry This course will cover aspects of mass spectrometric analysis as applied to clinical diagnostics. Rather than approach the use of diagnostic mass spectrometry from the ground-up, the course will focus on those components which are singular to the clinical diagnostics industry. Attendees will be provided with details and practical examples of laboratory operations in an environment which is regulated by the FDA Office of In Vitro Diagnostics (FDA-IVD), College of American Pathologists (CAP) and the Clinical Laboratory Improvement Act (CLIA). The various phases of utilizing mass spectrometry in the clinical diagnostics setting will be discussed with emphasis on the components of development (innovating a new test), validation of a laboratory-developed test (LDT), implementation of new assays and operationally analyzing samples. Particular care will be taken to address the regulatory aspects of clinical diagnostics under the most up-to-date guidance with consideration given to those guidance documents which are in draft. Prerequisites: Attendees are expected to have a fundamental understanding of liquid chromatographic and mass spectrometric techniques and practical experience validating and performing sample analysis in compliance with FDA bioanalytical guidance (May 2001) or previous experience with the requirements of clinical diagnostic testing. Instructors: Russell Grant and Chris Shuford (Laboratory Corporation of America); Brian Rappold (Essential Testing) 03 DMPK: Experimentation and Data Interpretation Mass spectrometry has become the dominant tool throughout the drug discovery / development continuum. This short course will provide a thesis on mass spectrometry in drug metabolism, pharmacokinetics (DMPK), and pharmacodynamics (biomarker) in support of R&D and the registration process. The course will use case studies to focus on the “why” and “how” knowledge base with regard to the use of mass spectrometry to measure small molecule drugs, biologics, and their conjugates in the discovery and development phases. Contents will include an introduction to the concepts / principles of DMPK, an overview of drug discovery / development processes, regulatory submission requirements, and common practices in DMPK studies. Current mass spectrometry technologies applied in ADME screening in lead optimization, drug quantification in PK studies, drug metabolite identification in animals and humans, as well as GLP bioanalysis quantification in clinical and toxicology studies will be discussed along with updated industry practices for experimental design, data interpretation, and data reporting. Practice sessions will be given to reinforce data analysis techniques learned in class

Prerequisites: Entry-level scientists with hands on experience in LC/ MS as well as advanced students who wish to learn more about mass spectrometry applications in drug discovery & development. The course is a unique opportunity for scientists already in the pharmaceutical, biotech, and other industries to broaden or enhance their expertise and knowledge. Due to the highly interactive format, managers and project leaders also may benefit from discussions on decision making, analytical technology, and emerging applications in DMPK and related topics. Instructors: Mark Hayward (ITSP Solutions and Active Ingredient Technologies); Mike S. Lee (Milestone Development Services); Naidong Weng (Janssen Research and Development); Mingshe Zhu (BristolMyers Squibb) 04 Glycans and Glycoproteins in Mass Spectrometry This course is designed for scientists who want to learn specific techniques for the MS and MS/MS characterization of glycans and glycoproteins. The course will address fundamental aspects of glycobiology, sample preparation and handling, mass spectrometry (hardware and software), and bioinformatic tools for interpretation of results. Real-world examples of the application of these techniques will include characterization of intact glycoproteins, characterization of released glycans, analysis of complex mixtures of glycoproteins and glycans. The role of MS-based methods in interdisciplinary efforts to solve these complex problems will also be addressed. Prerequisites: A basic knowledge of mass spectrometry and some rudimentary knowledge of biology and chemistry is desirable. Instructors: Jon Amster (University of Georgia); Carlito Lebrilla (University of California, Davis); Ron Orlando (University of Georgia); Joe Zaia (Boston University) 05 High Resolution Mass Spectrometry for Qualitative and Quantitative Analysis: An Introduction This introductory course is designed to teach participants the utility, advantages, and limitations of conducting LC-MSn analysis using high resolution mass spectrometry. The fundamental concepts of high resolution and accurate mass measurement will be developed, as will techniques for mass measurement and mass calibration. Current high resolution instrument options including time-of- flight (TOF) and Fourier transform mass spectrometry (FTMS) will be described and compared. Mass analyzer options will be extended into MS/MS platforms including hybrid instruments such as Q-TOF and LTQ-FTMS. Qualitative and quantitative applications of high resolution mass spectrometry will be discussed, especially for small-molecule pharmaceutical compounds, biologics (antibody drug conjugates, peptides, and proteins) and biomarkers. These applications will include molecular formula and structure analyses such as identification of metabolites, as well as effects of high resolution on sensitivity and specificity in quantitation. Instructors: Ragu Ramanathan, Matthew Blatnik, Cong Wei (Pfizer)

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TWO-DAY SHORT COURSES – Saturday and Sunday, June 4 and 5 06 LC-MS: Techniques of Electrospray, APCI and APPI: Understanding and Optimizing to Develop Successful LC-MS Methods This course is designed for the chromatographer / mass spectrometrist who wants to be successful in developing methods, optimizing methods and solving problems using LC-MS. The course covers the atmospheric pressure ionization (API) techniques of electrospray, pneumatically assisted electrospray and atmospheric pressure chemical ionization (APCI) and atmospheric pressure photo ionization (APPI) using single quadrupole, triple quadrupole, time-of-flight and ion trap mass analyzers. Discussions of sample preparation and modes of chromatography will target method development and optimization for the analysis of “real-world” samples by LC-MS. The course highlights the following topics with respect to optimization methods to achieve the best sensitivity, specificity and sample throughput. This course focuses on method development and application to small molecules that are pharmaceutical, peptide, clinically and agriculturally related. Prerequisites: Working knowledge of undergraduate analytical chemistry, including hands on experience with LC separations and mass spectrometry. This is a course for those using LC-MS and LC/MS/MS who want a deeper understanding of the technique to achieve better sensitivity, specificity and to improve their data interpretation skills. Instructor: Robert D. Voyksner (LCMS Limited) 07 LC-MS: Advanced Techniques and Applications This two-day course presents a comprehensive overview of technology and techniques of analytical mass spectrometry and from that foundation extends into exciting, disruptive recent developments. The course covers 1) sample preparation, 2) advanced separation techniques, 3) ionization techniques for ms, 4) mass analyzers, 5) imaging and profiling by MS, 6) high resolution MS, 7) miniaturization in MS, 8) synergistic Integration. Prerequisites: Students for this advanced course should either have five years or more of personal LC/MS/MS experience and familiarity with the scientific literature or have taken one or more introductory courses which cover atmospheric pressure ionization (API) techniques as well as the basics involved in routine LC/MS and LC/MS/MS analyses. Instructor: Jack Henion (Cornell University and Advion Biosciences) 08 MALDI Imaging Mass Spectrometry: Basic Tools and Techniques This course covers the general topic of using mass spectrometry to obtain molecular images from tissue sections, with a focus on MALDI imaging. An overview of different applications and mass analyzers commonly used for small and large molecule imaging will be presented. Discussion of various sample preparation methods, instrument selection and acquisition parameters, data processing tools, and image analysis strategies will be covered in detail. Additional topics such as tissue profiling, in situ chemistry, and 3-D imaging will also be discussed. Participants are encouraged to contribute individual applications for discussion. Instructors: Shannon Cornett (Bruker Daltonics); Michelle Reyzer (Vanderbilt University)

09 Mass Analyzers: Everything You Wanted to Know about Common Mass Spectrometers but Didn’t Know Who to Ask Measuring the mass of an ion requires a bit more sophistication than stepping on your bathroom scale. Thus, it is perhaps no surprise that mass spectrometers use sophisticated ways to measure ion masses, but they also can do so much more, depending on the needs of the analyst. There are a number of different mass analyzers in common use today. Which one is best for your experimental needs, and what performance can you expect from your existing instrument? Among the criteria to consider are: accuracy, precision, speed, sensitivity, limit of detection, and capabilities for multiple stages of mass spectrometry (MS/MS). Understanding the principles and operation of different mass analyzers with regard to these criteria is the foundation for choosing the analyzers(s) best suited for your needs. This course will provide basic knowledge related to time-of-flight, ion cyclotron resonance, quadrupole field based mass analyzers, and combinations thereof. The goal is for students to be able to maximize the potential of the mass spectrometers they currently use and to be able to critically evaluate the options for future instrument purchases. Prerequisites: An introductory level knowledge of physics, and some mass spectrometry experience are useful prerequisites. Instructors: Gary Glish (University of North Carolina); Michael Easterling (Bruker Daltonics); Kevin Owens (Drexel University); Richard Vachet (University of Massachusetts) 10 MS/MS: An Introduction to Instrumentation, Fundamentals, and Spectral Interpretation This course is designed for the student who wants to understand more about the fundamental, instrumental and practical aspects of tandem mass spectrometry. DAY 1 focuses on instrumentation (TOF, Q, QIT, LIT, Orbitrap, FTICR) with emphasis on combinations that are used for MS/ MS (QqTOF, QqQ, QIT, LIT, LIT-Orbitrap or QqFTICR), how these analyzers work together and a brief discussion of how ion mobility MS fits in. This is followed by descriptions of the different modes of scanning (product ion, precursor, neutral loss, SRM/MRM) with literature examples and interactive problem solving. There will be a brief discussion of sample effects, i.e. why you might derivatize and how you might avoid undesired interferences. DAY 2 begins with MS/MS rate theory and comparison of energy deposition mechanisms and dissociation times associated with popular instruments, including discussion of how these influence fragmentation patterns. Specific activation methods CID, IRMPD, ECD, ETD, and SID will be described. The remainder of the day will be spent on interpretation of MS/MS spectra, with sections devoted to small molecules and to peptides. The course material is designed for the general audience. We encourage participants to contact us in advance if there are particular molecule types or questions that we might address. Prerequisites: New users of MS/MS who have some basic knowledge of mass spectrometry (i.e. familiar with ESI and MALDI, and understand the basic principles of at least one type of mass analyzer.) Instructors: Linda Breci and George Tsaprailis (University of Arizona); Arpad Somogyi and Vicki Wysocki (Ohio State University)

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TWO-DAY SHORT COURSES – Saturday and Sunday, June 4 and 5

11 Peptides and Proteins in Mass Spectrometry Over the past two decades, mass spectrometry has become the key technology for the characterization of proteins. This course is designed as an introduction for researchers needing to expand their knowledge of the use of mass spectrometry-based methods for the identification, characterization, and quantification of peptides and proteins. Background material in basic protein chemistry will be provided along with a review of mass analyzers, acquisition types, and ionization sources used for protein and peptide analysis. Real-world examples will be used to illustrate protein sample preparation strategies, characterization of intact proteins, characterization of post-translational modifications (with emphasis on glycosylation and phosphorylation), identification of proteins via database searching, direct identification of proteins from gels and complex mixtures, and quantitative differential protein expression studies. The role of MS-based methods in interdisciplinary efforts to solve complex biomedical problems will also be addressed. Additionally, there will be tutorials on the use of open source proteomic software tools for interpretation of proteomic datasets. Problem sets will be used to emphasize practical aspects of comprehensive protein characterization, including peptide sequencing and protein identification by database searching. Prerequisites: Hands on experience in mass spectrometry and/or protein chemistry. Instructors: Kevin Blackburn (North Carolina State University); Arthur Moseley (Duke University); Douglas Sheeley (National Center for Research Resources/NIH) 12 Protein Structural Analysis by Mass Spectrometry: Hydrogen Exchange and Covalent Labeling This course is designed for those who wish to understand protein structure with mass spectrometry. We will focus on hydrogen exchange for half of the course and on covalent labeling for the other half. There will be a discussion of the theory behind each labeling method as it relates to proteins in solution, the general methodological steps one takes to do these analyses, and a guide on how to process and interpret the resulting data. There will be a discussion of the pitfalls one can encounter during these experiments. Specific examples of protein structure analysis will be provided in applications like enzymology, protein-ligand interactions, protein dynamics, and membrane proteins. Course participants will receive a copy of a new book from Wiley edited by the course instructors providing detailed additional background on all the approaches. Continuing education will be available through the hydrogen exchange and covalent labeling special interest group of ASMS, which will provide participants with additional learning opportunities during the meeting, as well as updates after the course. Prerequisites: Working knowledge of analytical chemistry, protein structure, and peptide/protein mass spectrometry. Instructors: John R. Engen and Thomas E. Wales (Northeastern University); Mark R. Chance and Janna Kiselar (Case Western Reserve)

13 Protein Therapeutics: Practical Characterization and Quantitation by Mass Spectrometry This introductory course is designed for practicing analytical scientists (new users, chromatographers, analytical chemists, protein chemists, and laboratory managers) performing/supporting recombinant protein characterization/analysis, in-process testing, quality control, quality assurance, research and development, and manufacturing. It covers the fundamental principles and practical applications of mass spectrometry (MS) for the characterization and quantitation of protein therapeutics with a focus on monoclonal antibodies including antibody-drug conjugates (ADCs). It emphasizes problem-solving skills in the areas where MS is used most extensively, for example, characterization of chemical modifications and post-translational modifications of protein therapeutics. The interpretation of protein therapeutics mass spectra will be illustrated with real case examples. This course will focus on electrospray ionization (ESI) and matrix-assisted laser desorption / ionization (MALDI) techniques in the analysis of protein therapeutics, and will survey various mass analyzer options for the characterization of protein therapeutics. A thorough coverage of approaches toward method development for both qualitative and quantitative analysis of protein therapeutics will provide a good starting point for understanding practical issues that are frequently encountered during implementation and applications of MS on protein therapeutics in the laboratory. Prerequisite: Working knowledge of mass spectrometry and/or protein chemistry Instructors: Guodong Chen and Li Tao (Bristol-Myers Squibb); David M. Hambly (Amgen) 14 Quantitative Mass Spectrometry This introductory/intermediate level course explores the principles of quantitative mass spectrometry as they apply primarily to small molecule analyses. Topics that will be discussed include: (a) MS instrumentation used for quantitative analyses; (b) how a quantitative assay is set up including statistics for quantitation, preparation and evaluation of standard curves and analysis of variance; (c) LC/MS/MS method development for target quantitative analysis of small molecules, including instrument and assay optimization procedures; (d) real world approaches to assessing problems and improving method ruggedness; (e) general guidelines for method validation in a regulated environment; (f ) an introduction to quantitation of endogenous compounds in biological matrices; and (g) an introduction to quantitation in proteomics. Ample time will be provided for general discussion of the topics presented and other current topics in quantitative MS. Prerequisite: Familiarity with the basic principles of mass spectrometry. Instructors: Robert Bethem (Independent Contractor); Dwight E. Matthews (University of Vermont)

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ONE-DAY SHORT COURSES Go to www.asms.org for more detailed descriptions, information, and online registration. 15 LC-MS Practical Maintenance and Troubleshooting Repeated Saturday and Sunday With expanding use of LC-MS in laboratories not specifically focused or trained in analytical chemistry, the analytical skills needed to maintain high instrument performance and data quality are less often part of the central curriculum or training of instrument operators in these laboratories.  Indeed, high-end LC-MS systems are now routinely found far outside of the traditional analytical chemistry laboratory, including labs focused on pharmaceutical, biochemical, biological, or environmental applications. This is a natural progression as LC-MS technologies become more mature and robust, however the increased use of LC-MS has not removed the need for basic troubleshooting, instrument maintenance and repair skills, which are critical for limiting downtime and providing cost savings to any laboratory. While an in-depth discussion of how to operate each individual instrument is surely outside the scope of any short course, this short course will seek to introduce a basic skill set for troubleshooting and repair of LC-MS instrumentation, including the following areas: basic “best practices,” system suitability and qualification, tuning, calibration, basic troubleshooting and maintenance. Importantly, the course will actively seek to remain as vendor-neutral as absolutely possible and will seek to cover nano-, micro- and analytical-scale chromatographic methods as well as general information on ion trap, quadrupole, FT, and TOF mass spectrometry platforms. The goal is to better equip the growing numbers of LC-MS practitioners for success in their use of the technology. Instructors: Susan Abbatiello (The Broad Institute); Thomas Blau (Ion Technology Support, Inc); Will Thompson (Duke University) 16 Metabolomics: Analyzing Chemical and Biological Diversity Repeated Saturday and Sunday This one-day short course is designed for investigators with minimal experience in metabolomics, but with background in mass spectrometry.  The course will focus on untargeted profiling of metabolites with LC/ MS approaches, providing a detailed overview of each step of the workflow.  The discussion will start with considerations related to experimental design and highlight advantages/disadvantages to using various extraction protocols, separation strategies, ionization methods, and data-analysis tools.  A survey of bioinformatic resources currently available to interpret metabolomic data will be provided and the application of these tools to translate raw data into biology explored.  Finally, the challenges of performing metabolomics will be outlined and technologies being developed to overcome these barriers highlighted.  The course will conclude with an overview of isotope-based approaches being used to investigate metabolic dynamics. Instructors: Gary Patti (Washington University School of Medicine)

pharmaceutical industry and CROs. The short course will also reflect the contents of the updated guidance and recently published white papers with regard to bioanalytical method validation using LC-MS/ MS. International harmonization of bioanalytical method validation guidance will be discussed. This one-day short course is for analytical chemists who use LC-MS/MS in GLPregulated labs, lab supervisors, QA managers and personnel, GLP auditors and CRO consultants, who need a fundamental understanding of GLP regulations and guidance. This course is also useful to all levels of management as a refresher on the GLP regulations. Instructor: Perry G. Wang, LC-MS Technical Expert 18 Ion Mobility Mass Spectrometry: An Introduction to Instrumentation, Applications, and Data Analysis Sunday only Realization of the value-added by combining ion mobility spectrometry (IMS) and mass spectrometry (MS) has generated significant interest in its use in range of fields including omics pursuits, threat detection, and fundamental studies of gas-phase ion chemistry. This course provides students with a basic understanding of the most common instrumental configurations, experimental sequence, best practices, and the theory underlying the different types of ion mobility systems employed both in academic and commercial settings. An overview of hyphenated IMS-MS instrumentation with multiple practical applications and experimental designs will be presented including comparative discussions on advantages and disadvantages between different stand-alone and hyphenated techniques. This course will provide a comprehensive look at ion mobility spectrometry and its use in modern analytical chemistry. While ion mobility systems are largely compatible with all mass spectrometry systems each IM-MS combination possesses a range of unique advantages and applications. These instrumental considerations and specific experimental sequences will be covered in this course. Graduates of the course will have gained a detailed understanding of IMS and an overview of its practical applications for both stand-alone and MS-coupled operation. Prerequisite: Working knowledge of mass spectrometry. Instructors: Brian H. Clowers (Washington State University); Erin Baker (PNNL); John A. McLean (Vanderbilt University)

17 Introduction to GLP Regulations and Bioanalytical Method Validation by LC-MS/MS Sunday only “GUIDANCE FOR INDUSTRY/Bioanalytical Method Validation” represents the Food and Drug Administration’s current thinking on this topic and was published in May 2001. Since then, almost all regulated bioanalytical methods have been validated based on the guidance even though it does not create or confer any rights for or on any person and does not operate to bind FDA or the public. In September 2013, FDA published a draft guidance, which provides general recommendations for bioanalytical method validation using advanced technologies including LC-MS/MS. The content of the original guidance issued in 2001 was revised to reflect advances in science and technology related to validating bioanalytical methods. This one-day short course will focus on the bioanalytical method validation for drugs and metabolites in biological matrices using LC-MS/MS. It will help audiences to comply with FDA’s regulations for drug discovery and development in the

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