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MSACL 2015 EU: Preliminary Conference Program

Salzburg Congress Center, AUSTRIA • September 8-11, 2015

With Thanks to Our Corporate Sponsors:
Thermo Shimadzu Agilent Chromsystems Tecan

TUESDAY
9:00 AM
10:00 AM
WELCOME COFFEE
@ Mozart 1-3
Enjoy coffee, a muffin and a chat with colleagues before the day starts.
10:00 AM
12:00 PM

SHORT COURSES: SESSION 1
Getting Started with Quantitative LC-MS/MS in the Diagnostic Laboratory
Judy Stone, PhD & Grace van der Gugten
Level: 1-2 (Beginner - Intermediate)
Location: Mozart 4
Breaking up with Excel: A Newbie's Introduction to the R Statistical Programming Language
Daniel Holmes, MD & Stephen Master, MD PhD
Level: 1-2 (Beginner - Intermediate)
Location: Mozart 5
Practical LC-MS Maintenance and Troubleshooting (Tuesday)
Erik J. Soderblom, PhD, Christopher Shuford, PhD, J. Will Thompson, PhD
Level: 2 (Intermediate)
Location: Trakl Hall
Registration for either day of this course (Tuesday or Wednesday) allows you to attend both days.
Development and Validation of Quantitative LC-MS/MS Assays for Use in Clinical Diagnostics
Russell Grant, PhD & Brian Rappold
Level: 3 (Advanced)
Location: Papageno Hall
12:00 PM
1:00 PM
LUNCH
@ Mozart 1-3
Lunch to be provided in Mozart 1-3 with the possibility to eat outside in front of the Congress Center on bistro tables, weather permitting.
1:00 PM
3:00 PM

SHORT COURSES: SESSION 2
3:00 PM
3:30 PM
COFFEE BREAK
@ Mozart 1-3
Take a break and get a coffee, water and/or snack. Commune with colleagues or perhaps go for a short walk outside to refresh for the next session.
3:30 PM
5:30 PM

SHORT COURSES: SESSION 3
5:30 PM
7:00 PM
WORKING DINNER
@ Mozart 1-3
An evening buffet working dinner with appetizers and drinks to allow you time to connect with your instructor and classmates before the last hour of class for the day!
7:00 PM
8:00 PM

SHORT COURSES: SESSION 4
8:00 PM
Your Decision
ENJOY THE CITY
@ Salzburg Old City
Explore the Old Town of Salzburg.
TUESDAY CLOSED

WEDNESDAY
8:00 AM
8:30 AM
WELCOME COFFEE
@ Mozart 1-3
Enjoy coffee, a muffin and a chat with colleagues before the day starts.
8:30 AM
10:30 AM

SHORT COURSES: SESSION 1
Getting Started with Quantitative LC-MS/MS in the Diagnostic Laboratory
Continued from Saturday
Judy Stone, PhD & Grace van der Gugten
Level: 1-2 (Beginner - Intermediate)
Location: Mozart 4
Breaking up with Excel: A Newbie's Introduction to the R Statistical Programming Language
Continued from Saturday
Daniel Holmes, MD & Stephen Master, MD PhD
Level: 1-2 (Beginner - Intermediate)
Location: Mozart 5
Detection Of Pathogens By Whole-Cell MALDI-TOF MS And Advanced Proteomics Approaches
Jean-Armengaud, PhD, Oliver Pible & Julia Chamot-Rooke, PhD
Level: 1-2 (Beginner - Intermediate)
Location: Paracelsus Hall
Practical LC-MS Maintenance and Troubleshooting (Wednesday)
Erik J. Soderblom, PhD, Christopher Shuford, PhD, J. Will Thompson, PhD
Level: 2 (Intermediate)
Location: Trakl Hall
Registration for either day of this course (Tuesday or Wednesday) allows you to attend both days.
Development and Validation of Quantitative LC-MS/MS Assays for Use in Clinical Diagnostics
Continued from Saturday
Russell Grant, PhD & Brian Rappold
Level: 3 (Advanced)
Location: Papageno Hall
10:30 AM
11:00 AM
COFFEE BREAK
@ Mozart 1-3
Take a break and get a coffee, water and/or snack. Commune with colleagues or perhaps go for a short walk outside to refresh for the next session.
11:00 AM
12:00 PM

SHORT COURSES: SESSION 2
12:00 PM
1:00 PM
LUNCH
@ Mozart 1-3
Lunch to be provided in Mozart 1-3 with the possibility to eat outside in front of the Congress Center on bistro tables, weather permitting.

Fresh air has been reported to assist in reduced lethargy and increased levels of Vitamin D (if accompanied by sun exposure).
1:00 PM
3:00 PM

SHORT COURSES: SESSION 3
3:00 PM
3:30 PM
COFFEE BREAK
@ Mozart 1-3
Take a break and get a coffee, water and/or snack. Commune with colleagues or perhaps go for a short walk outside to refresh for the next session. POSTER PRESENTERS: If you are presenting a poster today, it should be placed by the end of this break.
3:30 PM
5:30 PM

SHORT COURSES: SESSION 4
5:30 PM
8:30 PM
OPENING RECEPTION
@ Exhibit Hall / 1st Floor
Enjoy mingling with colleagues and Exhibitors. Take time to explore the Posters, which will be attended from 6:00 - 7:00 PM. Buffet dinner and drinks to be provided.
Thermo
6:00 PM
7:00 PM
POSTERS
@ Exhibit Hall / 1st Floor
ALL Posters Attended from 6:00 - 7:00 PM.
8:30 PM
Your Decision
ENJOY THE CITY
@ Salzburg Old City
Explore the Old Town of Salzburg.
WEDNESDAY CLOSED

THURSDAY
7:45 AM
8:45 AM
PLACE POSTERS
@ Exhibit Hall / 1st Floor
Poster presenters for Thursday must have their posters placed by 9 AM.
7:45 AM
8:45 AM
WELCOME COFFEE
@ Registration Foyer
Enjoy coffee, a muffin and a chat with colleagues before the day starts.
8:00 AM
8:40 AM

CORPORATE WORKSHOPS (8:00 - 8:40 AM)
Thermo Scientific
Papageno Hall

Time Matters. Simplify Workflow Routines
Edward Goucher, Thermo Fisher Scientific  

Pre-Register

Discover four medical devices for general clinical use: Thermo Scientific™ Prelude MD™ HPLC, Thermo Scientific™ Prelude LX-4 MD™ HPLC, Thermo Scientific™ Endura MD™ mass spectrometer, and Thermo Scientific™ ClinQuan MD™ Software. Clinical laboratories can use these devices to build their own lab developed tests (LDT). Various example compounds and workflows will be used to demonstrate the robustness and time efficiency of these new medical devices. For in vitro diagnostic use. Not available in all countries.
8:45 AMWELCOME, INTRODUCTION & ORIENTATION
@ Mozart Hall

Welcome to the 2nd Annual European MSACL Congress!

Congress Mobile Apps
Two apps to facilitate contact collection and Scientific Program browsing.

(1) The Mobile Program App
Online @ https://www.msacl.org/mobile

(2) BadgerScan™ for Contact Lead Collection.

Available on Google Play and the Apple App Store (iTunes).
PLENARY LECTURE SERIES
@ Mozart Hall
Chair: Michael Vogeser & Oleg Mayboroda
9:00 AM
9:45 AM
Mass Spectrometry as Metrological Anchor in Laboratory Medicine – a Meandering River
Linda MR Thienpont
Ghent University
Long Abstract | Biography | Financial Disclosure

Distinguished Contribution Awardee

The fundament of mass spectrometric (MS) work is the SI base unit “mole”, more in particular, quantitative MS establishes SI-traceability (mol/L) of measurement results. I will discuss the anchors and regulatory surrounding to implement SI-traceability in laboratory medicine. When I entered this field, I thought my scientific life would be a “fast-flowing river”. However, several obstacles forced it to meander, e.g., concepts like biological variation, analytical performance goals, commutability, and the introduction of MS in routine. Although I tried to cope with these obstacles, “my river” continues to meander. As an outlook, I will present some of my most recent work.
9:45 AM
10:30 AM
The Cellular Uptake of Pharmaceutical Drugs Is Transporter-Mediated and Is Thus a Problem Not of Biophysics But of Systems Biology
Douglas Kell
The University of Manchester
A fundamental question remains as to whether xenobiotic drugs cross cellular membranes mainly (or exclusively) by transporter-independent diffusion across whatever bilayer lipoidal parts of cellular membranes may be present, or whether they normally (or exclusively) ‘hitchhike’ rides using the carriers normally involved in the metabolism of natural metabolites. The former (for which, astonishingly, there is in fact no actual experimental evidence) would involve a biophysical mechanism, based mainly on lipophilicity, while the latter requires a mechanistic understanding of which carriers are involved, and is thus a problem of network or systems biology. In other words, “is carrier-mediated transport of pharmaceutical drugs the exception or the rule?” A huge amount of literature (see Long Abstract), that I shall summarise, indicates that there is no serious evidence against the view that trans-phosphobilayer-mediated transfer of pharmaceutical drugs across biological membranes is negligible (‘PBIN’), while there is abundant and increasing evidence for the carrier-mediated route. A recent approach in yeast illustrates this experimentally, while the digital availability of principled metabolic network models allows one to determine, consistent with this, that successful pharmaceutical drugs are much more like metabolites than are the ‘Lipinski-compliant’ molecules typically available in drug discovery libraries. This suggests (or is at least consistent with the view) that cellular drug uptake is more or less exclusively transporter-mediated, and that knowledge of both the metabolome and of the concentrations and activities of transporters used by individual xenobiotics will be of much value in designing better drugs and bioprocesses.
10:30 AM
11:15 AM
COFFEE BREAK
@ Exhibit Hall / 1st Floor
Visit the Exhibit Hall to procure coffee, juice, water and/or snacks. Explore what's on offer from the Exhibiting vendors, reconnect with colleagues, or go for a short walk outside to refresh for the next session. POSTER PRESENTERS: If you are presenting a poster today your poster should have been up 2 hours ago. If it is not up please put it up immediately.

Sponsored by:
Thermo Shimadzu Agilent
GENERAL SCIENTIFIC SESSION 1
Track 1
Mozart 1-3
Small Molecule Assay Development
Chair: Jody van den Ouweland		Track 2
Mozart 4-5
Early Life Metabolomics
Chair: Uta Ceglarek 		Track 3
Papageno Hall
Proteomics Assay Development
Chair: Steve Master		Track 4
Paracelsus Hall
DESI
Chair: David Phelps		Track 5
Trakl Hall
Intro to Mass Spec
Chair: Jane Yang
11:15 AM
11:40 AM
Design of Optimization: How to Systematically Improve the Performance of High Volume LC-MS/MS Clinical Assays
Brian Rappold
Essential Testing
Long Abstract
Method optimization is largely a colloquial term for LC/MS/MS users. The extent of optimization can range from cursory evaluations during core development experiments to substantive investigations of all variables associated with a method. Unlike design of experimentation which requires broad assumptions and large experimental parameter shifts, design of optimization relies on efficiently designed experimentation with the ability to produce valid conclusions. This paper will introduce a practical series of experimental frameworks to scientifically rationalize the parameters and processes involved in optimization of a clinical diagnostics assay. Examples will be shown from assays which analyze more than 500 specimen/day. 		Diagnosis of Inborn Metabolic Disorders via Absolute Enzymatic Activity
Zdenek Spacil

University of Washington
Long Abstract | Bio | View Video
The newborn screening for lysosomal storage disorders, such as sphingolipidoses or mucopolysacharidoses is critical to detect affected individuals and to timely initiate treatment. Over 15 screening assays developed in our lab measure activity of lysosomal enzymes in dried blood samples (DBS) by tandem mass spectrometry technology (SRM). These assays greatly outperform other screening tests, such as fluorometric assays in terms of sensitivity and specificity, are easy to implement and allow for multiplexing, which was collectively demonstrated in numerous pilot studies worldwide. Besides the latest development and clinical data from pilot studies, we are planning to present on the absolute enzyme activity measurement in highly purified leukocyte populations as a second-tier diagnostic test, which is transforming the field.		Development of Clinical Assays Based on Parallel Reaction Monitoring
Bruno Domon
Luxembourg Clinical Proteomics Center
Long Abstract | Bio | View Video
Targeted proteomics analyses of biomarker are routinely performed on triple quadrupole mass spectrometers, which present limited selectivity, due to low resolution of the mass filters. The targeted analyses of clinical samples carried out on a quadrupole-orbitrap mass spectrometer using parallel reaction monitoring (PRM) showed a significant gain in sensitivity and selectivity. A new acquisition method leveraging the presence of internal standards showed a dramatic improvement of the reliability and the precision of the analyses. The method has a broad applicability to the quantitative analysis of clinical samples, such as lung cancer plasma samples to discriminate the disease stages and subtypes; and to tissue samples to map driver mutations (EGFR and KRAS). 		Ovarian Tissue Identification with Rapid Evaporative Ionisation Mass Spectrometry (REIMS); the Surgical Intelligent Knife
David Phelps

Imperial College, London
Long Abstract | Bio | View Video
Ovarian cancer is common and 5-year survival is 43.5%. Surgeons use intra-operative frozen section for tissue identification but this is time-consuming and expensive. Microscopic non-descript lesions during surgery, which may be cancer can be difficult to correctly identify. The near-real-time tissue identification abilities of the iKnife were tested in this study. We have shown for the first time that ovarian, peritoneal and fallopian tube tissues have unique REIMS spectral signatures, which can be used to accurately classify tissue histopathology. Leave-one out patient cross-validation resulted in 100% sensitivity and 100% specificity in the separation of normal and cancerous ovary (n=189).		Introduction to Mass Spectrometry
Jane Yang
University of California, San Diego
Long Abstract
Mass spectrometry is a powerful analytical chemistry tool that detects molecules in the gas phase based on their mass to charge ratio. This session will cover the basic concepts of mass spectrometry, how it works, the components of a mass spectrum, tandem MS (or MS/MS), the relationship between chromatograms and mass spectra, tuning, and what your service representative will do.
11:40 AM
12:05 PM
What Is Wrong in Your LC-MS Analysis? Post Column Infusion as a Diagnosis Tool
Oskar Gonzalez

University of the Basque Country
Long Abstract | Bio | View Video
Continuous post-column infusion of standards is a very useful tool to obtain additional and very valuable information about a LC-MS method. Here we show multiple applications of this technique in method development and routine analysis based on our experience in the laboratory: detection of compounds causing ion suppression, aid in sample treatment choice, understanding of abnormal results, compensation of matrix effect and some others. Considering all these benefits, we encourage to use this technique in clinical analysis to improve the reliability of LC-MS methods and to obtain more accurate results.		Research into Lysosomal Storage Metabolism Using Plasma Lipid Characterization by LC-MS/MS
Dan Blake
SCIEX
Long Abstract | Bio | Financial Disclosure | View Video
Research into plasma sphingolipids and determining the concentrations of such is of growing importance in the clinical research laboratory, particularly within groups researching Lysosomal Storage Metabolism. Current methods of analysis primarily involve either enzyme activity procedures or derivatization of compounds prior to analysis. Direct analysis of these groups can be complex due to extensive structural homogeneity between individual compounds. We present here a method for a direct multi-compound approach to this analysis, employing modern advances in column technology to produce a rapid and sensitive LC-MS/MS method for these compounds.		MS-based Serum Proteomics in Clinical Chemistry: Requirements for Candidate Translation and Implementation as a Routine Assay
Christa Cobbaert
Leiden University Medical Center
Long Abstract | View Video
Numerous serum protein profiling efforts have aimed for biomarker discovery since the early days of mass spectrometry(MS)-based proteomics. These pipelines have yielded many promising protein candidates, but disappointingly none of these has been translated into a truly validated medical test. Alternatively, strategies have been followed to convert existing routine uniplex protein assays into an MS-based in-vitro diagnostic test, however also in this case the success rate has been low so far. In this presentation both approaches will be discussed with regard to requirements needed for translation or implementation these into a clinical chemistry laboratory.		The Mucosal Metabolome: Real-time Rapid Medical Swab Point-of-care Analysis Using TFME-DESI MS to Reveal Pathogenic and Inflammatory Metabolomic Markers
Pamela Pruski

Imperial College London
Long Abstract | Bio
The mucosal membrane, a protective layer responsible for trapping pathogens in the human body, is an easily accessible and highly clinically relevant sample to diagnose pathogenic and cancerous associated diseases. Since DESI MS is not applicable for in-vivo analysis due to the potential risk of electrical shock and the use of organic solvent, medical swabs are a standard collection device for mucosal membranes that can be directly analysed with DESI MS. Chemical signatures identification of specific bacteria within minutes on the surface of swabs would provide a rapid diagnosis of infections associated with preterm delivery in comparison to standard microbial testing.		Introduction to Ionization Modes in Mass Spectrometry
Jörg Hanrieder

University of Gothenburg
Long Abstract | View Video
Ionization of analytes into the gas phase is a critical step for accurate mass identification. Various types of ionization methods can be applied for optimal analyte identification. This session will involve a basic introduction to various ionization modes used in mass spectrometry and will discuss the advantages and disadvantages of each.
12:05 PM
12:30 PM
Fully Automated Multi-Method LC-MS: Application in Clinical Laboratory
Marco Cantù
Ente Ospedaliero Cantonale
Long Abstract | Bio | View Video
In the last 4 years we setup a flexible configuration able to do different analytical methods on a single LCMS. This configuration is able to load simultaneously: up to 24 solvents into the primary pump, up to 4 solvents into the secondary pump, up to 15 columns, divided into 2 independent thermostats. This system is able to manage all instrument’s changes (solvent line selection, column selection, etc.) directly from the work-list. This is a real huge result that open the possibility to process urgent samples just adding them into the work-list also while the LCMS is processing other methods, without any switch off, changes and restart.		Preeclampsia Risk Stratification Early in Pregnancy: First Results of a New LC-MS Based Multiplex Metabolite Assay
Robin Tuytten
Metabolomic Diagnostics
Long Abstract | Bio | Financial Disclosure
Preeclampsia is one of the major complications of pregnancy. First time pregnant women have an increased risk for preeclampsia, yet routine prenatal care fails to accurately identify the 1st time pregnant women at risk. Basic biomarker research revealed that blood borne metabolites bear potential to risk-stratify for preeclampsia early in pregnancy. In a dedicated translational research effort, a prototype multiplex metabolite LC-MS assay was developed and then applied to a large case:control study focusing on 1st time pregnant women. This study confirmed that the newly discovered metabolite biomarkers enable prediction of preeclampsia risk early in pregnancy. 		The Holy Trinity: The Inter-relationship of Standards, Matrix, and Internal Standardization in Protein Cleavage Isotope Dilution Mass Spectrometry (PC-IDMS)
Russell Grant
Laboratory Corporation of America
Long Abstract | Bio | Financial Disclosure
To better understand the inter-relationship of calibration (matrix and standard) and internal standardization on the accuracy of digestion-based protein quantification, the relative digestion efficiency of multiple internal standard types (peptides/protein), multiple standardized forms of unlabeled protein (recombinant/human-derived) and multiple matrices (true/surrogate) are evaluated for quantifying thyroglobulin in serum using multiple signature peptides. Comparing multiple matrices and protein standards will differentiate if the disparities/similarities between peptides are matrix effects and/or specific to the analytical standard used. A variety of digestion conditions are explored in combination with different internal standards to determine if disparities between peptides can be mitigated.		DESI-MS and REIMS as Excellent Techniques to Complement and Support Histology in Ovarian Cancer
Luisa Doria
Imperial College of London
Long Abstract | Bio | View Video
Ovarian cancer is the fifth most common cancer among women and one of the causes is the poor and vague prognosis and diagnosis. REIMS and DESI-MS are two mass spectrometry techniques with great potential to characterize and discriminate different cancer types and stages. Both techniques are excellent to complement and support histology, REIMS can identify different ovarian cancer types in real time and DESI can provide detailed spatial information within the sample giving the opportunity to investigate tumour biology from an entirely new perspective with accurate biochemical information about each tissue type. 		Introduction to Mass Analyzers: Quadrupole vs. Time-Of-Flight (TOF)
Jörg Hanrieder

University of Gothenburg
Long Abstract | View Video
Choosing a mass analyzer for clinical analysis is an important step in setting up a mass spectrometry laboratory. But what is a mass analyzer? Are all mass analyzers are created equal? What types of clinical tests can be validated on each? Each type of mass analyzer has its own benefits and caveats in mass resolution, sensitivity, and dynamic range for small molecule analysis. Choosing a mass analyzer to suit the needs of the clinical laboratory is an important consideration to make. This lecture will focus on quadruple and time of flight (TOF) mass analyzers for identification and quantification of small molecules. This lecture will also describe how these different mass analyzers actually create mass separations and will describe the various clinical applications available to each.
12:30 PM
2:30 PM
LUNCH
@ Exhibit Hall / 1st Floor
Lunch to be provided in the Exhibit Hall. • Get ready to join a Corporate Workshop after the poster session.

Sponsored by:
Thermo Shimadzu Agilent
1:30 PM
2:30 PM
POSTERS
@ Exhibit Hall / 1st Floor
All Posters to be attended from 1:30 - 2:30 PM.
2:30 PM
3:30 PM

CORPORATE WORKSHOPS (2:30 - 3:30 PM)
Shimadzu
 Mozart 1-3

Smart Solutions: Fully Automated Systems for Your Laboratory Applications
Pre-Register

1. TDMPREP - Centrifugation Free Sample Preparation with Magnetic Beads!
Mario WUTTKE, Magnamedics, Netherlands

2. New Developments in NBS using DBS for Metabolic Profiling including Steroids
Prof David C. KASPER, MedUni Vienna/ARCHIMEDlife, Austria

3. New LCMS-8060: Opens Up the Way for New Applications
Stephane MOREAU, Shimadzu Europa Gmbh, Germany

4. Fully Automated System including Sample Preparation
Stephane MOREAU, Shimadzu Europa Gmbh, Germany

Thermo Scientific
 Mozart 4-5

Identifying Unknown Unknowns with High Resolution Accurate Mass MS in Toxicology
Robert Mistrik, HighChem
Pre-Register

Despite the increasing use of modern high resolution mass spectrometers, toxicological analysis is hindered by an inability to identify detected compounds effectively. We will present an advanced computational and database framework leading to the much anticipated increase in coverage of toxicologically relevant compounds and their transformation products, taking into account all the important experimental and calculated information necessary for efficient and reliable identifications. Those methods are exploiting combination of library searching methods, both spectral (mzCloud) and structural (PubChem, ChemSpider), with computational techniques like quantum chemical methods, precursor ion fingerprinting, fragment ion search and others.

Agilent Technologies
Papageno Hall

Workshops on StreamSelect, Metabolic Workflows and Sample Prep with AssayMAP Bravo

1. Introducing the New Agilent StreamSelect LC/MS System
Pete Christensen, PhD, Agilent Technologies, UK

2. Metabolomic Workflows for Clinical Research-demonstrating a tool to address investigation of inborn metabolic errors
Dr. Holger Stalz, Agilent Technologies, Switzerland

3. Rethink your Protein Sample Preparation Strategy with Agilent AssayMAP Bravo
Moritz Wagner, PhD, Agilent Technologies, Germany

Spark Holland
 Paracelsus Hall

Scientific and Instrumental Advances in Dried Blood Spot Analysis
Christophe Stove and Bert Ooms
Dried blood spot analysis is emerging as a useful tool for quantitative bioanalysis, particularly for micro volumes of blood or plasma. DBS enables remote sampling in low resource settings or at-home sampling for out-patients. The micro-volume concept helps to reduce lab-animal use and allows use of finger prick instead of venous puncture, making the technique easier applicable for children. However, issues such as the effect of hematocrit on spot size have hindered broad acceptance of DBS for routine bioanalysis. Recent innovations have addressed these issues and will be presented and discussed in this workshop by professor Christophe Stove (Gent University). An introduction to the new DBS autosampler from Spark Holland will be presented by Bert Ooms, principal scientist(Spark Holland).
3:30 PM
4:30 PM
COFFEE BREAK
@ Exhibit Hall / 1st Floor
Visit the Exhibit Hall to procure coffee, juice, water and/or snacks. Explore what's on offer from the Exhibiting vendors, reconnect with colleagues, or go for a short walk outside to refresh for the next session.

Sponsored by:
Thermo Shimadzu Agilent
GENERAL SCIENTIFIC SESSION 2
Track 1
Mozart 1-3
Small Biomarkers
Chair: Laurent Decosterd		Track 2
Mozart 4-5
Steroid Metabolomics
Chair: Roland Geyer		Track 3
Papageno Hall
Quantitative Proteomics
Chair: Andy Hoofnagle		Track 4
Paracelsus Hall
Advanced Clinical Microbiology
Chair: Irene Burckhardt		Track 5
Trakl Hall
Basics of Mass Spectrometry
Chair: Steve Master
4:30 PM
4:55 PM
Endogenous Ouabain – LC-MS/MS Identifies a Fiction
Silvia Baecher
Hospital of the University of Munich
Long Abstract | Bio | Financial Disclosure | View Video
For decades immunoassays were applied in an attempt to quantify the cardiac glycoside ouabain in plasma of patients with cardiovascular conditions. This compound (CAS 630-60-4) was supposed to be of endogenous origin as a counterpart of exogenous cardiac glycosides such as digoxin. Using a fully validated, highly specific and extremely sensitive LC-MS/MS method we were able to show that ouabain cannot be present in relevant concentrations in human plasma - as claimed by immunoassay studies. 		Quantitation of Steroid Hormones in Different Biological Matrices – One Method to Rule Them All
Alexander Gaudl

Leipzig University
Long Abstract | Bio
The quantitative analysis of endogenous steroid hormones cuts across plasma, serum, saliva, urine, dried blood and hair rendering immunological analysis laborious due to the need of different methods. We developed a unifying, highly adaptive LC-MS/MS-method covering the most important endogenous steroid hormones. With excellent inter day imprecision (3 15%), very high recovery (89 103%) and a total runtime of 5.0 min we enabled rapid, multi-matrix analysis utilizing MRM and MS³ in modified, positive and negative electrospray ionization. Online sample clean-up via automatic column switching combined with an extremely simple dilute-and-shoot sample preparation allows high throughput analysis in clinical routine diagnostics and epidemiological studies.		Targeted Quantification of 97 Proteins in Dried Blood Spots
Christoph Borchers
UVic Genome BC Proteomics Centre
Long Abstract | View Video
We report a multiplexed LC-MRM-MS method for the precise quantification of 97 endogenous proteins in human DBS samples, suitable for biomedical research applications. Standard curves were generated for each of the 173 target peptides (representing the 97 proteins) to fully characterize the assay’s analytical merits. Furthermore, the stability of each target peptide was investigated in DBS samples stored at -20°C, 4°C, RT and 37°C over a duration of 154 days. Finally, we will present a simple strategy for managing the hematocrit effect across samples and discuss the implications on the DBS-MRM workflow.		Automated Rapid Evaporative Ionisation Mass Spectrometry (REIMS) for the Characterisation and Identification of Microbes
Frances Bolt
Imperial College
Long Abstract | Financial Disclosure
The introduction of mass spectrometry for clinical microbiology laboratories has revolutionised work flows and provided timely and accurate clinical diagnoses. Rapid evaporative ionisation mass spectrometry (REIMS) has previously been show to allow the differentiation of fungi and bacterial species based upon their lipidome (Strittmatter et al., 2013; Strittmatter et al., 2014). In order to utilise this tool for clinical diagnostics and research we are creating a spectral library comprising over 50,000 isolates and 4000 species. A novel customised TECAN platform which incorporates automated colony imaging, colony picking and REIMS analysis has been developed to provide a reproducible system for high throughput workflows. REIMS on the customised TECAN EVO platform provides an ideal method for different applications including clinical diagnostics, lipidome and metabonomic analysis. 		Tuning Your Mass Spectrometer: How to Make It Sing
Michael Chen
McGill University
| View Video
Tuning is fundamental to operating a mass spectrometer and involves mass calibration and mass resolution. This overview will focus on what happens when the mass spectrometer is tuned and introduces basic chemical concepts to help novice users understand what it means when your instrument representative says “I need to tune the mass spectrometer”.
4:55 PM
5:20 PM
Quantitation of Desmosine in Plasma by LC-MS/MS as Biomarkers for Elastin Degradation
Jody van den Ouweland
Canisius-Wilhelmina Hospital
Long Abstract | Bio | View Video
Desmosine is a promising biomarker for estimating activity of elastin degradation in patients with COPD, although its clinical validity remains uncertain as reliable and sensitive assays are lacking. An LC-MS/MS method for measuring plasma desmosine was developed and validated. Sample preparation consisted of acid hydrolysis, cellulose SPE using D4-DES as IS, followed by C18 chromatography and MS-analysis using SRM. Measuring range was 0.1-10 ng/ml, imprecision <10%, with recoveries within 80-120%. Plasma desmosine levels from COPD patients were significantly higher compared with healthy individuals. The LC-MS/MS assay appears a valuable tool to assess the potential of desmosine as a biomarker for monitoring disease activity in COPD and to study the effect of therapeutic interventions.		Strategy to Identify and Validate Urinary Steroid Biomarkers Obtained by Untargeted LC-MS/MS Metabolomics: Application to Human Cases of Dioxin Exposure
Fabienne Jeanneret
University of Geneva
Long Abstract | Bio | View Video
In vitro metabolic reactions were investigated to improve the identification rate from urinary biomarkers obtained by untargeted metabolomic approaches. A previous study performed with UHPLC-QTOF highlighted a subset of 24 urinary steroid and bile acid biomarkers in human cases of acute dioxin exposure. Biosynthesis of glucuronide and sulfate conjugates of biomarker candidates, respectively produced in human liver microsomes and cytosolic fractions, was demonstrated as a successful approach to identify urinary metabolites when authentic chemical standards are not commercially available. Analysis of the biomarkers subset in an independent human cohort exposed to dioxins strengthened the hypothesis of dysregulated profiles of steroids and bile acids.		Quantification of Human Receptor Tyrosine-protein Kinase ErbB-2 (HER2) by Targeted Mass Spectrometry in Formalin-Fixed Paraffin-Embedded Breast Cancer Tissue
Axel Ducret
F. Hoffmann-La Roche Ltd
Long Abstract | Bio | Financial Disclosure | View Video
We describe in this study a selected reaction monitoring (SRM) assay for the quantification of HER2 in FFPE tissues. The assay’s six best candidate peptides showed a linear response over a calibration range of 0.012 to 100 fmol on column (R2: 0.99–1.00) and a lower limit of quantification of 0.155 fmol on column. HER2 peptides were quantified in a cohort of 40 breast tumors expressing different HER2 levels (FISH ratio: 0-2, 2-4, 4-10 and >10 respectively). The SRM assay showed good analytical performance and a high agreement with IHC and FISH data. Furthermore, after normalization for tissue sample size, SRM peptide measurements were able to correctly predict 90% of HER2 amplification status as defined by American Society of Clinical Oncology and College of American Pathologists.		Skin Imprinting in Silica Plates: A Potential Diagnostic Methodology for Leprosy Using High-Resolution Mass Spectrometry
Estela de Oliveira Lima
Universidade Estadual de Campinas
Long Abstract | Bio | View Video
Leprosy is an infectious disease caused by Mycobacterium leprae, primarily present at skin macrophages and Schwann cells. Presently, the available laboratorial diagnostic methods for Leprosy are invasive, expensive, and present low sensibility for the asymptomatic cases. Therefore, this work intended to develop a noninvasive, fast and sensible method for leprosy diagnosis, associated to high-resolution mass spectrometry. Our data analysis has elected two mycobacterial biomarkers at leprosy skin patients, absent at control samples. These results indicate that our new methodology can be candidate as a fast and sensible leprosy diagnostic method, even for patients without clinical skin manifestations. 		Varying Those Vexing Voltages: Compound Specific Tuning
Daniel Holmes
St. Paul's Hospital, Vancouver
| View Video
After ensuring your instrument’s resolution and mass accuracy are appropriately set, the next step in developing a quantitative LC-MS/MS multiple reaction monitoring (MRM) assay is to perform compound-specific tuning. Well-defined signal-optimization experiments are used to determine appropriate ion source electronics and gas flow parameters to specifically quantify the compound of interest. This overview will introduce basic concepts of so-called "compound optimization". In other words, we will explain what is meant when people say that they have "developed mass spectrometric method."
5:20 PM
5:45 PM
Assaying Protein Unbound Drugs Using a Highly Sensitive LC-MS/MS Method
Heike Bittersohl

Klinikum rechts der Isar
Long Abstract | Bio
Therapeutic monitoring of protein unbound (free) immunosuppressants has the potential to better predict the clinical outcome compared to conventional monitoring of drug levels in whole blood or plasma. Only a small proportion of drugs are free in the patient blood, hence, requiring a sensitive analytical method for their determination. We established an LC-MS/MS method able to simultaneously measure levels of cyclosporine A and mycophenolic acid in the picomolar range. This procedure is currently applied in a study on samples from kidney transplant recipients taking both drugs as a combination therapy.		Not Always CAH. Urine Steroid Profiling in the Investigation and Diagnosis of Adrenal Causes of Neonatal Hyponatraemia and Failure to Thrive
Francis Lam

UCLH NHS Foundation Trust
Long Abstract | Bio | View Video
A baby presented at a local hospital with failure to thrive. Initial biochemistry showed hyponatraemia and hyperkalaemia. Other blood analyses were inconclusive. A spot urine sample was sent for urine steroid profile (USP) analysis. The USP showed a relative abundance of corticosterone metabolites with undetectable tetrahydroaldosterone, a pattern indicative of aldosterone synthase deficiency, subsequently confirmed by genetic testing in this laboratory. Where a steroid disorder is suspected, a USP has great utility since the specimen is easily accessible and can identify/exclude a variety of disorders. Where urgent samples are involved, analyses can be prioritised with relatively rapid turnaround time.		Calculated HbA1c from Total Glycated Hemoglobin by Quantitative MALDI-TOF Mass Spectrometry
Jane Yang
UCSD
Long Abstract | Financial Disclosure
Percent HbA1c, the standard measure of glycated hemoglobin used to diagnose and monitor diabetes mellitus, correlates linearly with total glycated hemoglobin (tGHb). MALDI-TOF mass spectrometry of whole blood hemolysates allows the direct quantitation of the total glycated hemoglobin (tGHb) ratios of alpha and beta chains from a single mass spectrum. Sample preparation for this approach is minimal, analysis is rapid, 80 spots in 20 min (15 sec/spot), and hemoglobin variants are also detected. tGHb by MALDI is reproducible with intra-plate CVs < 1.4%, linear vs. cation exchange HPLC (y = 1.20x – 1.07; R2 = 0.99) from 2.7% to 22% A1c. MALDI method is faster and less expensive than HPLC.		Discrimination and Relative Quantitation of Closely Related Pathogens in Complex Clinical Samples
Olivier Pible
CEA
Long Abstract | Bio
Mass spectrometry is a powerful tool to identify pathogens. However some issues such as mixture handling are usually beyond reach of whole-cell MALDI-TOF approaches. We developed a tandem mass spectrometry approach which not only can address complicated samples such as mixtures of any organisms, but can also give access to relative quantitation of pathogens. It is based on the analysis of the peptide content of the sample and the extraction of phylogenetic information. An universal organism signature has been characterized using this molecular information. The identification problem is then reduced to the search of the linear combination of organism signatures which best matches the overall mass spectrometry signal. 		Building your Assay, Breaking your Compound: Developing MRM Transitions
Stephen Master
Weill Cornell Medical College
| View Video
Now that you understand how to perform compound-specific tuning, you are ready to build your assay. In this session, we will explore how to take a compound that you're interested in measuring by mass spectrometry, break it apart, and pick appropriate product ions for quantitation. This is the final step that allows you to sensitively and specifically measure the abundance of your desired analyte.
5:45 PM
7:45 PM
RECEPTION
@ Exhibit Hall / 1st Floor
Enjoy mingling with colleagues and Exhibitors. Take time to browse the posters. Buffet dinner, appetizers and drinks to be provided. POSTER PRESENTERS: Remove posters between 7:30 - 7:45 PM.
Sponsored by:
Thermo
PLENARY LECTURE SERIES
@ Mozart Hall
Chair: Russell Grant
7:45 PM
8:30 PM
Why Patients Adore Mass Spectrometry
Andy Hoofnagle
University of Washington
Physicians rely on high quality measurements of small molecules and proteins for the diagnosis, prognosis, and management of disease. Health care providers have become reliant on relatively inexpensive, high-throughput immunoassays to capture biomarker data from patient samples. Unfortunately, these assays can be misleading. Clinical mass spectrometric assays bring sensitivity and specificity together in previously unthinkable ways and span the range of medical specialties. This talk will describe the ways in which mass spectrometry can improve the care of our patients.
8:30 PM
Your Decision
ENJOY THE CITY
@ Salzburg Old City
Explore the Old Town of Salzburg.
THURSDAY CLOSED

FRIDAY
8:00 AM
9:00 AM
PLACE POSTERS
@ Exhibit Hall / 1st Floor
Poster presenters for Friday must have their posters placed by 9 AM.
8:00 AM
9:00 AM
WELCOME COFFEE
@ Registration Foyer
Enjoy coffee, a muffin and a chat with colleagues before the day starts.
PLENARY LECTURE SERIES
@ Mozart Hall
Chair: TBA
9:00 AM
9:45 AM
Steroid Metabolomics as a Discovery Tool
Wiebke Arlt
Institute of Metabolism & Systems Research, University of Birmingham, UK
Our group employs steroid metabolomics to reveal the pathogenesis and identify diagnostic and prognostic biomarkers in steroid-producing and steroid-dependent disease. Our approach uses gas chromatography - mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with computational data analysis by machine learning-based approaches. I will present a number of examples including adrenal disorders, hypertension and steroid-dependent cancer to illustrate the power of this approach.
9:45 AM
10:30 AM
Innovative Instrumentation and Methods for the Identification of Intact Proteins in Mixtures and for Sequence Analysis of Antibodies and Posttranslationally-Modified, Intact Proteins on a Chromatographic Time-Scale
Donald Hunt
University of Virginia
This lecture will focus on data generated with a new ion source that facilitates simultaneous generation of positively charged sample ions by electrospray ionization and negatively charged reagent ions for both electron transfer dissociation (ETD) and ion-ion proton transfer (IIPT) reactions on Orbitrap mass spectrometers. Implementation of multiple C-trap fills for enhanced sensitivity will be discussed and both parallel peak parking, and ion ejection strategies to facilitate protein separation and enhanced sequence coverage of intact proteins will be described. Use of IIPT/ETD facilitates near complete sequence coverage on many intact proteins and is ideally suited for locating multiple posttranslational modifications on the same protein molecule. Sequence analysis of antibodies with an enzyme reactor that generates 3-10 KDa fragments in seconds will also be discussed. If time permits, the lecture will also provide an update on the use Class I MHC phosphopeptides for the immunotherapy of cancer.
10:30 AM
11:15 AM
COFFEE BREAK
@ Exhibit Hall / 1st Floor
Visit the Exhibit Hall to procure coffee, juice, water and/or snacks. Explore what's on offer from the Exhibiting vendors, reconnect with colleagues, or go for a short walk outside to refresh for the next session. POSTER PRESENTERS: If you are presenting a poster today your poster should have been up 2 hours ago. If it is not up please put it up immediately.

Sponsored by:
Thermo Shimadzu Agilent
GENERAL SCIENTIFIC SESSION 3
Track 1
Mozart 1-3
Sample Prep
Chair: Christoph Seger		Track 2
Mozart 4-5
Renal Metabolomics
Chair: Rochat Bertand		Track 3
Papageno Hall
Cancer Biomarkers
Chair: Bruno Domon		Track 4
Paracelsus Hall
Imaging
Chair: Theodore Alexandrov		Track 5
Trakl Hall
Case Histories in Basic Method Development
Chair: Judy Stone
11:15 AM
11:40 AM
Ferromagnetic Particles as a Rapid and Robust Sample Preparation for Absolute Quantification of Eicosanoids
Anna Catharina Suhr

Laboratory Medicine, Munich (LMU)
Long Abstract | Bio | View Video
We used ferromagnetic particles as a novel technique to deproteinize plasma samples prior to UHPLC-MS/MS analysis for the quantification of important lipid mediators. A combination of "ferromagnetic particle enhanced deproteination" and subsequent "on-line solid phase extraction" realises quick and convenient sample preparation as well as it provides high sensitivity and robustness. We were able to show that this approach allows accurate and precise quantification of seven exemplary eicosanoids (TXB2, PGE2, PGD2, 5-HETE, 11-HETE, 12-HETE, and arachidonic acid). The use of this semi-automated sample preparation facilitates the screening of large study cohorts. In general ferromagnetic particles might smooth the trail to automation in UHPLC-MS/MS.		Development of Quantitative UPLC-MS/MS Method for Clinical Diagnostic of Rare Kidney Stones and Kidney Failure
Finnur Eiriksson

University of Iceland
Long Abstract | Bio | View Video
Adenine phosphoribosyltransferase (APRT) deficiency results in excessive urinary excretion of poorly soluble 2,8-dihydroxyadenine (DHA) causing kidney disease. Treatment with allopurinol and febuxostat prevents the disease in patients with APRT deficiency. Therapeutic drug monitoring is currently performed by urine microscopy but a more sensitive and reliable method is needed. A UPLC-MS/MS method was developed and utilized to measure the concentration of DHA in urine samples from 28 patients, before and after treatment with allopurinol or febuxostat. Significant changes were observed in urinary excretion of DHA after pharmacotherapy was initiated. A decrease in the DHA-to-creatinine ratio was observed with both allopurinol and febuxostat therapy. The developed UPLC-MS/MS assay will greatly facilitate clinical diagnosis and therapeutic drug monitoring in patients with APRT deficiency.		Determination of EGFR and VEGF Signaling Pathway Activity by Immuno-MALDI Targeting Akt1 and Akt2 in Colorectal Cancer Tumours
Robert Popp

UVic - Genome BC Proteomics Centre
Long Abstract | Bio | View Video
Targeted treatment of colorectal cancer (CRC) only works in a minority of patients, and reliable methods to quantify signaling pathway activity are lacking. We therefore set out to develop immuno-MALDI (iMALDI) assays to determine expression levels and stoichiometry of critical phosphorylation sites in Akt1 (P31749) and Akt2 (P31751) in cancer cells and tumours. We quantified non-phosphorylated Akt1 from 100 µg protein of breast and CRC cells (MDA-231, SW480 and HCT116), and breast cancer tumours. Non-phosphorylated Akt2 has been quantified in 100 µg MDA-231 breast cancer cells. Recently, we improved sensitivity by 10-fold, which allowed decreasing the sample amount from 100 µg to 10 µg. The current lower limit of detection is 0.35 pg Akt1/µg lysate protein. We have developed iMALDI Akt1 and Akt2 assays for quantitation of non-phosphorylated Akt1 and Akt2.		Molecular Annotation and Mapping of Big Data from Spatial Metabolomics
Theodore Alexandrov
European Molecular Biology Laboratory
Long Abstract | Bio | Financial Disclosure | View Video
Spatial metabolomics is emerging as a powerful approach to localize hundreds of metabolites directly from sections of biological samples with the grand challenge to be in the molecular annotation of big data generated. Existing bioinformatics tools cannot be applied directly because of the sheer data size and high complexity of spectra. We developed algorithms for molecular annotation for High Resolution Imaging Mass Spectrometry that integrates both spectral and spatial filters and map the results onto metabolic pathways. We will present our efficient implementation using modern big data technologies and apply it to 3D cell spheroids, microbial agar plates, and biological tissues. We will present the European project METASPACE on Bioinformatics for Spatial Metabolomics.		Brief LC Topic Review - The Basics of Modifying Mobile Phase and Stationary Phase Chemistries to Achieve Resolution
Judy Stone
University of California, San Diego Health System
| View Video
With modern liquid chromatography-tandem mass spectrometry (LC-MSMS)– it is sometimes said that tandem MS alone conveys sufficient selectivity and therefore LC separation is not always necessary. However LC has tremendous capability to improve MSMS quantitation by reducing positive interference from isobaric compounds and negative interference from ion suppression. When you need that additional specificity it is useful to understand how and when stationary and mobile phase chemistries versus gradient, flow rate, column and particle dimensions can be manipulated to improve resolution. We will review the practicalities of optimizing the mobile phase organic component and modifiers and interrogating different stationary phases.
11:40 AM
12:05 PM
Solid Nanostructure Matrices for Small Molecule Detection by MALDI-TOF MS for Clinical Applications
Jo-Il Kim
Yonsei University
Long Abstract | Bio
Two kinds of solid nanostructure matrices, TiO2 nanowires and functional nanowebs, were synthesized to detect small molecules from human serum and dairy milk samples by MALDI-TOF MS. TiO2 nanowires were synthesized by top-down hydrothermal process, and functional nanowebs were synthesized by electrospinning on the metal plate. The feasibility of applying solid nanostructure matrices to MALDI-TOF MS was demonstrated by the detection of short peptides and amino acids. For the real sample analysis, amino acids and antibiotics were spiked into human sera and milk, respectively. The spiked analytes in serum and milk were detected qualitatively and quantitatively using solid nanostructure matrices.		Urinary Metabolic Profiling as Method to Assess Kidney Functionality
Tiziana Pacchiarotta

LUMC (Leiden University Mdical Centre)
Long Abstract | Bio | View Video
Ischemia/reperfusion injury (IRI) is an inevitable event after kidney transplantation, leading to extensive injury and poor graft survival. Current markers for the evaluation of IRI are considered insensitive and reliable markers are highly needed. Here we use a well-established rat model to document the protective role of MBL in renal IRI as target for therapeutic intervention in kidney transplantation and we show that the urinary metabolic pattern can be used to evaluate an in-tissue IRI event. Using a combination of statistical models, we link the metabolic trajectory to the recovery process under different antibody treatment. We demonstrate that “protective” urinary metabolic signature correlates with expression of GRP 78. 		The C-terminal Fragment of Prostate-specific Antigen, a 2331 Da Peptide, as a New Urinary Pathognomonic Biomarker Candidate for Diagnosing Prostate Cancer
Kenji Nakayama
Shimadzu Techno-Research (Kyoto Univ. Hospital)
Long Abstract | Bio | Financial Disclosure
Prostate cancer (PCa) is one of the most common cancers and leading cause of cancer-related deaths in men. Mass screening has been carried out since the 1990s using prostate-specific antigen (PSA) levels in the serum as a PCa biomarker. However, PSA is not a cancer-specific marker. Therefore, our aim was to discover new biomarkers for the diagnosis of PCa. We focused on urine samples voided following prostate massage and conducted a peptidomic analysis of them using MALDI-TOF/MS. Multivariate analysis of the mass spectra of urinary extracts revealed a 2331 Da peptide as a C-terminal PSA fragment. Their quantitative analyses using MALDI-TOF/MS revealed that the peptide may be a new pathognomonic biomarker candidate that can differentiate PCa patients from non-cancer subjects. Those results indicate that the peptide may become a new pathognomonic biomarker for the PCa diagnosis.		Mass Spectrometry Based Molecular Imaging for Probing Aβ-Plaque Pathology in Transgenic Alzheimer’s Disease Mice
Jörg Hanrieder

University of Gothenburg
Long Abstract | View Video
The major pathological hallmarks of Alzheimer’s disease (AD) is the progressive accumulation and aggregation of beta-amyloid (Aβ) and phospho-tau, into neurotoxic deposits. However, the exact molecular processes underlying protein aggregation and plaque pathology remains unknown. The primary goal of this project was to employ advanced molecular imaging mass spectrometry (IMS) to probe the chemical and structural aspects of Aβ plaque pathology in experimental AD. MALDI IMS followed by multivariate image analysis reveal region specific accumulation of differently truncated amyloid peptides in various regions of the brain. Moreover, different protein species were selectively regulated in plaque proximity. In conclusion, MS imaging is a promising approach to probe plaque chemistry in Alzheimer's disease.		Serum Aldosterone - An LC Method Development Case Study for a Difficult Analyte (Part 1)
Grace van der Gugten
Providence Health
| View Video
For the most part, LC-MS/MS assay development and validation is, and should be, a within-laboratory project. However, the new user can find assay development a daunting task. We will describe the LC method development for one of our more challenging to measure endogenous steroids: serum/plasma aldosterone. We will discuss selection of mobile phases, column selection, gradient program development, interference testing, and addition of cortisol to the method. Importantly, we will cover unexpected workflow challenges and continual improvements and discoveries we have made while the assay has been in clinical production.
12:05 PM
12:30 PM
Development of a UPLC-MS/MS Method to Measure Neurotransmitters and Trace Amines in Human Plasma
Antonina Gucciardi

University of Padova, Italy
Long Abstract | Bio
We developed and validated a method for measurement of plasma neurotransmitters (dopamine, epinephrine, norepinephrine, serotonin) and trace amines (tyramine, octopamine, synephrine, β-phenylethylamine and tryptamine) by UPLC-MS/MS. Analytes were extracted using weak cation exchange SPE and separated by a PFP column and water-acetonitrile-methanol gradient as mobile phase, into the ACQUITY UPLC system. The metabolites were measured in MRM mode with positive electrospray ionization using a Waters Xevo TQ-S mass spectrometer. Commercially quality control (QC) materials from Chromsystems were used for validation. The method showed good precision, specificity, sensitivity and linearity, and will be useful for clinical research of neurodegenerative disease.		Nanoelectrospray High Resolution Mass Spectrometry or Liquid Chromatography High Resolution Mass Spectrometry for Urinary Metabolic Profiling?
Elena Chekmeneva

Imperial College London
Long Abstract | Bio | View Video
We present a practical comparison of nESI-HRMS and reversed-phase UPLC-MS methods for multiplexed targeted and global metabolic profiling of the urine specimens from the INTERMAP study. The test set consisted of 132 randomly selected samples which included 22 pairs of blinded replicated and different types of quality control samples. Selected metabolites were quantified using the stable isotope labelled internal standards (IS). Both methods were validated according to the FDA guidelines. The quantification results, sensitivity and dynamic ranges were assessed and compared between two methods. The classification ability was examined on the basis of the multivariate analysis of the global profiles. 		CESI-MS as a Tool for Glycosylation Analysis of PSA and Improved Ionization Efficiency with Acetonitrile-enriched Nebulizer Gas
Guinevere S. M. Kammeijer

Leiden University Medical Center
Long Abstract | Bio | Financial Disclosure | View Video
A powerful analytical platform for the analysis of biomolecules is capillary electrophoresis – electrospray ionization – mass spectrometry (CESI-MS). Implementation of an acetonitrile-enriched nebulizer gas on the CESI-MS system yielded a more robust platform as well as a gain in sensitivity compared to the conventional platform. The gain in sensitivity could aid in the analysis of samples which are only available in minute amounts. The conventional CESI-MS method was used for the analysis of the glycoprotein prostate specific antigen (PSA). We were able to identify 50 different N-glycans on a single N-glycosylation site (N69). The used method shows great potential for studying PSA in prostate cancer research to identify disease related alterations of glycosylation in an early stage and therefore could be a promising tool for diagnostic and prognostic evaluation.		Diagnostic and Prognostic Biomarker Discovery of Soft Tissue Sarcomas by Mass Spectrometry Imaging
Sha Lou

Leiden University Medical Center
Long Abstract | Bio | View Video
Ability of Matrix-assisted laser desorption/ionization Mass Spectrometry Imaging to distinguish between the most encountered but clinically challenging high grade Soft Tissue Sarcomas (four subtypes) were investigated (leading to diagnostic biomarkers discovery) and if there are individual proteins (signatures) that are statistically associated with patient survival and development of metastases were also investigated (thus would be prognostic biomarkers). Twenty protein peaks were found as diagnostic biomarkers. Fourteen protein peaks were found as prognostic biomarkers. Based on comparisons with databases, Acyl-CoA-binding protein, Macrophage migration inhibitory factor, Thioredoxin and Galectin-1 were tentatively assigned among diagnostic biomarkers; Thymosin beta-10, Proteasome activator complex subunit 1, two modified Histone H4 were tentatively assigned among prognostic biomarkers.		Serum Aldosterone - An LC Method Development Case Study for a Difficult Analyte (Part 2)
Grace van der Gugten
Providence Health
For the most part, LC-MS/MS assay development and validation is, and should be, a within-laboratory project. However, the new user can find assay development a daunting task. We will describe the LC method development for one of our more challenging to measure endogenous steroids: serum/plasma aldosterone. We will discuss selection of mobile phases, column selection, gradient program development, interference testing, and addition of cortisol to the method. Importantly, we will cover unexpected workflow challenges and continual improvements and discoveries we have made while the assay has been in clinical production.
12:30 PM
2:30 PM
LUNCH
@ Exhibit Hall / 1st Floor
Lunch to be provided in the Exhibit Hall. • Get ready to join a Corporate Workshop.

Sponsored by:
Thermo Shimadzu Agilent
1:30 PM
2:30 PM
POSTERS
@ Exhibit Hall / 1st Floor
All Posters to be attended from 1:30 - 2:30 PM.
2:30 PM
3:30 PM

CORPORATE WORKSHOPS (2:30 - 3:30 PM)
Waters
 Mozart 1-3

UPLC-MS/MS-Based Studies of Vitamin D Metabolism: Lessons Learned from CYP24A1 Knockout Mice and Men
Martin Kaufmann, Queen’s University, Kingston Canada
Pre-Register

Vitamin D function in the body is partly regulated by catabolism of 1,25-(OH)2D and 25-OH-D by CYP24A1. We have developed a sensitive assay using ACQUITY/TQ-S instruments to quantitate metabolites formed by CYP24A1 including 24,25-(OH)2D3, in addition to 25-OH-D. The talk will focus on the application of our assay to a unique combination of samples from CYP24A1-null mice and patients with inactivating mutations to CYP24A1 (Idiopathic Infantile Hypercalcemia) as well as subjects taking vitamin D supplements. We reveal that the ratio of 25-OH-D3:24,25-(OH)2D3 is a useful tool to predict the presence of CYP24A1 mutations as well vitamin D deficiency.

SCIEX
 Mozart 4-5

Mass Spectrometry Applications for Clinical Research
Dr Robert Graham and Dr Michael Wright
Pre-Register

In this workshop we bring together scientists, clinicians and biochemists that hold an interest in the use of Mass Spectrometry in the areas of Clinical Research as we discuss what can achieved with today’s technology and what we can expect in the future. We have two speakers delivering this workshop. Dr Robert Graham is Senior Lecturer in Clinical Proteomics at the University of Manchester, UK. Dr Graham will discuss the use of advanced accurate mass MS technologies in ovarian cancer biomarker research We also have Dr Michael Wright, from the Prince of Wales Hospital, Sydney, Australia Dr Wright is Senior Leader of clinical Chemistry and Endocrinology. In the workshop he will discuss novel and advanced MS technologies specifically with respect to their use in reducing background and matrix effects in sample analysis.

Phenomenex
 Papageno Hall

Workshops on Navigating Sample Prep Techniques & Leveraging the Power of Microsampling
1. Navigating Sample Preparation Techniques in the Clinical LCMS Laboratory
Sean Orlowicz Manager- PhenoLogix
Have you ever asked yourself, "Which Sample Prep Technique is right for this analysis?" If so, you are not alone. In this talk we will investigate, through case studies, some differences between common sample preparation techniques.

2. Leverage the Power of Microsampling to Benefit Both Your Patient and Your Laboratory
James Rudge Ph.D. -Senior Business Development Manager – Europe
Hospital, clinical, and wellness/biomarker screening labs are using Mitra Microsampling to provide a better, more convenient experience to their patients, while at the same time reducing costs and streamlining workflows. In this workshop, we will discuss the details of an at-home sampling workflow, from collection through analysis, including the optimization and automation of extractions for clinical samples

Tecan
 Paracelsus Hall

Workshops on Method Automation
1. Automation and Validation of a SPE Method for the Analysis of 23 Opioids, Cocaine, and Metabolites in Urine with UHPLC-MS/MS
Maria del Mar Ramirez Fernandez, National Institute of Criminology and Criminalistics, Belgium

2. Automated LC-MS/MS methods for Clinical Diagnostics - Realized for Therapeutic Drug Monitoring and Vitamins
Dr. Silvia Bächer, R&D, RECIPE Chemicals + Instruments GmbH

3. Automated SPE Method Development using StrataTM – X 96-Well SPE Method Development Plates in Conjunction with a Tecan Freedom EVO® Liquid Handling Platform Sean Orlowicz, Manager- Phenomenex

Customer experience and commercial insights will be presented for applying automated MS sample preparation in areas of clinical and forensic sample testing, such as therapeutic drug monitoring, DOA- and Vitamin testing. We have pulled together speakers that will present most relevant automated methods and provide guidance on successfully implementing lab established and kit based methodologies.

3:30 PM
4:00 PM
COFFEE BREAK
@ Registration Foyer & Front Patio
Visit the Registration Foyer to procure coffee, juice, water and/or snacks. Reconnect with colleagues, or go for a short walk outside to refresh for the next session.

Sponsored by:
Thermo Shimadzu Agilent
GENERAL SCIENTIFIC SESSION 4
Track 1
Mozart 1-3
Endocrine
Chair: Brian Keevil		Track 2
Mozart 4-5
Advances in Metabolomics
Chair: Oleg Mayboroda 		Track 3
Papageno Hall
Proteomics
Chair: Donald Hunt		Track 4
Paracelsus Hall
Metabolomics
Chair: Dietrich Matern		Track 5
Trakl Hall
Intro to Sample Prep II
Chair: Grace van der Gugten
4:00 PM
4:25 PM
The Importance of Accurate and Sensitive Oestradiol Measurement & Strategies to Achieve this Difficult Task
Laura Owen
Univeristy Hospital of South Manchester
Long Abstract | Bio
Oestradiol measurement is traditionally performed by immunoassays which have poor performance at lower concentrations. It is important to accurately measure oestradiol at low concentrations in certain patient groups e.g. females with breast cancer. Mass spectrometry is an attractive alternative due to its better specificity and performance at the low concentrations. We have investigated different mechanisms for improving the sensitivity of our current oestradiol assay. Further investigation into the interference of breast cancer medications has shown that one in particular, fulvestrant causes direct interference in two immunoassays. The other medications (anastrozole, exemestane and tamoxifen) do not appear to directly interfere. 		Metabolic Phenotyping Reveals a Lipid Mediator Response to Ionizing Radiation
Giuseppe Astarita
Georgetown University
Long Abstract | Financial Disclosure | View Video
Exposure to ionizing radiation has dramatically increased in modern society, raising serious health concerns. The molecular response to ionizing radiation, however, is still not completely understood. Here we screened mouse serum for metabolic alterations following an acute exposure to gamma radiation using a multi-platform, mass-spectrometry-based strategy. A global, molecular profiling revealed that mouse serum undergoes a series of significant molecular alterations following radiation exposure. We identified and quantified bioactive metabolites belonging to key biochemical pathways and eicosanoids, which could be utilized as an indicator of radiation exposure and as novel target for therapeutic intervention. Monitoring such a molecular response to radiation exposure might have implications not only for radiation pathology but also for countermeasures and personalized medicine.		Novel Insights into Apolipoprotein Metabolism in Mice and Man by Application of a Targeted Peptide-based LC-MS/MS Assay
Uta Ceglarek
University Hospital Leipzig
Long Abstract | Bio | Financial Disclosure | View Video
Apoproteins are key components of lipoproteins and mediate diverse functions in lipid transport and metabolism. A LC-MS/MS methods to analyze apoproteins using species-unique peptide sequences for Apo A-I, Apo A-II, Apo A-IV, Apo B48, Apo B100, Apo C-I, Apo E and Apo J in 3 µl human or murine plasma. In human plasma Apo A-I and Apo A-II showed the highest concentrations. The simultaneous analysis of apoproteins in only 3 µl murine and human plasma allows us a comprehensive characterization of apolipoprotein levels using normo- and hyperlipidemic patients and different mice strains under different nutritional conditions.		Why High-resolution-MS Is Becoming the New Gold Standard MS Analyzer in Clinical Labs
Bertrand Rochat
CHUV - qMSF
Long Abstract | Bio | View Video
As we see more and more demonstrations of the capability of high-resolution (HR)-MS instruments to perform sensitive and reliable quantification of a large variety of analytes in HR-full scan mode, we can consider that HRMS is the new gold standard MS analyzer for LCMS analyses (quantitative and qualitative). Examples showing the versatility and the performances of HRMS in HR-full scan will be presented: 1) routine quantification of drugs and endogenous metabolites in plasma extracts, 2) Qual/Quan analysis in a study of the fate of tamoxifen drug in humans and 3) targeted and untargeted metabolomics. 		Brief Sample Preparation Topic Review - Extraction Options for Neutral, Non-Polar Analytes in Serum - Say Good-By to Phospholipids
Judy Stone
University of California, San Diego Health System
| View Video
Extraction protocols that concentrate neutral, non-polar serum analytes to pg/mL lower limits of quantitation and remove enough matrix to produce a robust method are challenging to develop. Serum phospholipids are present at mg/mL concentrations and contain a charged phosphate group and non-polar fatty acids. This amphipathic structure complicates removal of phospholipids during extraction. Insufficient removal of phospholipids may cause substantial LC-MSMS ion suppression and compromised method robustness -- shortened column lifetimes, shifting retention times, high baselines, and decreased MSMS sensitivity. We will review commonly used LC-MSMS extraction protocols for the ability to concentrate non-ionizable analytes while optimally removing serum phospholipids.
4:25 PM
4:50 PM
Eve’s Curse: How Best to Measure Estradiol Across a Broad Spectrum of Reproductive Endocrine Disorders in Women
Michael Wright
Prince of Wales Hospital
Long Abstract | Bio | View Video
Estradiol is arguably the most potent sex steroid in human biology requiring relatively small concentrations to exert large physiological effects in comparison to its precursor, testosterone. However, the delivery of a clinical diagnostic estradiol service is a significant challenge facing many laboratories. In this study we look at a number of sample extraction options, chromatographic techniques and multistage fragmentation (MRM3) using quadrupole linear ion trap instruments to create robust LC-MS methods that meet the requirements of low level measurement. In this presentation we propose three different methods to meet the needs of our diverse patient population.		Targeting Resistance to Proteasome Inhibitor Therapy: A Metabolomics Approach
Celia Berkers

Utrecht University
Long Abstract | Bio
Proteasome inhibition has emerged as an important strategy for the treatment of cancer. However, treatment with proteasome inhibitors is often hampered by the occurrence of both primary and acquired resistance. We embarked on unravelling the metabolic mode of resistance to the proteasome inhibitor bortezomib. To this end, we profiled bortezomib-sensitive and -resistant cell lines, combining steady-state metabolomics screens with metabolic flux studies using stable isotope labelling approaches. Our studies revealed that the metabolic profiles of bortezomib-sensitive and resistant cells differed significantly. In particular, resistant cells were more dependent on the uptake of specific nutrients from their environment. Together, our data indicate a potential role for nutrient starvation in the treatment of bortezomib-resistant tumours. 		Top-down Mass Spectrometry Methods for Hemoglobin Disorders Diagnosis
Didia Coelho Graça

University of Geneva
Long Abstract | Bio
Low and high-resolution top-down mass spectrometry (MS) methods were developed for hemoglobin disorders characterization. An automated workflow using an ion-trap with ETD capabilities allowed to identify the most clinically significant hemoglobin variants and to quantify hemoglobin chains. Analytical performances achieved with this method were compared to gold standard assays used in clinical labs. A high-resolution method was also developed for hemoglobin variants identification. Selected diagnostic product ions were used for fast and reliable data interpretation by non-expert users. MS brings more precise information at the protein level than protein analysis methods currently used for hemoglobin disorders diagnosis.		Identification and Characterisation of Oxygenated Porphobilinogen Derivatives and their Amino Acid/peptide Conjugates Using Liquid Chromatography-accurate Mass
Christopher Benton
Agilent Technologies
Long Abstract | Financial Disclosure
Studying the modification of porphobilinogen is important when trying to understand the pathogenesis of the peripheral neuropathy, chronic renal failure and hepatocellular carcinoma observed in the AHP. Using LC/HRMS we demonstrate how PBG can form various oxygenated species, derived from reactive epoxide, hydroperoxide and endoperoxide intermediates. Furthermore, these intermediates formed adducts with amino acids and peptides, highlighting their reactivity. The mechanisms of formation of these conjugates are postulated to be via epoxyporphobilinogen and PBG endoperoxide intermediates. If formed in vivo, such compounds could covalently bind to cellular macromolecules, such as proteins and nucleic acids, altering their biological function. 		A Sample Preparation Method Development Case Study - Trials and Tribulations with Serum Testosterone (Part 1)
Grace van der Gugten
Providence Health
| View Video
Use of liquid chromatography-mass spectrometry for analysis of small molecules in clinical laboratories has increased in the past few years. An important component of developing a mass spectrometry method is the sample preparation technique employed in order to sufficiently clean up the sample without sacrificing analyte recovery. A number of references out there detail sample preparation techniques but which one do you choose? This presentation will detail the thought process behind the sample preparation technique choice for measurement of serum total testosterone in one laboratory, including the balancing act of what you want to, and what you can realistically achieve.
(Courtesy of Deborah French)
4:50 PM
5:15 PM
Clinical Utility of Reporting 3-Methoxytyramine as Part of a Plasma Metanephrines Profile
Sarah Pitkin

UCL Hospitals NHS Foundation Trust
Long Abstract | Bio | View Video
The utility of reporting the dopamine metabolite 3-methoxytyramine (3-MT) in plasma free metanephrines profiles has been questioned due to analytical and clinical concerns. We developed and validated an LC-MS/MS assay for plasma free metanephrines, including 3-MT, and reviewed the analytical and clinical utility of reporting 3-MT over a 1 year period. Performance of the assay in an external quality assurance scheme was excellent. Clinically, 3-MT was found to be a valuable addition to the profile, providing a useful tumour marker for long term monitoring in one patient. Cabergoline therapy was found to decrease plasma 3-MT, whereas levodopa raised it.		Combining Dried Blood Spots with Stable-isotope Tracers to Profile Dynamics of Glucose Metabolism in Human Subjects
Jean-Pierre Trezzi
University of Luxembourg, LCSB
Long Abstract | Bio | View Video

This talk will be presented by Jean-Pierre Trezzi, in place of Karsten Hiller

Here, we present time-resolved dynamics of glucose metabolism in blood after oral administration of 13C stable-isotope labeled glucose in human subjects. To extract absolute flux values for glucose production (GP), glucose disappearance (GD) and gluconeogenesis (GNG)on a whole organism scale in diabetes patients, we developed a simple mathematical ODE based dynamic model that considers the main fluxes of (13C labeled) glucose into and out of the blood. In combination with the time-resolved enrichment patterns (Mass Isotopomer Distributions / MIDS) and absolute concentrations of the target metabolites obtained from DBS sampling and GC/MS measurement, we could determine quantitative and robust values for GP and GNG for each studied subject. 		Linkage-specific Sialic Acid Derivatization for MALDI-TOF-MS Profiling of Glycoconjugates
Noortje de Haan

Leiden University Medical Center
Long Abstract | Bio | View Video
Glycosylation is among the most common co- and post-translational protein modifications, affecting the physiological and biochemical properties of a protein in various ways. High-throughput protein glycosylation analysis may be performed by MALDI-TOF-MS of either released glycans or glycopeptides. This approach, however, biases the analysis of sialylated glycoconjugates by metastable decay and variations in ionization. Here, we present approaches for the linkage-specific sialic acid derivatization of both released glycans and glycopeptides followed by MALDI-TOF-MS analysis. The methods are fast and have an excellent intra- and inter-day repeatability. We envision the use of these methods for the monitoring of protein glycosylation, both in the analysis of biopharmaceuticals and for the detection of glycomic disease biomarkers.		Enzyme Assays for the Diagnosis of Inborn Errors of Galactose Metabolism Using Liquid Chromatography Tandem Mass Spectrometry
Dietrich Matern
Biochemical Genetics Laboratory, Mayo Clinic
Long Abstract | Bio | View Video
Galactosemias are autosomal recessive disorders that results from a deficiency of one of three enzymes catalyzing the conversion of galactose to glucose: galactose-1-phosphate uridyltransferase (GALT), galactokinase (GALK), and uridine diphosphate galactose-4-epimerase (GALE). We implemented LC-MS/MS assays for each enzyme that can be performed from the same specimen. 843 controls and samples from known patients (GALT: n=161; GALK: n=1; GALE: n=6) and asymptomatic mutation carriers (GALT: n=92) were analyzed to determine reference ranges. These tests provide a comprehensive and cost-effective laboratory evaluation to quickly resolve abnormal NBS results for galactosemia or a diagnosis of other at-risk patients.		A Sample Preparation Method Development Case Study - Trials and Tribulations with Serum Testosterone (Part 2)
Grace van der Gugten
Providence Health
Use of liquid chromatography-mass spectrometry for analysis of small molecules in clinical laboratories has increased in the past few years. An important component of developing a mass spectrometry method is the sample preparation technique employed in order to sufficiently clean up the sample without sacrificing analyte recovery. A number of references out there detail sample preparation techniques but which one do you choose? This presentation will detail the thought process behind the sample preparation technique choice for measurement of serum total testosterone in one laboratory, including the balancing act of what you want to, and what you can realistically achieve.
(Courtesy of Deborah French)
5:15 PM
7:15 PM
CLOSING RECEPTION
@ Registration Foyer & Front Patio
Enjoy some last bits of time mingling with colleagues. Located in Registration Foyer and Front Patio, weather permitting. Appetizers and drinks to be provided.
Sponsored by:
Thermo
FRIDAY CLOSED