Mass Spectrometry for Lipidomics E-Book

• Herausgeber: Wiley-VCH GmbH
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch

Seitenzahl: 1403

Veröffentlichungsjahr: 2023

Beschreibung

Mass Spectrometry for Lipidomics

All-in-one guide to successful lipidomic analysis, combining the latest advances and best practices from academia, industry, and clinical research

Mass Spectrometry for Lipidomics presents a systematic overview of lipidomic analysis, covering established standards of lipid analysis, available technology, and key lipid classes, as well as applications in basic research, medicine, pharma, and the food industry.

Through connecting recent technological advances with key application areas, this unique guide bridges the gap between academia and industry by translating the vast body of knowledge that has been gained in the past decade into much-needed practical advice for novices as well as routine users.

Edited by the president and vice-president of the International Lipidomics Society with contributions from the top experts in lipid analysis, Mass Spectrometry for Lipidomics covers a wide range of key topics, including:

• Aspects of sample preparation, separation methods, different mass spectrometry modes, as well as identification and quantitation, including the use of bioinformatics tools for data analysis
• Identification, quantitation and profiling of lipids in different types of biological samples
• Analytical approaches for all major classes of biological lipids, from fatty acids to phospholipids to sterols
• Novel applications in biological research, clinical diagnostics, as well as food and crop science

For analytical chemists, biochemists, clinical chemists, and analytical laboratories and hospitals, Mass Spectrometry for Lipidomics presents a comprehensive and authoritative overview of the subject, with unmatched expertise from practicing professionals actively involved in the latest research.

Leseprobe

Mass Spectrometry for Lipidomics

Methods and Applications

Edited by Michal Holčapek and Kim Ekroos

Volume 1

Mass Spectrometry for Lipidomics

Methods and Applications

Edited by Michal Holčapek and Kim Ekroos

Volume 2

Editors

Dr. Michal HolčapekUniversity of PardubiceFaculty of Chemical TechnologyStudentská 57353210 PardubiceCzech Republic

Dr. Kim EkroosLipidomics Consulting Ltd.Irisviksvägen 31D02230 EspooFinland

Cover Design: WileyCover Images: © Kateryna Kon/Shutterstock; Courtesy of Michaela Chocholoušková

All books published by WILEY‐VCH are carefully produced. Nevertheless, authors, editors, and publisher do not warrant the information contained in these books, including this book, to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate.

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© 2023 Wiley‐VCH GmbH, Boschstraße 12, 69469 Weinheim, Germany

All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form – by photoprinting, microfilm, or any other means – nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law.

Print ISBN: 978‐3‐527‐35221‐0ePDF ISBN: 978‐3‐527‐83649‐9ePub ISBN: 978‐3‐527‐83650‐5oBook ISBN: 978‐3‐527‐83651‐2

Editors

Dr. Michal HolčapekUniversity of PardubiceFaculty of Chemical TechnologyStudentská 57353210 PardubiceCzech Republic

Dr. Kim EkroosLipidomics Consulting Ltd.Irisviksvägen 31D02230 EspooFinland

Cover Design: WileyCover Images: © Kateryna Kon/Shutterstock; Courtesy of Michaela Chocholoušková

All books published by WILEY‐VCH are carefully produced. Nevertheless, authors, editors, and publisher do not warrant the information contained in these books, including this book, to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate.

Library of Congress Card No.: applied for

British Library Cataloguing‐in‐Publication DataA catalogue record for this book is available from the British Library.

Bibliographic information published by the Deutsche NationalbibliothekThe Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available on the Internet at <http://dnb.d-nb.de>.

© 2023 Wiley‐VCH GmbH, Boschstraße 12, 69469 Weinheim, Germany

All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form – by photoprinting, microfilm, or any other means – nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law.

Print ISBN: 978‐3‐527‐35222‐7ePDF ISBN: 978‐3‐527‐83649‐9ePub ISBN: 978‐3‐527‐83650‐5oBook ISBN: 978‐3‐527‐83651‐2

Preface

The field of lipidomics has undergone an enormous growth in recent years, which can be illustrated by the number of published articles and other bibliometric parameters. This highlights the renewed interest in lipids, now driven by the enthusiasm to explore the world of lipidomes and how these, among others, impact health and disease. The excitement is enormous, prompting many newcomers to enter the field. However, training and education in lipidomics are still scarce or even lacking. A successful lipidomics study requires appropriate expertise in all aspects of the lipidomic workflow, covering experimental design, sample preparation, analytical measurement using mass spectrometry techniques, data processing, and finally correct reporting of lipidomic results. The large discrepancy in know‐how and lipidomics assessments causes confusion in the field that is also mirrored in the literature. Recently, the International Lipidomics Society was established to fill this gap and to unite researchers around the world interested in all aspects of lipidomics research and collectively start creating urgently needed textbook chapters in lipidomics. This situation prompted us to start working on this book project, where we have assembled the content covering three sections: analytical methodologies in lipidomics, lipidomic analysis according to lipid categories and classes, and finally lipidomic applications. We invited leading experts for particular topics, and, after more than a year of tedious work, we are proud to present the result.

We believe that this book can serve as a valuable tool and resource for anyone interested in lipidomics, from beginners to field leaders, because everyone should be able to find something new in these 27 chapters. The methodological section describes the most common methods used in lipidomic analysis, such as the preanalytical phase, sample preparation, shotgun mass spectrometry, coupling with chromatography, mass spectrometry imaging, ion mobility, advanced tools for structural characterization, approaches for the right identification and quantitation, and finally bioinformatics, software, and databases. The second section is prepared from a different view, targeting selected lipid categories and classes and then sorting convenient methods for their analysis. We believe that this point of view is important for researchers looking for the best method for their lipids of interest. Finally, we present an application section to illustrate a wide range of lipidomics, which covers, for example, clinical diagnostics, biobanking, nutritional aspects, plant science, fluxomics, multiomics, cell biology, microbial lipidomics, and research on serious diseases, such as cancer, Alzheimer's disease, and aging. We hope that these chapters provide an interesting inspiration for new possible applications of lipidomics.

We greatly appreciate the great effort and the extensive time invested by all authors in the preparation of their chapters. Last but not least, we appreciate the support of the publisher in compiling this up‐to‐date book on lipidomic analysis. We hope that you enjoy reading and that the book will be an everyday companion rather than a dust‐covered item on the bookshelf.

Michal Holčapek and Kim EkroosPardubice and Esbo31 July 2022

1Introduction to Lipidomics

Harald C. Köfeler1, Kim Ekroos2, and Michal Holčapek3

1 Medical University Graz, Center for Medical Research, Stiftingtalstrasse 24, 8010, Graz, Austria

2 Lipidomics Consulting, Esbo, Finland

3 University of Pardubice, Faculty of Chemical Technology, Department of Analytical Chemistry, Pardubice, Czech Republic

1.1 Preface

We are entering a new era in lipidomic analysis. Technology advances in conjunction with community‐wide collaboration efforts have prompted new ways to investigate the world of lipids. These developments have revoked interest in lipids, creating new opportunities to study lipids in different biological and biomedical settings in the hope of improving health and disease. Today, technologies allow us to dive deep into the lipid content and dissect the lipid makeup in detail, providing quantitative numbers of hundreds of lipid molecules. Lipid measurements no longer circle just around cholesterol in the context of LDL or HDL, but now the typical target is to determine the comprehensive lipidome of these particles. The new previously unseen lipid details spark curiosity and interest in reactivating research on cellular membranes, signaling cascades, and metabolic networks, among others, to shed new insights into the dysfunctions underlying a disease or a disorder. The objectives are clear. Can lipid details untangle disease biology, provide improved predictive or diagnostic biomarkers, and deliver new therapeutic strategies? However, opportunities extend further beyond, as a detailed lipid fingerprint can be envisioned, serving as a health status map of individuals. Our unique lipid code, which all of us possess, becomes a tool for precision health and medicine, which we are only beginning to explore.

The study of lipids using lipidomics can be rephrased as mass spectrometry (MS)‐based lipid analysis. Until now, the field has been living its Wild West era where everything has been allowed. Although this has provided significant development, the downside is that it has resulted in inaccurate and irreproducible research results, preventing science from moving forward. With the establishment of the International Lipidomics Society (ILS), we have taken an active role in further maturing, harmonizing, and developing the lipidomics field to meet the current and future needs. By connecting the worldwide lipid community and focusing on transparent communication and collaboration, we aim to identify the common language for the entire discipline. Simply, the focus is to guide, educate, collaborate, and provide services to the academic and medical communities, industries, and the public in lipidomics. We have established several interest groups (see https://lipidomicssociety.org/working-groups) with different focuses to accelerate various angles of the field. A central program is briefly described here with the focus on the standardization of lipidomics, where we are preparing a new reporting checklist for any future lipidomics study. This is a true game changer that is needed to unlock the full potential of lipidomics. Now, we can meet the regulatory requirements for use in clinical research and diagnostics and enhance the comparability of data and understanding of the functional roles of specific lipid species. A new order in lipidomics has begun.

1.2 Historical Perspective

Although the determination of individual lipids by MS goes back to the 1970s (e.g. prostaglandins by GC/MS), the term lipidomics was introduced in 2003 by Xianlin Han and Richard Gross, defined as the system‐level analysis of lipid species' abundance, biological activities, subcellular localization, and tissue distribution [1]. Lipidomics became possible by the introduction of new technologies in MS, particularly electrospray ionization (ESI), matrix‐assisted laser desorption/ionization (MALDI), and Orbitrap instrumentation, resulting in a broader scope of analysis with increased sensitivity and selectivity. Fueled by these technical prerequisites and the concomitant increased biological usability of lipid data, a growing number of scientific groups have joined the field. In parallel, it soon became clear that the fast growing lipidomics field would need some sort of guidance for standards. In the early new millennium, LIPID MAPS was funded by NIH as a huge “glue grant” that included multiple labs in the United States. The most important achievement of the LIPID MAPS consortium was a comprehensive classification scheme of lipids into eight categories subdivided into dozens of lipid classes and subclasses [2, 3]. Based on this classification scheme, the LIPID MAPS Structure Database (LMSD) became the most important and comprehensive international lipid database containing 46 843 lipid structures as of December 2021, 24 815 of them experimentally proven and curated, and 22 028 of them generated in silico[4]. In parallel, a large‐scale European grant LipidomicNET was awarded by the European Union and started to develop annotation rules for lipids detected by MS [5]. These rules culminated in the slogan: “Only annotate what is experimentally proven.” According to this motto, a shorthand nomenclature for lipids was designed, where it is possible to simply infer the degree of annotation certainty by the nomenclature level used. In 2020, the shorthand notation for lipidomic data got a major overhaul, and now, e.g. also includes oxidized lipids and sphingolipids beyond ceramides and sphingomyelins [3]. The whole shorthand nomenclature project was performed according to the lipid categories developed by LIPID MAPS [2]. In the direct legacy of the shorthand nomenclature project, the Lipidomics Standards Initiative (LSI) was established in 2018 by Gerhard Liebisch and Kim Ekroos together with an informal group of lipidomics scientists who care for the development of standards in lipidomics (Figure 1.1). In 2019, the LSI led to the foundation of the ILS, in which the LSI constitutes one of the most important interest groups. Besides LSI, ILS hosts seven additional interest groups (applied bioinformatics, clinical lipidomics, global networking, instrumental and methodology development, lipid function, lipid ontology, reference materials, and biological reference ranges) and coordinates their activities. Some of the aforementioned interest groups and their activities will serve as a structure template for this chapter. Other community‐wide standardization endeavors of the past decade worth mentioning are ring trials. Between 2014 and 2017, a ring trial organized by John A. Bowden at the National Institute of Standards and Technology (NIST) occurred [6]. The aim of this ring trial was limited to an interlaboratory lipidomics precision comparison on NIST Standard Reference Material (SRM)‐1950, a reference plasma collected by NIST, because the true quantitative values of lipids in this biological material were unknown, and thus, it was impossible to determine the accuracy of the experimentally determined values. Furthermore, several community‐wide position papers recently clearly defined the necessity and demand for standardization in lipidomics, including further steps to be taken toward achieving this goal [7–9].

Figure 1.1 The Lipidomics Standards Initiative (LSI) and its various fields of action within the lipidomics workflow, ranging from sample collection to data analysis.

1.3 Sampling and Preanalytics

“Without a community‐wide consensus on best practices for the prevention of lipid degradation and transformations through sample collection and analysis, it is difficult to assess the quality of lipidomics data and hence trust results” [10]. Keeping this quote in mind, monitoring and documentation of the sampling step in the lipidomics workflow are of utmost importance because whatever is lost at sampling cannot be regained even by the most sophisticated analysis methods. Because of its importance in the workflow for lipidomics analysis, the LSI dedicates a separate chapter on this topic in its lipidomics guidelines (manuscript in preparation). Although stability is not as critical as when, e.g. handling RNA, there are nevertheless two big stability issues to be specifically considered when working with lipids: hydrolysis and oxidation [10, 11]. While hydrolysis affects esterified fatty acids, lipid peroxidation can occur at the methylene groups spacing two adjacent double bonds, e.g. C11 in linoleic acid. Both mechanisms may result in extensive fragmentation, truncation, and modification of lipids [12]. In contrast to lipid peroxidation, which is, in the context of sample stability, primarily a nonenzymatic chemical reaction, the threat of lipid hydrolysis also arises from enzymatic reactions catalyzed by lipases in the sample matrix. Thus, the most important measure to be taken against sample degradation is a short storage time and keeping the samples at as low temperatures as possible if storage of samples is needed. Sample workup immediately after collection is recommended because this would at least eliminate any enzymatic degradation, or, if this is not possible, the addition of methanol before freezing, to precipitate enzymes, and therefore minimize biological degradation processes. When already extracted samples are stored in organic solvents, a neutral pH avoids the hydrolysis of fatty acids, and the coverage of the extracts by an inert gas, such as nitrogen or argon, aids in preventing lipid peroxidation. Nevertheless, it is highly recommended to store samples at least at −80 °C for not too long periods. All listed recommendations and issues have to be particularly emphasized when working with lipids such as oxidized phospholipids or lysophospholipids, which are inherent degradation products of other lipids and occur only in small amounts. In such a case, only the slightest degradation could already immensely distort the results. Finally, above all, the most important point stipulated in the lipidomics guidelines is the proper documentation of preanalytics in a comprehensive way, which then even allows retrospectively evaluating the quality of the final data. In summary, the lipidomics community represented by LSI and ILS is well aware of the above‐mentioned points, and recommendation guidelines for standardization of preanalytics are close to publishing.

1.4 Reference Materials and Biological Reference Ranges

The first concerted approach toward the determination of reference ranges in biological samples was undertaken by the LIPID MAPS consortium in 2010. In two consecutive publications, they quantitatively determined the lipidome of human plasma [13] and mouse macrophages [14] in great detail. From a technical perspective, it is worth mentioning that these were the first harmonized interlaboratory approaches in which each contributing laboratory was responsible for one lipid category; for example, glycerolipids were determined in Denver (Murphy group), sphingolipids in Atlanta (Merrill group), fatty acids in San Diego (Dennis group), etc. Thus, the studies were organized as a multisite trial and resulted in the first broad high‐quality lipidomic analysis of both biological matrices. The second concerted approach in this field was performed by John A. Bowden from NIST in 2017, but this time, it was designed to be a ring trial using, as the LIPID MAPS trial described above, again NIST SRM‐1950, a standardized NIH plasma pool, with 31 international laboratories contributing to this endeavor (Figure 1.2) [6]. As the true values for the 339 lipids analyzed were not known, it was just possible to determine the consensus values for each lipid, including the interlaboratory precision. Furthermore, not every laboratory determined each lipid species but rather contributed whatever was in its quantitative lipidomics portfolio by this time. Figure 1.3 shows the consensus values and the interlaboratory spread of the lipid classes analyzed. The graph clearly shows that certain lipid classes, such as free fatty acids (FFAs) or oxylipins, are analyzed by a handful of laboratories, while others, such as the membrane lipid class phosphatidylcholine (PC), are analyzed by almost every laboratory. Although the spread of quantitative numbers is considerable, most of the mean quantities correlated quite well with the LIPID MAPS study on the same reference material and thus could be considered close to the real values of individual lipids. However, the issue of “real value” in biological reference materials remains untouched in its core and could only be solved by future inclusion of complementary analysis methods with quantitative properties better than ESI, e.g. NMR. The second important point when talking about reference materials are lipid standard compounds, whether nonlabeled reference standards or stable isotope‐labeled internal standards [9]. In this regard, the interest group reference materials and biological reference ranges are the central coordination hub for lipid synthesis companies and also academic groups working on novel concepts for the biotechnological bulk generation of total isotope‐labeled lipidomes.

Figure 1.2 HRMS, High Resolution Mass Spectrometry; PRM, Parallel Reaction Monitoring; LDA, Lipid Data Analyzer; MDMS‐SL, Multi‐Dimensional Mass Spectrometry‐based‐Shotgun Lipidomics; IS, Internal Standard.

Figure 1.3 Consensus values for individual lipid classes as calculated from the lipidomics ring trial initiated by John A. Bowden (NIST, Gaithersburg, MD, USA). As not every participating laboratory performed the same panel of analysis, not every lipid class has the same number of data points. DG, diacylglycerol; TG, triacylglycerol; LPC, lysophosphatidylethanol; PE, phosphatidylethanol; PI, phosphatidylinositol; PG, phosphatidylglycerol; SM, sphingomyelin; BA, bile acid; CE, cholesterylester.

1.5 Clinical Lipidomics

Clinical lipidomics aims at the application of lipidomics to clinical diagnostics. Based on the harmonization study initiated by Bowden et al. [6], a position paper organized by the Wenk group in Singapore together with 16 additional internationally recognized lipidomics laboratories wrapped up the state of the art in the field of lipidomics with regard to clinical applications [7]. The article also lists the most crucial prerequisites that must be met by lipidomics analysis to make an impact in clinical diagnostics. Among these, the most important are reproducibility, accuracy, and precision. While reproducibility and precision are easy to get under control, as long as sufficient resources are invested into quality assurance, accuracy is a factor that still poses a problem in handling. In real‐life samples, such as human plasma, the quantity of each individual lipid cannot be known a priori, and thus, it is per definition impossible to calculate the accuracy. This shortcoming is circumvented by taking the consensus values from the Bowden et al. study for NIST SRM‐1950 and assuming that the concordant values from 31 laboratories are close to the “true” values [6]. Furthermore, this publication lists the full workflow of lipidomics from preanalytics to data analysis, discussing all relevant steps and a number of key issues for each step of the workflow. The next topic on the agenda of this group of principal investors was an international ring trial that monitored ceramide concentrations in human plasma, performed in 2019 (manuscript in preparation). In this case, the organizers, according to an already published methodology, predetermined the LC/MS methodology. This was in contrast to the previous study conducted by John Bowden, where each laboratory was free to choose its method [6]. Based on these pieces of preliminary work, the Interest Group Clinical Lipidomics led by Michal Holčapek picked up the topic and is currently underway in organizing a lipidomics ring trial that includes 30 academic groups and corporate laboratories, distributed all over the globe. Regarding the methodology, this round robin will neither be completely open like the Bowden et al. study [6], nor will it be restricted to just one predetermined method. It will rather give a choice from four internationally established lipidomics workflows, i.e. lipid class separation, lipid species separation, and shotgun approaches either with low or high resolving power MS. The workflows by themselves try to keep a balance between parameters strictly demanded by the protocol, parameters just recommended, and parameters open to choose freely. In summary, the organizers anticipate that this clinical lipidomics ring trial on SRM‐1950 will give a good idea where the lipidomics field stands regarding the clinical application of this methodology.

1.6 Identification and Annotation

The identification of lipids by MS and their subsequent correct annotation are what could be called the core business of lipidomics. The most important issue with respect to the identification of lipids by MS and their further annotation is that the annotation nomenclature used always must reflect the identification status of the individual lipid. During the EU FP7 large‐scale grant LipidomicNET (2008–2012), it became evident that the various analytical laboratories involved in this endeavor do use different styles of annotating the same molecular compound, which in turn was detrimental to database generation, where each compound needs one unique ID. The root of this issue is the fact that the overwhelming majority of lipid identification generated by MS never reaches the level at which each molecular detail of a compound, including double‐bond positions and double‐bond stereochemistry, is known and where the nomenclature designed by the LIPID MAPS consortium could be applied. Although this level of detail could basically be obtained by MS and aligned technologies such as chromatography, the degree of analytical effort required can hardly be justified in an omics setting, where hundreds of lipids need to be identified in each sample. Kim Ekroos already proposed a hierarchy of lipid annotation back in 2011 [15]. Figure 1.4 shows the scheme based on this hierarchy jointly proposed by LipidomicNET and the LIPID MAPS consortium in accordance with the International Lipid Classification and Nomenclature Committee (ILCNC) in 2013 and updated in 2020. The leading figure in this endeavor has been Gerhard Liebisch from Regensburg. This hierarchy correlates the level of structure details elucidated by mass spectrometric/chromatographic/ion mobility spectrometric analysis with certain annotation requirements. Because of the high degree of isomerism that inherently arises in many lipid classes because of the variations in fatty acyl composition, each annotation in the nomenclature hierarchy reflects a subset of isomeric lipids, unless the fully defined LIPID MAPS structure level is used. In this case and only in this case, it is possible to pinpoint one unique lipid structure in the LMSD, while the molecular species level in Figure 1.4 leaves the sn‐positions of the corresponding fatty acyls, their double‐bond location, and the double‐bond configuration unresolved. Furthermore, each level of depth of structural identification is closely related to certain analytical techniques. While it may be sufficient for annotation at the species level to involve just reversed‐phase liquid chromatography and a low‐resolution precursor ion scan on the phospholipid head group, further levels of the pyramid will require MS/MS spectra, high mass resolution, and additional advanced techniques such as OzID, chiral chromatography, or ion mobility spectrometry. At the end of the day, it will always come down to a tradeoff between the available resources (manpower, instrument quality, etc.) and the minimum structural depth needed for answering a certain scientific question.

Figure 1.4 The hierarchical lipid shorthand nomenclature pyramid depicted for a phosphatidyl choline species on the left side of the figure integrates with the various levels of this nomenclature on the right‐hand side. This example shows that not all annotation levels are applicable for every lipid. In this case, the phosphate position level, structure‐defined level, and full structure level are skipped because the lipid does neither have an inositol phosphate group nor any other additional functional group in the fatty acyls.

1.7 Quantitation

When identification issues are resolved, the immediately subsequent question usually is about the quantity of individual lipid species or, in some cases, whole lipid classes. Again, the quantitative aspects depend heavily on the scientific questions to be answered. Although in some cases it might be good enough to state that a knockout mouse model accumulates some lipids roughly by a factor of 10, in other cases such as clinical diagnostics, exact molar numbers of highly reliable quality might be required. To deal with such a wide spectrum of quality requirements, LSI recommends protocols for three levels of quantitation. For all the three levels of quantitation, it is necessary to use an internal standard, which has to be a nonendogenous compound added to the sample at the beginning of the lipid extraction process. The reason for the importance of internal standards in lipidomics is the tendency of ESI toward ion suppression effects, which may vastly distort quantitative results. Despite these shortcomings, ESI is still the ionization of choice because it allows coupling with liquid chromatography and has the ability to ionize a large spectrum of various lipids. Ideally, the internal standard should be of the same chemical nature as the target lipid but be separable by its mass, which naturally results in stable isotope‐labeled lipids as the premier choice for internal standards. The superiority of stable isotope‐labeled internal standards is reflected in Level 1 and Level 2 quantitation, both of which rely on stable isotope‐labeled internal standards and can be considered as the gold standard in quantitative lipidomics. Preferably, the internal standard should coionize (coelute in the case of chromatography) with the target lipid compound with known response factors. Alternatively, when no coionizing internal standard is available or the applied internal standard is from another lipid class, Level 3 quantitation has to be used. The development of this standardized three‐level system reflects the quality of quantitative data and should thus provide a standardized quality assessment at a glance for journals and readers alike. Further important quantitative aspects are isotopic correction [16] and one‐point calibration versus multipoint calibration [17]