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The Minimum Information Required for a Glycomics Experiment (MIRAGE) Project: Improving the Standards for Reporting Mass-spectrometry-based Glycoanalytic Data

The Minimum Information Required for a Glycomics Experiment (MIRAGE) Project: Improving the... Technological Innovation and Resources © 2013 by The American Society for Biochemistry and Molecular Biology, Inc. This paper is available on line at http://www.mcponline.org The Minimum Information Required for a Glycomics Experiment (MIRAGE) Project: Improving the Standards for Reporting Mass-spectrometry-based Glycoanalytic Data Daniel Kolarich‡, Erdmann Rapp§, Weston B. Struwe¶, Stuart M. Haslam, Joseph Zaia**, Ryan McBride‡‡, Sanjay Agravat§§, Matthew P. Campbell¶¶, a b a,c , Carsten Kettner , and William S. York Masaki Kato, Rene Ranzinger The MIRAGE guidelines are being developed in response depend on comprehensive meta-data describing the in- to a critical need in the glycobiology community to clarify strumentation, instrument setup, and data acquisition glycoanalytic results so that they are more readily evalu- protocols. The MIRAGE guidelines for MS-based glycomics ated (in terms of their scope and depth) and to facilitate have been designed to facilitate the collection and sharing the reproduction of important results in the laboratory. of this critical information in order to assist the glycoana- The molecular and biological complexity of the glycosyl- lyst in generating data sets with maximum information ation process makes thorough reporting of the results of content and biological relevance. Molecular & Cellular a glycomics experiment a highly challenging endeavor. Proteomics 12: 10.1074/mcp.O112.026492, 991–995, 2013. The resulting data specify the identity and quantity of complex structures, the precise molecular features of REPORTING GUIDELINES FOR GLYCOMICS—WHY BOTHER? which are sometimes inferred using prior knowledge, The increasing importance of glycoscience in modern biol- such as familiarity with a particular biosynthetic mecha- ogy was recently described in the publication Transforming nism. Specifying the exact methods and assumptions that Glycoscience: A Roadmap for the Future, prepared by the were used to assign and quantify reported structures al- U.S. National Academy of Sciences (1). Glycomics, which is lows the interested scientist to appreciate the scope and one emerging discipline of glycoscience, utilizes diverse an- depth of the analysis. Mass spectrometry (MS) is the most alytical and computational techniques aimed at comprehen- widely used tool for glycomics experiments. The interpre- tation and reproducibility of MS-based glycomics data sively identifying and characterizing the repertoire of glycan structures present in an organism, cell, or tissue at a defined time. Recent technical advances have enabled glycan analy- From the ‡Department of Biomolecular Systems, Max Planck In- ses to proceed with increased depth, speed, and efficiency stitute of Colloids and Interfaces, 14424 Potsdam, Germany; §Max and have led to both the increased publication of glycomics Planck Institute for Dynamics of Complex Technical Systems, Bio- data in carbohydrate-related journals and the accumulation of process Engineering, 39106 Magdeburg, Germany; ¶Department of large data sets on a global scale. Chemistry, Chemistry Research Laboratory, University of Oxford, Ox- ford, OX1 3QZ, UK; Division of Molecular Biosciences, Faculty of The application of data mining techniques and analytical Natural Sciences, Imperial College London, London SW7 2AZ, UK; software tools make it possible to identify relationships **Center for Biomedical Mass Spectrometry, Department of Biochem- among distinct data sets in a way that generates new knowl- istry, Boston University School of Medicine, Boston, Massachusetts edge. However, the annotation and archiving of information 02118; ‡‡Department of Chemical Physiology, The Scripps Research are often carried out in a retrospective way (e.g. by manually Institute, 10550, N. Torrey Pines Road, La Jolla, California 92037; §§Center for Comprehensive Informatics, Research and Woodruff extracting it from the literature and importing it into data- Health Sciences IT, Emory University, 1784 N. Decatur Rd, Atlanta, bases). Therefore, database quality is highly dependent on the Georgia 30322; ¶¶Biomolecular Frontiers Research Centre, Macqua- reliability and depth of literature reports, which can be judged rie University, Sydney, NSW 2109, Australia; Structural Glycobiology only if the experiments that generate the data are adequately Team, Riken Advanced Science Institute, 2-1 Hirosawa, Wako, described. Thus, in both publications and databases, the Saitama 351–0198, Japan; Complex Carbohydrate Research Cen- ter, University of Georgia, 315 Riverbend Road, Athens, Georgia prerequisite for high information quality is comprehensive re- 30602; Beilstein-Institut, Trakehner Str. 7-9, 60487 Frankfurt am porting of the experimental context in which the data were Main, Germany generated. Received December 5, 2012, and in revised form, February 1, Unfortunately, a large proportion of published glycomics data do not meet this criterion. Although experimental data Published, MCP Papers in Press, February 1, 2013, DOI 10.1074/mcp.O112.026492 are highly dependent on the experimental conditions applied, Molecular & Cellular Proteomics 12.4 991 This is an Open Access article under the CC BY license. Guidelines for Reporting MS Data the descriptions of experimental conditions in the Materials and Methods sections of many publications are often inad- vertently or deliberately incomplete. This issue has been recognized previously by diverse bio- logical and biomedical initiatives that promote reporting standards for analytical data. These include MIAME (2), MIAPE (3), and STRENDA (4). To make it easier for authors to identify appropriate guidelines, a platform project called Min- imum Information for Biological and Biomedical Investigations has been developed to provide descriptions for each guide- line, including the type of information that is required in order to thoroughly report each particular experiment (5). The need for and success of these initiatives are clearly indicated by the fact that many of these guidelines are already recommended by journals, and the submission of these vital sets of informa- FIG.1. Process used within the MIRAGE project for the devel- tion is often mandatory in order for a manuscript to be con- opment of guidelines. The multistep process established for this sidered for publication. purpose includes drafting within the subgroups, refinement within the However, the field of glycomics currently lacks such guide- entire working group, and reviews by the advisory board. Finally, the lines. This is likely partly because of the diverse number of scientific community is invited to comment so as to achieve broad preparative and analytical methods applied in characterizing agreement and minimize potential mistakes and misunderstandings. glycans and differences in the intended depths of analyses. For example, protein-bound glycans such as N-linked or O- tion is required in order to integrate glycomics data that are linked glycans require sample preparation steps that differ widely spread among diverse databases and thereby facilitate quite significantly from protocols used in the analysis of bac- the development and application of bioinformatic tools for the terial or plant polysaccharides. Glycans often have very com- analysis of these data. This initiative gained significant mo- plex structures that cannot be directly inferred from genomics mentum when international leaders in the development of data, as is frequently done to obtain protein sequences. glycomics analysis techniques and software tools for glycoin- Therefore, diverse analytical techniques are used for glyco- formatics were joined by the editors of the major journals that mics analyses, including those that exclusively utilize HPLC or publish glycomics and glycoproteomics research in express- MS and those that combine more than one method, such as ing their willingness to support a standardization initiative. LC-MS/MS analysis. In some cases, minimal information is This resulted in the creation of the MIRAGE (Minimum Infor- obtained, as in some glycan mass profiling experiments (e.g. mation Required for a Glycomics Experiment) initiative, led by MALDI compositional analysis). In rare cases, detailed struc- experts in the fields of glycobiology, glycoanalytics, and gly- ture characterization is performed using NMR. coinformatics with the goal of creating minimum information The application of these techniques can result in varying guidelines for glycomics. The organization of this international levels of structural information that, when combined with ad- group and their recent conclusions are published on the pro- ditional information, such as knowledge of the underlying ject website (http://glycomics.ccrc.uga.edu/MIRAGE/). Mem- biosynthetic pathways, often allows a defined structure to be bership is open for additional scientists who would like to par- proposed. However, the degree of structural definition and ticipate in the work, and input from the scientific community is the assumptions that have been made in order to assign each welcome. Additionally, proposals will be presented and discussed structure are not always well reported. at the biennial Beilstein Symposia on Glyco-Bioinformatics In summary, the exact experimental conditions for sample (http://www.beilstein-institut.de/en/symposia/overview/). preparation and analysis, in combination with the techniques Because glycobiology covers a wide range of different mol- and equipment used, have profound influences on the quali- ecules and all the peculiarities of glycan sample preparation tative and quantitative results generated by a glycomics anal- and analysis need to be considered, a new set of guidelines is ysis. Therefore comprehensive description of conditions, being generated to address diverse information-reporting re- techniques and results is required to enable researchers to quirements. The working group has initiated the development evaluate and unambiguously interpret the results of these of guidelines that take into account the generation, sampling, analyses and to reproduce them when necessary. and storage of glycomics data obtained using MS. These The MIRAGE Project—In 2009, at the Workshop on Analyt- guidelines are derived from the MIAPE-MS guidelines and ical and Bioinformatic Glycomics, organized by the Consor- have been extended to address issues that are unique to tium for Functional Glycomics, an international group of gly- glycomics data. The initial version of these MIRAGE-MS coscientists concluded that there is an urgent need for the guidelines has been reviewed and approved by the MIRAGE standardization of data reporting in this area (6). Standardiza- advisory board (Fig. 1) and has been made available online so 992 Molecular & Cellular Proteomics 12.4 Guidelines for Reporting MS Data that the scientific community can offer further comments and database developers would undoubtedly impose additional refinement. Similar to the MIAPE and MIAMI concepts, data submission requirements of their own. Conversely, jour- MIRAGE identifies specific metadata that significantly in- nals are likely to have less formal requirements for data sub- crease the value of the associated experimental data. The mission than those described in the MIRAGE guidelines. In MIRAGE guidelines will facilitate the collection of this informa- this context, this journal’s guidelines for the submission of tion, for example, by stimulating the development of computa- glycomics data serve as a good use case: many explicit tional methods to automatically extract this information using details regarding instrumental setup parameters are not re- software supplied by mass spectrometer manufacturers. quired, but the spirit of the MIRAGE guidelines is maintained. MIRAGE-MS Guidelines—The MIAPE committee has had a That is, the Journal requires information that will allow an major effect on proteomics analysis by addressing diverse expert to judge the quality of the results and to reproduce the aspects related to the preparation, analysis, and identification overall conclusions of the reported study. Although not fully of proteins. Despite the fact that similar methods and instru- and formally implemented by the Journal, the MIRAGE guide- ments are used in glycomics and that many instrument pa- lines can serve a critical role by prompting the analyst and the rameters are equally applicable to MS-based proteomics and reviewer to consider experimental parameters that have a glycomics, unique experimental requirements differentiate profound effect on the data and their interpretation. glycan analysis from protein analysis in several respects. The types of metadata recommended for reporting by the These differences are related to the very distinct nature of MIRAGE-MS guidelines are divided into five sections (Fig. 2). glycan structures, chemistry, and biosynthesis. One of the Overall, Sections 1–3 deal mainly with the instrumental hard- major differences is that glycans undergo vibrational dissoci- ware used to generate, fragment, and detect ions, whereas ation at lower energies than do peptides. For this reason, it is Sections 4 and 5 are focused on data interpretation and han- important to include information regarding the mass spec- dling issues. Section 1, “General Features,” serves as the basis trometer settings used for glycan ion analysis. This informa- for the required metadata, with global descriptions on the used tion should include the ion source, ion transfer, and ion iso- instrumentation, any particular customizations, and general instru- lation appropriate for glycan classes. The necessity of the ment control parameters such as instrument control software. specification of this information is emphasized by the fact that Section 2, “Ion Sources,” continues to summarize all crucial glycans may be analyzed in native or derivatized forms, in parameters for ion generation such as controls of in-source positive or negative ionization modes, as cation or anion fragmentation or the degree of prompt fragmentation, in ad- adducts, or as unattached/unlinked ions. The ability to extract dition to other, more common parameters (e.g. capillary volt- and interpret structural information from the data in a repro- age or laser intensity settings). Glycans contain several types ducible manner depends on the accurate communication of of labile bonds, including bonds to fucose and sialic acid information regarding sample and instrumental conditions. residues and to sulfate and phosphate substituents. It is thus Therefore, it is particularly important to include minimal infor- very important to determine whether biologically or chemically mation regarding the experimental conditions used for MS- significant ions observed in full scan mass spectra arise as a based glycomics. result of prompt fragmentation during the ionization process. The MIRAGE guidelines for mass spectral glycoanalysis are The extent of prompt fragmentation can be established by relevant to both database deposition and the submission of examining data obtained using purified standard glycans, results to a journal. However, the MIRAGE guidelines are which allows one to demonstrate that the mass spectrometer intended to be neither comprehensive nor absolute. In gen- is tuned properly for analysis of the glycan class in question. eral, the deposition of data to a database requires highly For example, if analyzing native N-glycans, one can show formal parameter descriptions because of the necessity of data obtained using a commercial sialylated N-glycan stand- controlling vocabulary, digital data formats, and other techni- ard to show that sialic acid residues are not lost during cal characteristics of the information. Nevertheless, restrictive ionization under the conditions used. Such data are impor- vocabularies and explicit digital data formats are beyond the tant in order for readers and database users to evaluate the current scope of the MIRAGE guidelines for mass spectral instrumental conditions under which data were acquired. data, as such requirements are best determined by the data- The MIRAGE-MS guidelines do not require that the experi- base developers. An example of the appropriate application mentalist perform an analysis of prompt fragmentation, but he of the MIRAGE guidelines for MS analysis would be the pop- or she must report whether such an analysis was done and, if ulation of a specialized database with the mass spectra of so, provide the resulting data. well-characterized standard molecules (i.e.“gold standard” Section 3, “Ion Transfer and Post-source Components,” spectra). The usefulness of such data collection would most asks for instrumental details associated with the transport, likely depend on compliance with the MIRAGE guidelines (e.g. gas phase reactions, and detection of ions once they are reporting of instrument setup parameters) so that users of the generated. This critical step is selective for the respective data could design experiments to obtain spectra that were instrument(s) and thus is more difficult to generalize; there- comparable to the standard spectra in the database. The fore, a clear separation into major detection and ion transport Molecular & Cellular Proteomics 12.4 993 Guidelines for Reporting MS Data FIG.2. The five major sections of the MIRAGE-MS guidelines. Sections 1–3 mainly deal with the instrumental hard- ware used to generate, fragment, and detect ions, and Sections 4 and 5 focus on data interpretation and handling issues. categories currently found on the market has been complex mixture. A similar state of affairs exists for glycomics introduced. experiments. Nevertheless, with certain limitations and cave- Section 4, “Spectrum and Peak List Generation and Anno- ats, glycomics analysis using single-stage MS can provide tation,” and Section 5, “Interpretation and Validation,” sum- data that are adequate to answer a specific experimental marize the crucial parameters that form the basis of the ana- question. Examples (e.g. modulation of the N-glycosylation of lytical results that are generated after spectra have been a very well-defined plant protein or control of the incorpo- recorded by the instrument. Detailed description of these ration/absence of particular sugar residues such as fucose) parameters is vital because, to date, robust, widely distributed clearly show that laborious in-depth experiments are not (and thus commonly used) tools for this step are not available. always required in order to answer a well-defined question Parameters such as software, software customizations, and (7, 8). databases (if used) are overviewed in these sections. Quan- Overall, these MIRAGE-MS guidelines summarize a list of titative aspects, which often play a considerable role in gly- instrumental and experimental parameters that are consid- comics experiments, are also considered within the guide- ered critical in describing MS-related conditions for the ac- lines, and a set of parameters judged to be crucial is listed in quisition and interpretation of glycoanalysis data. Based on Subsection 4.d. these MIRAGE-MS guidelines, the committee is now working Important aspects such as assignment validation and “de- on developing guidelines for various other techniques and duced structure(s)” are specifically listed in order to further approaches (including sample preparation methods) that are emphasize that the interpretation of many MS experiments is commonly used in glycomics analysis. The lack of adequate highly connected to the well-established (mammalian) glyco- and generally applicable software tools as utilized in pro- sylation pathways, and therefore particular structural details teomic research adds an additional challenge for glycoconju- are often inferred rather than confirmed via orthogonal tech- gate structural determination and structure reporting. niques. This constitutes one of the major differences existing CONCLUSIONS between MS-based glycomics and proteomics and is clearly reflected in the MIRAGE-MS guidelines. It is clear that mass The MIRAGE-MS guidelines have been proposed in order profiling (without the use of tandem MS) can provide valuable to encourage authors, editors, and reviewers to gather and information for both peptide and glycan samples. However, report all essential information describing a glycomics exper- mass profiling of complex samples generated during bot- iment that is being reported. The guidelines can be viewed in tom-up proteomics analysis can lead to unacceptably high their entirety in the supplementary material or at the project false discovery rates, given the statistical probability that un- web page. It is important to note that these guidelines are related peptides might have the same mass. Thus, tandem intended neither to dictate the use of particular methods MS sequence tags are often required in order for one to (which should be decided by the experimentalist) nor to serve confidently identify a large number of distinct proteins in a as a substitute for the review process. The goal of the guide- 994 Molecular & Cellular Proteomics 12.4 Guidelines for Reporting MS Data lines is to provide a summary of the information describing an R. J., Paulson, J. C., Sasisekharan, R., Varki, A. P., and Wong, C.-H.) (2012) Transforming Glycoscience: A Roadmap for the Future, The Na- MS experiment at a level that allows it to be understood, tional Academies Press Washington, D.C. evaluated, and reproduced. Furthermore, the guidelines pro- 2. Brazma, A., Hingamp, P., Quackenbush, J., Sherlock, G., Spellman, P., vide authors with a framework and standard for defining the Stoeckert, C., Aach, J., Ansorge, W., Ball, C. A., Causton, H. C., Gaaster- land, T., Glenisson, P., Holstege, F. C., Kim, I. F., Markowitz, V., Matese, depth of structural analysis that supports the structural mod- J. C., Parkinson, H., Robinson, A., Sarkans, U., Schulze-Kremer, S., els reported in the manuscript. This is important for enabling Stewart, J., Taylor, R., Vilo, J., and Vingron, M. (2001) Minimum infor- both expert glycoscientists and readers who are less familiar mation about a microarray experiment (MIAME)—toward standards for microarray data. Nat. Genet. 29, 365–371 in this area to understand the conclusions of the publication 3. Taylor, C. F., Paton, N. W., Lilley, K. S., Binz, P. A., Julian, R. K., Jr., Jones, based on a rigorous and comprehensive description of the A. R., Zhu, W., Apweiler, R., Aebersold, R., Deutsch, E. W., Dunn, M. J., materials and methods used and the results obtained. As the Heck, A. J., Leitner, A., Macht, M., Mann, M., Martens, L., Neubert, T. A., Patterson, S. D., Ping, P., Seymour, S. L., Souda, P., Tsugita, A., Vande- reader depends on the judgment of the reviewers to set high kerckhove, J., Vondriska, T. M., Whitelegge, J. P., Wilkins, M. R., Xe- standards for the publication of glycoanalytic data, this infor- narios, I., Yates, J. R., 3rd, and Hermjakob, H. (2007) The minimum mation is absolutely required in order for the reviewers to information about a proteomics experiment (MIAPE). Nat. Biotechnol. 25, 887–893 evaluate the results reported for each experiment described in 4. Apweiler, R., Cornish-Bowden, A., Hofmeyr, J.-H. S., Kettner, C., Leyh, the manuscript. T. S., Schomburg, D., and Tipton, K. T. (2005) The importance of uni- formity in reporting protein-function data. Trends Biochem. Sci. 30, * In 2011, the Beilstein-Institut, a non-profit foundation located in 11–12 Frankfurt am Main, Germany, assumed financial responsibility for 5. Taylor, C. F., Field, D., Sansone, S. A., Aerts, J., Apweiler, R., Ashburner, M., Ball, C. A., Binz, P. A., Bogue, M., Booth, T., Brazma, A., Brinkman, MIRAGE and the advancement of this standardization initiative. This R. R., Michael Clark, A., Deutsch, E. W., Fiehn, O., Fostel, J., Ghazal, P., work has also been supported by several other funding agencies. Gibson, F., Gray, T., Grimes, G., Hancock, J. M., Hardy, N. W., Hermja- These include the Max Planck Society (D. Kolarich and E. Rapp), the kob, H., Julian, R. K., Jr., Kane, M., Kettner, C., Kinsinger, C., Kolker, E., European Union’s Seventh Framework Programme (FP7-Health-F5– Kuiper, M., Le Nove` re, N., Leebens-Mack, J., Lewis, S. E., Lord, P., 2011) under Grant No. 278535 (E. Rapp), the Biotechnology and Mallon, A. M., Marthandan, N., Masuya, H., McNally, R., Mehrle, A., Biological Sciences Research Council (Grant Nos. SF19107, B19088, Morrison, N., Orchard, S., Quackenbush, J., Reecy, J. M., Robertson, and BBF008309/1 to S. Haslam), and the Australian government D. G., Rocca-Serra, P., Rodriguez, H., Rosenfelder, H., Santoyo-Lopez, through the project NeCTAR, financed by the Education Investment J., Scheuermann, R. H., Schober, D., Smith, B., Snape, J., Stoeckert, Fund (M. Campbell). The authors also acknowledge NIGMS (National C. J., Jr., Tipton, K., Sterk, P., Untergasser, A., Vandesompele, J., and Institutes of Health) Grant No. P41GM104603 (J. Zaia) and funding Wiemann, S. (2008) Promoting coherent minimum reporting guidelines for biological and biomedical investigations: the MIBBI project. Nat. received from the NIGMS-funded National Center for Glycomics and Biotechnol. 26, 889–896 Glycoproteomics (National Institutes of Health) (Grant No. 6. Workshop on Analytic and Bioinformatic Glycomics , April 16–18, 2009, 8P41GM103490 to M. J. Pierce and W. S. York). Bethesda, MD To whom correspondence should be addressed: William S. York, 7. Kolarich, D., and Altmann, F. (2000) N-Glycan analysis by matrix-assisted Tel.: 1 706 542 4628; FAX: 1 706 542 4412; E-mail: will@ laser desorption/ionization mass spectrometry of electrophoretically ccrc.uga.edu. separated nonmammalian proteins: application to peanut allergen Ara h 1 and olive pollen allergen Ole e 1. Anal. Biochem. 285, 64–75 REFERENCES 8. Rillahan, C. D., Antonopoulos, A., Lefort, C. T., Sonon, R., Azadi, P., Ley, K., 1. Committee on Assessing the Importance and Impact of Glycomics and Dell, A., Haslam, S. M., and Paulson, J. C. (2012) Global metabolic Glycosciences (Walt, D. A., Aoki-Kinoshita, A. F., Bendiak, B., Bertozzi, inhibitors of sialyl- and fucosyltransferases remodel the glycome. Nat. C. R., Boons, G.-J., Darvill, A., Hart, G., Kiessling, L. L., Lowe, J., Moon, Chem. Biol. 8, 661–668 Molecular & Cellular Proteomics 12.4 995 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Molecular & Cellular Proteomics American Society for Biochemistry and Molecular Biology

The Minimum Information Required for a Glycomics Experiment (MIRAGE) Project: Improving the Standards for Reporting Mass-spectrometry-based Glycoanalytic Data

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American Society for Biochemistry and Molecular Biology
Copyright
Copyright © 2013 Elsevier Inc.
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1535-9476
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1535-9484
DOI
10.1074/mcp.o112.026492
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Abstract

Technological Innovation and Resources © 2013 by The American Society for Biochemistry and Molecular Biology, Inc. This paper is available on line at http://www.mcponline.org The Minimum Information Required for a Glycomics Experiment (MIRAGE) Project: Improving the Standards for Reporting Mass-spectrometry-based Glycoanalytic Data Daniel Kolarich‡, Erdmann Rapp§, Weston B. Struwe¶, Stuart M. Haslam, Joseph Zaia**, Ryan McBride‡‡, Sanjay Agravat§§, Matthew P. Campbell¶¶, a b a,c , Carsten Kettner , and William S. York Masaki Kato, Rene Ranzinger The MIRAGE guidelines are being developed in response depend on comprehensive meta-data describing the in- to a critical need in the glycobiology community to clarify strumentation, instrument setup, and data acquisition glycoanalytic results so that they are more readily evalu- protocols. The MIRAGE guidelines for MS-based glycomics ated (in terms of their scope and depth) and to facilitate have been designed to facilitate the collection and sharing the reproduction of important results in the laboratory. of this critical information in order to assist the glycoana- The molecular and biological complexity of the glycosyl- lyst in generating data sets with maximum information ation process makes thorough reporting of the results of content and biological relevance. Molecular & Cellular a glycomics experiment a highly challenging endeavor. Proteomics 12: 10.1074/mcp.O112.026492, 991–995, 2013. The resulting data specify the identity and quantity of complex structures, the precise molecular features of REPORTING GUIDELINES FOR GLYCOMICS—WHY BOTHER? which are sometimes inferred using prior knowledge, The increasing importance of glycoscience in modern biol- such as familiarity with a particular biosynthetic mecha- ogy was recently described in the publication Transforming nism. Specifying the exact methods and assumptions that Glycoscience: A Roadmap for the Future, prepared by the were used to assign and quantify reported structures al- U.S. National Academy of Sciences (1). Glycomics, which is lows the interested scientist to appreciate the scope and one emerging discipline of glycoscience, utilizes diverse an- depth of the analysis. Mass spectrometry (MS) is the most alytical and computational techniques aimed at comprehen- widely used tool for glycomics experiments. The interpre- tation and reproducibility of MS-based glycomics data sively identifying and characterizing the repertoire of glycan structures present in an organism, cell, or tissue at a defined time. Recent technical advances have enabled glycan analy- From the ‡Department of Biomolecular Systems, Max Planck In- ses to proceed with increased depth, speed, and efficiency stitute of Colloids and Interfaces, 14424 Potsdam, Germany; §Max and have led to both the increased publication of glycomics Planck Institute for Dynamics of Complex Technical Systems, Bio- data in carbohydrate-related journals and the accumulation of process Engineering, 39106 Magdeburg, Germany; ¶Department of large data sets on a global scale. Chemistry, Chemistry Research Laboratory, University of Oxford, Ox- ford, OX1 3QZ, UK; Division of Molecular Biosciences, Faculty of The application of data mining techniques and analytical Natural Sciences, Imperial College London, London SW7 2AZ, UK; software tools make it possible to identify relationships **Center for Biomedical Mass Spectrometry, Department of Biochem- among distinct data sets in a way that generates new knowl- istry, Boston University School of Medicine, Boston, Massachusetts edge. However, the annotation and archiving of information 02118; ‡‡Department of Chemical Physiology, The Scripps Research are often carried out in a retrospective way (e.g. by manually Institute, 10550, N. Torrey Pines Road, La Jolla, California 92037; §§Center for Comprehensive Informatics, Research and Woodruff extracting it from the literature and importing it into data- Health Sciences IT, Emory University, 1784 N. Decatur Rd, Atlanta, bases). Therefore, database quality is highly dependent on the Georgia 30322; ¶¶Biomolecular Frontiers Research Centre, Macqua- reliability and depth of literature reports, which can be judged rie University, Sydney, NSW 2109, Australia; Structural Glycobiology only if the experiments that generate the data are adequately Team, Riken Advanced Science Institute, 2-1 Hirosawa, Wako, described. Thus, in both publications and databases, the Saitama 351–0198, Japan; Complex Carbohydrate Research Cen- ter, University of Georgia, 315 Riverbend Road, Athens, Georgia prerequisite for high information quality is comprehensive re- 30602; Beilstein-Institut, Trakehner Str. 7-9, 60487 Frankfurt am porting of the experimental context in which the data were Main, Germany generated. Received December 5, 2012, and in revised form, February 1, Unfortunately, a large proportion of published glycomics data do not meet this criterion. Although experimental data Published, MCP Papers in Press, February 1, 2013, DOI 10.1074/mcp.O112.026492 are highly dependent on the experimental conditions applied, Molecular & Cellular Proteomics 12.4 991 This is an Open Access article under the CC BY license. Guidelines for Reporting MS Data the descriptions of experimental conditions in the Materials and Methods sections of many publications are often inad- vertently or deliberately incomplete. This issue has been recognized previously by diverse bio- logical and biomedical initiatives that promote reporting standards for analytical data. These include MIAME (2), MIAPE (3), and STRENDA (4). To make it easier for authors to identify appropriate guidelines, a platform project called Min- imum Information for Biological and Biomedical Investigations has been developed to provide descriptions for each guide- line, including the type of information that is required in order to thoroughly report each particular experiment (5). The need for and success of these initiatives are clearly indicated by the fact that many of these guidelines are already recommended by journals, and the submission of these vital sets of informa- FIG.1. Process used within the MIRAGE project for the devel- tion is often mandatory in order for a manuscript to be con- opment of guidelines. The multistep process established for this sidered for publication. purpose includes drafting within the subgroups, refinement within the However, the field of glycomics currently lacks such guide- entire working group, and reviews by the advisory board. Finally, the lines. This is likely partly because of the diverse number of scientific community is invited to comment so as to achieve broad preparative and analytical methods applied in characterizing agreement and minimize potential mistakes and misunderstandings. glycans and differences in the intended depths of analyses. For example, protein-bound glycans such as N-linked or O- tion is required in order to integrate glycomics data that are linked glycans require sample preparation steps that differ widely spread among diverse databases and thereby facilitate quite significantly from protocols used in the analysis of bac- the development and application of bioinformatic tools for the terial or plant polysaccharides. Glycans often have very com- analysis of these data. This initiative gained significant mo- plex structures that cannot be directly inferred from genomics mentum when international leaders in the development of data, as is frequently done to obtain protein sequences. glycomics analysis techniques and software tools for glycoin- Therefore, diverse analytical techniques are used for glyco- formatics were joined by the editors of the major journals that mics analyses, including those that exclusively utilize HPLC or publish glycomics and glycoproteomics research in express- MS and those that combine more than one method, such as ing their willingness to support a standardization initiative. LC-MS/MS analysis. In some cases, minimal information is This resulted in the creation of the MIRAGE (Minimum Infor- obtained, as in some glycan mass profiling experiments (e.g. mation Required for a Glycomics Experiment) initiative, led by MALDI compositional analysis). In rare cases, detailed struc- experts in the fields of glycobiology, glycoanalytics, and gly- ture characterization is performed using NMR. coinformatics with the goal of creating minimum information The application of these techniques can result in varying guidelines for glycomics. The organization of this international levels of structural information that, when combined with ad- group and their recent conclusions are published on the pro- ditional information, such as knowledge of the underlying ject website (http://glycomics.ccrc.uga.edu/MIRAGE/). Mem- biosynthetic pathways, often allows a defined structure to be bership is open for additional scientists who would like to par- proposed. However, the degree of structural definition and ticipate in the work, and input from the scientific community is the assumptions that have been made in order to assign each welcome. Additionally, proposals will be presented and discussed structure are not always well reported. at the biennial Beilstein Symposia on Glyco-Bioinformatics In summary, the exact experimental conditions for sample (http://www.beilstein-institut.de/en/symposia/overview/). preparation and analysis, in combination with the techniques Because glycobiology covers a wide range of different mol- and equipment used, have profound influences on the quali- ecules and all the peculiarities of glycan sample preparation tative and quantitative results generated by a glycomics anal- and analysis need to be considered, a new set of guidelines is ysis. Therefore comprehensive description of conditions, being generated to address diverse information-reporting re- techniques and results is required to enable researchers to quirements. The working group has initiated the development evaluate and unambiguously interpret the results of these of guidelines that take into account the generation, sampling, analyses and to reproduce them when necessary. and storage of glycomics data obtained using MS. These The MIRAGE Project—In 2009, at the Workshop on Analyt- guidelines are derived from the MIAPE-MS guidelines and ical and Bioinformatic Glycomics, organized by the Consor- have been extended to address issues that are unique to tium for Functional Glycomics, an international group of gly- glycomics data. The initial version of these MIRAGE-MS coscientists concluded that there is an urgent need for the guidelines has been reviewed and approved by the MIRAGE standardization of data reporting in this area (6). Standardiza- advisory board (Fig. 1) and has been made available online so 992 Molecular & Cellular Proteomics 12.4 Guidelines for Reporting MS Data that the scientific community can offer further comments and database developers would undoubtedly impose additional refinement. Similar to the MIAPE and MIAMI concepts, data submission requirements of their own. Conversely, jour- MIRAGE identifies specific metadata that significantly in- nals are likely to have less formal requirements for data sub- crease the value of the associated experimental data. The mission than those described in the MIRAGE guidelines. In MIRAGE guidelines will facilitate the collection of this informa- this context, this journal’s guidelines for the submission of tion, for example, by stimulating the development of computa- glycomics data serve as a good use case: many explicit tional methods to automatically extract this information using details regarding instrumental setup parameters are not re- software supplied by mass spectrometer manufacturers. quired, but the spirit of the MIRAGE guidelines is maintained. MIRAGE-MS Guidelines—The MIAPE committee has had a That is, the Journal requires information that will allow an major effect on proteomics analysis by addressing diverse expert to judge the quality of the results and to reproduce the aspects related to the preparation, analysis, and identification overall conclusions of the reported study. Although not fully of proteins. Despite the fact that similar methods and instru- and formally implemented by the Journal, the MIRAGE guide- ments are used in glycomics and that many instrument pa- lines can serve a critical role by prompting the analyst and the rameters are equally applicable to MS-based proteomics and reviewer to consider experimental parameters that have a glycomics, unique experimental requirements differentiate profound effect on the data and their interpretation. glycan analysis from protein analysis in several respects. The types of metadata recommended for reporting by the These differences are related to the very distinct nature of MIRAGE-MS guidelines are divided into five sections (Fig. 2). glycan structures, chemistry, and biosynthesis. One of the Overall, Sections 1–3 deal mainly with the instrumental hard- major differences is that glycans undergo vibrational dissoci- ware used to generate, fragment, and detect ions, whereas ation at lower energies than do peptides. For this reason, it is Sections 4 and 5 are focused on data interpretation and han- important to include information regarding the mass spec- dling issues. Section 1, “General Features,” serves as the basis trometer settings used for glycan ion analysis. This informa- for the required metadata, with global descriptions on the used tion should include the ion source, ion transfer, and ion iso- instrumentation, any particular customizations, and general instru- lation appropriate for glycan classes. The necessity of the ment control parameters such as instrument control software. specification of this information is emphasized by the fact that Section 2, “Ion Sources,” continues to summarize all crucial glycans may be analyzed in native or derivatized forms, in parameters for ion generation such as controls of in-source positive or negative ionization modes, as cation or anion fragmentation or the degree of prompt fragmentation, in ad- adducts, or as unattached/unlinked ions. The ability to extract dition to other, more common parameters (e.g. capillary volt- and interpret structural information from the data in a repro- age or laser intensity settings). Glycans contain several types ducible manner depends on the accurate communication of of labile bonds, including bonds to fucose and sialic acid information regarding sample and instrumental conditions. residues and to sulfate and phosphate substituents. It is thus Therefore, it is particularly important to include minimal infor- very important to determine whether biologically or chemically mation regarding the experimental conditions used for MS- significant ions observed in full scan mass spectra arise as a based glycomics. result of prompt fragmentation during the ionization process. The MIRAGE guidelines for mass spectral glycoanalysis are The extent of prompt fragmentation can be established by relevant to both database deposition and the submission of examining data obtained using purified standard glycans, results to a journal. However, the MIRAGE guidelines are which allows one to demonstrate that the mass spectrometer intended to be neither comprehensive nor absolute. In gen- is tuned properly for analysis of the glycan class in question. eral, the deposition of data to a database requires highly For example, if analyzing native N-glycans, one can show formal parameter descriptions because of the necessity of data obtained using a commercial sialylated N-glycan stand- controlling vocabulary, digital data formats, and other techni- ard to show that sialic acid residues are not lost during cal characteristics of the information. Nevertheless, restrictive ionization under the conditions used. Such data are impor- vocabularies and explicit digital data formats are beyond the tant in order for readers and database users to evaluate the current scope of the MIRAGE guidelines for mass spectral instrumental conditions under which data were acquired. data, as such requirements are best determined by the data- The MIRAGE-MS guidelines do not require that the experi- base developers. An example of the appropriate application mentalist perform an analysis of prompt fragmentation, but he of the MIRAGE guidelines for MS analysis would be the pop- or she must report whether such an analysis was done and, if ulation of a specialized database with the mass spectra of so, provide the resulting data. well-characterized standard molecules (i.e.“gold standard” Section 3, “Ion Transfer and Post-source Components,” spectra). The usefulness of such data collection would most asks for instrumental details associated with the transport, likely depend on compliance with the MIRAGE guidelines (e.g. gas phase reactions, and detection of ions once they are reporting of instrument setup parameters) so that users of the generated. This critical step is selective for the respective data could design experiments to obtain spectra that were instrument(s) and thus is more difficult to generalize; there- comparable to the standard spectra in the database. The fore, a clear separation into major detection and ion transport Molecular & Cellular Proteomics 12.4 993 Guidelines for Reporting MS Data FIG.2. The five major sections of the MIRAGE-MS guidelines. Sections 1–3 mainly deal with the instrumental hard- ware used to generate, fragment, and detect ions, and Sections 4 and 5 focus on data interpretation and handling issues. categories currently found on the market has been complex mixture. A similar state of affairs exists for glycomics introduced. experiments. Nevertheless, with certain limitations and cave- Section 4, “Spectrum and Peak List Generation and Anno- ats, glycomics analysis using single-stage MS can provide tation,” and Section 5, “Interpretation and Validation,” sum- data that are adequate to answer a specific experimental marize the crucial parameters that form the basis of the ana- question. Examples (e.g. modulation of the N-glycosylation of lytical results that are generated after spectra have been a very well-defined plant protein or control of the incorpo- recorded by the instrument. Detailed description of these ration/absence of particular sugar residues such as fucose) parameters is vital because, to date, robust, widely distributed clearly show that laborious in-depth experiments are not (and thus commonly used) tools for this step are not available. always required in order to answer a well-defined question Parameters such as software, software customizations, and (7, 8). databases (if used) are overviewed in these sections. Quan- Overall, these MIRAGE-MS guidelines summarize a list of titative aspects, which often play a considerable role in gly- instrumental and experimental parameters that are consid- comics experiments, are also considered within the guide- ered critical in describing MS-related conditions for the ac- lines, and a set of parameters judged to be crucial is listed in quisition and interpretation of glycoanalysis data. Based on Subsection 4.d. these MIRAGE-MS guidelines, the committee is now working Important aspects such as assignment validation and “de- on developing guidelines for various other techniques and duced structure(s)” are specifically listed in order to further approaches (including sample preparation methods) that are emphasize that the interpretation of many MS experiments is commonly used in glycomics analysis. The lack of adequate highly connected to the well-established (mammalian) glyco- and generally applicable software tools as utilized in pro- sylation pathways, and therefore particular structural details teomic research adds an additional challenge for glycoconju- are often inferred rather than confirmed via orthogonal tech- gate structural determination and structure reporting. niques. This constitutes one of the major differences existing CONCLUSIONS between MS-based glycomics and proteomics and is clearly reflected in the MIRAGE-MS guidelines. It is clear that mass The MIRAGE-MS guidelines have been proposed in order profiling (without the use of tandem MS) can provide valuable to encourage authors, editors, and reviewers to gather and information for both peptide and glycan samples. However, report all essential information describing a glycomics exper- mass profiling of complex samples generated during bot- iment that is being reported. The guidelines can be viewed in tom-up proteomics analysis can lead to unacceptably high their entirety in the supplementary material or at the project false discovery rates, given the statistical probability that un- web page. It is important to note that these guidelines are related peptides might have the same mass. Thus, tandem intended neither to dictate the use of particular methods MS sequence tags are often required in order for one to (which should be decided by the experimentalist) nor to serve confidently identify a large number of distinct proteins in a as a substitute for the review process. The goal of the guide- 994 Molecular & Cellular Proteomics 12.4 Guidelines for Reporting MS Data lines is to provide a summary of the information describing an R. J., Paulson, J. C., Sasisekharan, R., Varki, A. P., and Wong, C.-H.) (2012) Transforming Glycoscience: A Roadmap for the Future, The Na- MS experiment at a level that allows it to be understood, tional Academies Press Washington, D.C. evaluated, and reproduced. Furthermore, the guidelines pro- 2. Brazma, A., Hingamp, P., Quackenbush, J., Sherlock, G., Spellman, P., vide authors with a framework and standard for defining the Stoeckert, C., Aach, J., Ansorge, W., Ball, C. A., Causton, H. C., Gaaster- land, T., Glenisson, P., Holstege, F. C., Kim, I. F., Markowitz, V., Matese, depth of structural analysis that supports the structural mod- J. C., Parkinson, H., Robinson, A., Sarkans, U., Schulze-Kremer, S., els reported in the manuscript. This is important for enabling Stewart, J., Taylor, R., Vilo, J., and Vingron, M. (2001) Minimum infor- both expert glycoscientists and readers who are less familiar mation about a microarray experiment (MIAME)—toward standards for microarray data. Nat. Genet. 29, 365–371 in this area to understand the conclusions of the publication 3. Taylor, C. F., Paton, N. W., Lilley, K. S., Binz, P. A., Julian, R. K., Jr., Jones, based on a rigorous and comprehensive description of the A. R., Zhu, W., Apweiler, R., Aebersold, R., Deutsch, E. W., Dunn, M. J., materials and methods used and the results obtained. As the Heck, A. J., Leitner, A., Macht, M., Mann, M., Martens, L., Neubert, T. A., Patterson, S. D., Ping, P., Seymour, S. L., Souda, P., Tsugita, A., Vande- reader depends on the judgment of the reviewers to set high kerckhove, J., Vondriska, T. M., Whitelegge, J. P., Wilkins, M. R., Xe- standards for the publication of glycoanalytic data, this infor- narios, I., Yates, J. R., 3rd, and Hermjakob, H. (2007) The minimum mation is absolutely required in order for the reviewers to information about a proteomics experiment (MIAPE). Nat. Biotechnol. 25, 887–893 evaluate the results reported for each experiment described in 4. Apweiler, R., Cornish-Bowden, A., Hofmeyr, J.-H. S., Kettner, C., Leyh, the manuscript. T. S., Schomburg, D., and Tipton, K. T. (2005) The importance of uni- formity in reporting protein-function data. Trends Biochem. Sci. 30, * In 2011, the Beilstein-Institut, a non-profit foundation located in 11–12 Frankfurt am Main, Germany, assumed financial responsibility for 5. Taylor, C. F., Field, D., Sansone, S. A., Aerts, J., Apweiler, R., Ashburner, M., Ball, C. A., Binz, P. A., Bogue, M., Booth, T., Brazma, A., Brinkman, MIRAGE and the advancement of this standardization initiative. This R. R., Michael Clark, A., Deutsch, E. W., Fiehn, O., Fostel, J., Ghazal, P., work has also been supported by several other funding agencies. Gibson, F., Gray, T., Grimes, G., Hancock, J. M., Hardy, N. W., Hermja- These include the Max Planck Society (D. Kolarich and E. Rapp), the kob, H., Julian, R. K., Jr., Kane, M., Kettner, C., Kinsinger, C., Kolker, E., European Union’s Seventh Framework Programme (FP7-Health-F5– Kuiper, M., Le Nove` re, N., Leebens-Mack, J., Lewis, S. E., Lord, P., 2011) under Grant No. 278535 (E. Rapp), the Biotechnology and Mallon, A. M., Marthandan, N., Masuya, H., McNally, R., Mehrle, A., Biological Sciences Research Council (Grant Nos. SF19107, B19088, Morrison, N., Orchard, S., Quackenbush, J., Reecy, J. M., Robertson, and BBF008309/1 to S. Haslam), and the Australian government D. G., Rocca-Serra, P., Rodriguez, H., Rosenfelder, H., Santoyo-Lopez, through the project NeCTAR, financed by the Education Investment J., Scheuermann, R. H., Schober, D., Smith, B., Snape, J., Stoeckert, Fund (M. Campbell). The authors also acknowledge NIGMS (National C. J., Jr., Tipton, K., Sterk, P., Untergasser, A., Vandesompele, J., and Institutes of Health) Grant No. P41GM104603 (J. Zaia) and funding Wiemann, S. (2008) Promoting coherent minimum reporting guidelines for biological and biomedical investigations: the MIBBI project. Nat. received from the NIGMS-funded National Center for Glycomics and Biotechnol. 26, 889–896 Glycoproteomics (National Institutes of Health) (Grant No. 6. Workshop on Analytic and Bioinformatic Glycomics , April 16–18, 2009, 8P41GM103490 to M. J. Pierce and W. S. York). Bethesda, MD To whom correspondence should be addressed: William S. York, 7. Kolarich, D., and Altmann, F. (2000) N-Glycan analysis by matrix-assisted Tel.: 1 706 542 4628; FAX: 1 706 542 4412; E-mail: will@ laser desorption/ionization mass spectrometry of electrophoretically ccrc.uga.edu. separated nonmammalian proteins: application to peanut allergen Ara h 1 and olive pollen allergen Ole e 1. Anal. Biochem. 285, 64–75 REFERENCES 8. Rillahan, C. D., Antonopoulos, A., Lefort, C. T., Sonon, R., Azadi, P., Ley, K., 1. Committee on Assessing the Importance and Impact of Glycomics and Dell, A., Haslam, S. M., and Paulson, J. C. (2012) Global metabolic Glycosciences (Walt, D. A., Aoki-Kinoshita, A. F., Bendiak, B., Bertozzi, inhibitors of sialyl- and fucosyltransferases remodel the glycome. Nat. C. R., Boons, G.-J., Darvill, A., Hart, G., Kiessling, L. L., Lowe, J., Moon, Chem. Biol. 8, 661–668 Molecular & Cellular Proteomics 12.4 995

Journal

Molecular & Cellular ProteomicsAmerican Society for Biochemistry and Molecular Biology

Published: Apr 1, 2013

References