Andrea Geistanger is Head of Systems Data Analytics, at Roche Diagnostics in Germany. Her department of biostatisticians supports system and assay development through the whole life cycle of Roche’s cobas products. Her team is involved in the early development phases, including biomarker search projects with machine learning and multivariate statistics analysis. During product development phases, Andrea’s data analysts support scientists in experimental planning with Design of experiments, as well as in the experiment of validation studies according to regulatory requirements. Furthermore, they develop standardization schemes and calibration concepts for cobas analyzers. Throughout the development phase, software tools are designed and developed as needed. These programs are also made available to a broader community through open software projects.

Trueness and precision are the key quality attributes of a diagnostic assay and have to be proven in validation experiments throughout each assay development. CLSI does also acknowledge the importance of these criteria, having two guidelines in place, EP9 for method comparison, and EP5 for precision studies describing the design and the analysis of the corresponding experiments. The statistical methodology for both experiments is quite advanced and cannot be operated in a bread and butter software such as Excel. For method comparison studies a Deming regression is required and in some cases also a robust Passing-Bablok regression is state-of-the-art. Classical linear regression methods are not appropriate here, as measurement errors occur for both measurement methods. For precision studies, an appropriate variance-components design should be used and statistically analyzed accordingly.

The mcr R-package is a free available open source R package, which incorporates all analysis methods for method comparison studies, with special focus on the regression methods as Deming or Passing-Bablok regression.

The VCA package is the pendant for precision experiments, where different measurement designs can be analyzed. It is also freely available as open source R-package. Both R packages have been developed and are maintained by the Roche Diagnostics R&D biostatistics department.
The talk will cover the major aspects of the analysis requirements for method comparison and variance-components studies. In addition, we show the features of both R packages, their calculation capabilities as well as the graphical representation possibilities.

Dr. Robert B. (Chip) Cody received his Ph.D. from Purdue University in 1982 under the direction of Prof. Ben S, Freiser. After graduate school, he worked at Nicolet Instruments developing methods for Fourier Transform Mass Spectrometry until 1989 when he joined JEOL USA, Inc. where he is presently Product Manager for Mass Spectrometry. Among other achievements, Dr. Cody is responsible for developing the trapped-ion tandem-in-time MS/MS and MSn techniques, laser-desorption FTICR, and is coinventor of the DART ion source. He served as Vice-President for Arrangements for the American Society of Mass Spectrometry and was awarded the 2011 Anachem Award and a 2012 Purdue University Distinguished Alumni Award. He has over 100 publications and several patents, edited (with Marek Domin of Boston College) the book Ambient Ionization Mass Spectrometry, and is author of the Mass Mountaineer software suite.

It has now been 17 years since a patent was filed describing the Direct Analysis in Real Time (DART) ion source, yet no clinical applications of DART MS are currently in use. This is not to say that DART has no potential for clinical applications! As an ambient ionization method, DART has several attractive characteristics for clinical chemistry. DART analysis is rapid and robust, and can be applied to a wide range of analytes. In combination with a high-resolution and/or tandem mass spectrometer, DART can be quite sensitive and selective. Point-of-care applications are possible if DART is combined with a compact mass spectrometer.
Several promising DART applications have been reported. Because it produces a broad profile of small-molecule biomarkers, DART is well matched with chemometric analysis for speciation and classification. Two published feasibility studies have shown the potential for microbial identification using DART MS. The first (from CDC and GA Tech) used in-situ methylation and DART to identify bacterial fatty acid profiles. The second study found that free fatty acids from a simple extraction method could identify ten different pathogens. Another study from the Fernandez lab at GA Tech showed a DART method for ovarian cancer screening with statistics that showed 100% accuracy!
Clinical toxicology is another area of potential application. DART is well established for forensic drug screening. That same capability could be used to screen for drugs and toxins to guide treatment in victims of poisoning or overdose. With relatively simple sample handling methods, detection limits for drugs in body fluids are suitable for rapid screening. DART has demonstrated the potential for monitoring drug excretion kinetics and in at least one case, detection of biomarkers for disease conditions. In a recent study, we have found that DART can be combined with another ambient ionization method (Coated Blade Spray) to provide complementary data from minimal sample volumes.
So, why has DART not yet found a place in clinical chemistry? Commercially available laboratory systems have been on the market for 15 years, and portable systems are also now commercially available. Perhaps the answer is just a need for early adopters who are willing to carry out clinical validation studies, much as the VA DFS did for forensic drug screening.

Dr. Rosano is board certified in forensic toxicology by the American Board of Forensic Toxicologists and in clinical chemistry by the American Board of Clinical Chemistry. For over 35 years Dr. Rosano has been director of Clinical Chemistry and Toxicology at the Albany Medical Center Hospital where in 1993 he expanded toxicology services to include postmortem toxicology services for a 22 county region. During his career at the Albany Medical Center Dr. Rosano has progressed to tenured professor of Laboratory Medicine at the Albany Medical College where he has lectured and mentored medical and graduate students along with residents and fellows. In 2018 Dr. Rosano transitioned to toxicologist and director of the National Toxicology Center at the Center for Medical Science in Albany, New York and was promoted to Professor Emeritus in Laboratory Medicine at the Albany Medical College. His current focus is on court-ordered and addiction medicine casework and the advancement of clinical and forensic toxicology through innovations in high-volume definitive screening and applications of high-resolution mass spectroscopy.

Advancing analytical technology serves as the foundation of our toxicology practice and the explosion in pharmaceutical and illicit drug use now mandates the application of definitive testing technology in both our screening and confirmatory test protocols. While nominal mass GC-MS traditionally served as the analytical technology for confirmatory drug testing, the transition to liquid chromatography coupled with tandem mass spectroscopy has largely occurred and has brought with it an emerging application of high resolution mass spectroscopy. As definitive methods further the molecular identification and certainty of drug and metabolite confirmation work, our screening protocols in many areas of clinical toxicology still rely on presumptive methods with their high false negative rates and lack of selectivity. Conversion to definitive methods of screening with expanded drug panels is clearly needed but the challenges of high-volume screening with mass spectrometry has slowed the conversion to definitive screening across many areas of clinical toxicology. The hurdles on the way to definitive screening include automated sample preparation, rapid chromatography separation, analyte-specific matrix normalization, data management, alternative confirmatory methodology and interpretive reporting of findings. The presentation will focus on one laboratory’s journey and experience with definitive screening and confirmation protocols using a novel calibration technique for matrix normalization and application of high resolution mass spectroscopy for confirmation testing. Findings in addiction medicine and pain management casework will be presented and compared with the authors experience in court-ordered and postmortem casework.

John R. Yates is the Ernest W. Hahn Professor in the Departments of Molecular Medicine and Neurobiology at The Scripps Research Institute. His research interests include development of integrated methods for tandem mass spectrometry analysis of protein mixtures, bioinformatics using mass spectrometry data, and biological studies involving proteomics. He is the lead inventor of the SEQUEST software for correlating tandem mass spectrometry data to sequences in the database and developer of the shotgun proteomics technique for the analysis of protein mixtures. His laboratory has developed the use of proteomic techniques to analyze protein complexes, posttranslational modifications, organelles and quantitative analysis of protein expression for the discovery of new biology. Many proteomic approaches developed by Yates have become a national and international resource to many investigators in the scientific community. He has received the American Society for Mass Spectrometry research award, the Pehr Edman Award in Protein Chemistry, the American Society for Mass Spectrometry Biemann Medal, the HUPO Distinguished Achievement Award in Proteomics, Herbert Sober Award from the ASBMB, and the Christian Anfinsen Award from The Protein Society, the 2015 ACS’s Analytical Chemistry award, 2015 The Ralph N. Adams Award in Bioanalytical Chemistry, the 2018 Thomson Medal from the International Mass Spectrometry Society, and the 2019 John B. Fenn Distinguished Contribution to Mass Spectrometry award from the ASMS. He was ranked by Citation Impact, Science Watch as one of the Top 100 Chemists for the decade, 2000-2010. He was #1 on a List of Most Influential in Analytical Chemistry compiled by The Analytical Scientist 10/30/2013 and is on the List Of Most Highly Influential Biomedical Researchers, 1996-2011, European J. Clinical Investigation 2013, 43, 1339-1365 and the Thomson Reuters 2015 List of Highly Cited Scientists. He has published over 950 scientific articles with >125,000 citations, and an H index of 174 (Google Scholar). Dr. Yates served as an Associate Editor at Analytical Chemistry for 15 years and is currently the Editor in Chief at the Journal of Proteome Research.

Developments in mass spectrometers over the last decade have been numerous, but there are some clear trends. The drive to increase confidence in the identification of peptides and post-translational modifications pushed the development of high-resolution and high-mass accuracy instruments, most notably Orbitrap and time-of-flight (TOF) mass analyzers. Improvements in mass resolution in these instruments resulted in an increase in the mass range for effective analysis, precipitating greater interest in the “top down” proteomics which now could be performed without expensive high-field magnets previously required for ion cyclotron resonance MS of intact proteins. Additionally, the emergence of biological therapeutics has fueled a greater need to characterize intact proteins to verify structure, sequence, and modifications. Fragmentation of the amide bonds in intact proteins requires more robust methods than fragmentation of peptides to obtain sequence information. Two methods in particular—electron transfer dissociation (ETD) and ultraviolet photodissociation (UVPD)—have been used to achieve more efficient fragmentation of intact proteins, especially when used in combination. These substantial improvements in MS capability have led to greatly improved prospects for top-down MS.

I am an analytical chemist focused on infection metallomics and clinical mass spectrometry (MS). From 1991 I have had the main contract with the Institute of Microbiology in Prague, where I run an analytical lab with 25 employees now benefitting from mass spectrometry, nuclear magnetic resonance spectroscopy, and electron microscopy (https://ms.biomed.cas.cz/). As a professor of analytical chemistry, I am also active at Palacky University in Olomouc, CZ, providing me with access to elemental imaging mass spectrometry and infection models. I have a scientific profile with 3582 citations (access date May 21, 2020), 1517 citations since 2015. My current h-index is 30 (Google Scholar). In 2020 I contributed to 11 peer-reviewed papers, nine of them I corresponded.

Background: The detection and quantitation of siderophores in urine represents an innovative early indication of pathogenic microorganisms in a host [1]. In this presentation, we document why the general application of siderophores in invasive infectious diagnostics will soon represent the same clinical breakthrough as ribosomal protein typing ten years ago. In addition to handling co-infections or polymicrobial frenemies, with mass spectrometry, we can monitor the antimicrobial drug metabolism [2].

Methods: A database of 700 microbial siderophores was used for screening a patient’s status with invasive infections caused by Aspergillus fumigatus, Pseudomonas aeruginosa, and zygomycetes. Human urine samples were examined by liquid chromatography and Fourier-transform ion cyclotron resonance (FTICR) mass spectrometer [3]. The biomarkers were quantified against matrix-matched standards, and our in-house tool called CycloBranch (https://ms.biomed.cas.cz/cyclobranch) was used for compound dereplication [4].

Results: For the diagnosis of aspergillosis, we used an array of ferri-triacetylfusarinine C (TAFC)/ferri-ferricrocine (FC)/gliotoxin (GTX) in a cohort of 28 patients (14 positive and 14 negative controls). LOD for TAFC in human urine was 0.1 ng/mL. The maximum experienced concentration was 1.7 ug/mL. In the working dataset of critically ill, mostly non-neutropenic humans, the TAFC/FC/GTX sensitivity in urine was 93% compared to standard GM in serum (36%). The kinetic profiles upon antimycotic treatment showed a quick drop in siderophore production in vivo. In the same cohort, ferri-pyoverdines and pyochelin were detected as urinal biomarkers in the patients with Pseudomonas aeruginosa. The LOD for Fe-PVD in urine was 10 ng/mL. In serum samples, the LOD and LOQs were 1 and 5 ng/mL, respectively. In the seminar I will also show the co-infecting frenemies studied in animal models of bacterial [5] or fungal infections [6].

Conclusion: Urinal samples are analytically attractive due to less chemical background compared to serum and clinically interesting due to non-invasive applications. Optimized sample preparation could soon move siderophore analysis from FTICR equipment to standard MALDI-Biotyper-type labs.

References:

1. Luptáková, D.; Pluhá?ek, T.; Pet?ík, M.; Novák, J.; Palyzová, A.; Sokolová, L.; Škríba, A.; Šedivá, B.; Lemr, K. and Havlí?ek, V. Non-invasive and invasive diagnoses of aspergillosis in a rat model by mass spectrometry. Sci Rep 2017, 7, 16523.

2. Houst, J.; Spizek, J. and Havlicek, V. Antifungal Drugs. Metabolites 2020, 10, article No. 106.

3. Pluhacek, T.; Skriba, A.; Novak, J.; Luptakova, D. and Havlicek, V. Analysis of Microbial Siderophores by Mass Spectrometry. In: SK Bhattacharya (ed) Metabolomics: Methods and Protocols 2019: 131-153.

4. Novák, J.; Škríba, A. and Havlí?ek, V. CycloBranch 2: Molecular Formula Annotations Applied to imzML Data Sets in Bimodal Fusion and LC-MS Data Files. Anal Chem 2020, 92, 6844-6849.

5. Petrik, M.; Umlaufova, E.; Raclavsky, V.; Palyzova, A.; Havlicek, V.; Haas, H.; Novy, Z.; Dolezal, D.; Hajduch, M. and Decristoforo, C. Imaging of Pseudomonas aeruginosa infection with Ga-68 labelled pyoverdine for positron emission tomography. Sci Rep 2018, 8, 15698.

6. Skriba, A.; Pluhacek, T.; Palyzova, A.; Novy, Z.; Lemr, K.; Hajduch, M.; Petrik, M. and Havlicek, V. Early and Non-invasive Diagnosis of Aspergillosis Revealed by Infection Kinetics Monitored in a Rat Model. Front Microbiol 2018, 9, 2356.

Michael H. Gelb is Professor of Chemistry and Barbara L. Weinstein Endowed Chair in Chemistry, Adjunct Professor of Biochemistry at the University of Washington. Major developments in the Gelb lab include discovery of protein prenylation, development of ICAT proteomic reagents, identification of phospholipases involved in lipid mediator generation, development of anti-parasite drugs, and development of mass spectrometry for newborn screening. Awards include: Repligen Award in Chemistry of Biological Processes (Amer. Chem. Soc.), Univ.of Washington Faculty Lecture Award, Gustavus John Esselen Award (Harvard Univ.), AAAS Fellow, NIH Merit Award, Medicines for Malaria Project of the Year Award, Pfizer Award in Enzyme Chemistry, ICI Pharmaceuticals Award for Excellence in Chemistry. The Gelb lab has published more than 500 papers and 100 patents in biological chemistry. The Gelb laboratory has developed mass spectrometry for worldwide newborn screening of lysosomal storage diseases (the latest expansion of newborn screening panels).

Mass spectrometry has a strong presence in newborn screening laboratories because of the ability to quantify numerous metabolites in dried blood spots on newborn screening cards. Over the past decade mass spectrometry has been developed to measure enzymatic activities in dried blood spots. We will present an 18-plex mass spectrometry assay that provides enzymatic activities and biomarkers for lysosomal diseases and allows consolidation with existing mass spectrometry newborn screening panels. In the second part of the presentation we will show the feasibility of using mass spectrometry for proteomics-based newborn screening. This allows screening for a panel of treatable diseaes for which no other methods exists.
As more and more inborn errors become treatable, in part due to gene therapy, it will be important to develop a universal screening platform that allows high throughput, consolidated newborn screening. Despite great progress in DNA sequencing technology, this will not replace biochemical newborn screening in the foreseable future.

INTRODUCTION: Cystic Fibrosis is a disease caused by mutations in the cystic fibrosis ion transport regulator gene (CFTR). The most common mutation, deletion of Phe at position 508 (DF508), results in a loss of function phenotype that causes a large number of clinical symptoms including chronic respiratory infections. At the protein level, the DF508 mutation causes the protein to misfold which results in rapid degradation of the protein in the ER.

OBJECTIVES: The objective of this study is to understand the mechanisms involved in regulating protein misfolding to learn how to rescue the misfolded protein. A detailed understanding of the mechanism should lead to the identification of drug targets for treatment of the disease.

METHODS: Mass spectrometry-based proteomics was used to identify the proteins interacting with wild type CFTR and DF508 CFTR to identify those proteins interacting with the mutant protein, but not the wild type. Proteins interacting uniquely with the mutant were knocked down to identify those proteins whose “inhibition” could potentially rescue the misfolded protein. In addition, regulation of the trafficking process by post translational modifications was also determined using mass spectrometry.

RESULTS: A much better understanding of the mechanisms behind rescue of the mutant protein has been derived from these studies. A disease specific interactome was identified. Proteins interacting with DF508 whose knockdown rescue the mutant protein have been identified. A PTM code has been identified that must be present for the protein to exit the ER. In addition, structural details of the misfolded protein have been determined using a surface accessibility method.

CONCLUSION: By understanding the mechanism behind the loss of function phenotype of DF508 CFTR we now have better routes to rescue the protein and treat CF disease. This knowledge can be used to understand other mutations in the gene that also result non-trafficking of the protein.