There Are No Facts, Only Interpretations - Journal of Proteome

There Are No Facts, Only Interpretations. Nicolle H. Packer, and Niclas G. Karlsson. J. Proteome Res. , 2006, 5 (6), pp 1291–1292. DOI: 10.1021/ ...
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E ditor - in - chief

editorial

William S. Hancock

Barnett Institute and Department of Chemistry Northeastern University 360 Huntington Ave. 341 Mugar Bldg. Boston, MA 02115 617-373-4881; fax 617-373-2855 [email protected]

Associate E ditors Joshua LaBaer Harvard Medical School György Marko-Varga AstraZeneca and Lund University Cons u lting E ditor Jeremy Nicholson Imperial College London E ditorial adv isory board Ruedi H. Aebersold ETH Hönggerberg Leigh Anderson Plasma Proteome Institute Ettore Appella U.S. National Cancer Institute Rolf Apweiler European Bioinformatics Institute Ronald Beavis Manitoba Centre for Proteomics John J. M. Bergeron McGill University Richard Caprioli Vanderbilt University School of Medicine Christine Colvis U.S. National Institutes of Health R. Graham Cooks Purdue University Thomas E. Fehniger AstraZeneca Catherine Fenselau University of Maryland Daniel Figeys University of Ottawa Sam Hanash Fred Hutchinson Cancer Research Center Stanley Hefta Bristol-Myers Squibb Denis Hochstrasser University of Geneva Michael J. Hubbard University of Melbourne Donald F. Hunt University of Virginia Barry L. Karger Northeastern University Daniel C. Liebler Vanderbilt University School of Medicine Matthias Mann Max Planck Institute of Biochemistry David Muddiman North Carolina State University Robert F. Murphy Carnegie Mellon University Gilbert S. Omenn University of Michigan Aran Paulus Bio-Rad Laboratories Jasna Peter-Katalini´c University of Muenster Clifford H. Spiegelman Texas A&M University Ruth VanBogelen Pfizer Global Research & Development Peter Wagner Zyomyx Scot R. Weinberger GenNext Technologies Keith Williams Proteome Systems John R. Yates, III The Scripps Research Institute

© 2006 American Chemical Society

There Are No Facts, Only Interpretations

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riedrich Nietzsche did not coin the phrase “There are no facts, only interpretations” to refer to the analysis of the mass spectra obtained from the analysis of carbohydrates (!), but the quote does encapsulate a point that we would like to make. The interpretation of peptide mass data derived from the tryptic digestion of unknown proteins using published genomes enabled the proteomics “revolution” to occur. It has taken 10 years, however, for that interpretation to be standardized and for guidelines to be established for the publication of protein identities deduced from mass spectra (Wilkins, M. R.; et al. Proteomics 2006, 6, 4–8). Now, before the glycomics revolution begins, we have the unique opportunity to set some guidelines for the interpretation of mass spectrometric data acquired from the analysis of the oligosaccharides attached to glycoproteins. In proteomic MS, there is essentially a choice of 19 masses arranged in linear sequences; the assignment of a sequence to a particular peptide mass is enabled by knowledge of the genomic template. This is not the case in glycomic MS. Many different sugar compositions can correspond to the same mass, but there is no blueprint with which to distinguish among the possibilities. This is a consequence of having only seven main mass constituents of glycoprotein oligosaccharides: Hex (mannose, glucose), HexNAc (N-acetylglucosamine, N-acetylgalactosamine), dHex (fucose, rhamnose), NeuAc (N-acetyl neuraminic acid), NeuGc (N-glycolyl neuraminic acid), HexA (glucu­ ronic, galacturonic, and iduronic acids), and S (sulfate). The importance of these mass combinations to the interpretation of mass spectra becomes apparent when the residue masses of some oligosaccharide substructures and their adducts are compared in the table below.

Residue composition 1 Hex + Na – H Hex3 NeuAc + K – H Fuc2

Mass 184.03029 486.15399 329.04682 292.11134

Residue composition 2 dHex + K – H HexNAc2 + S NeuGc + Na – H NeuAc

Mass 184.00931 486.11108 329.06780 291.09094

To some extent, the accurate mass spectrometers being developed (e.g., FTICR instruments) may be able to differentiate these compositions. However, these instruments need to be carefully calibrated to give any confidence to this type of assignment, and they are not available to most laboratories for routine analysis. Other misinterpretations may occur due to the chemical conditions used to release oligosaccharides from proteins. For example, reductive b-elimination may result in incorrect compositional assignments because of the potential formation of sugar peptides from the reductive cleavage around the glycosylation site and the reduction of the peptide carboxyterminus, chemical degradation of oligosaccharides during the release, N-deacetylation (especially NeuAc), methylation (if removing borates with methanol), and/or acetylation (if removing borates with methanol and acetic acid). The addition of adducts in MS is another trap for young players because the difference between a sodium adduct (M + Na = M+23) and a potassium adduct (M + K = M+39) is 16, which is the same as the mass difference between NeuGc and NeuAc and between Hex and dHex. The use of 2,5-dihydroxybenzoic acid (DHB) as a matrix for MALDI MS could also make it hard to distinguish between sodiated DHB adducts [M + DHB + Na] = M+177 and hexuronic-containing oligosaccharides [M + HexA + H] = M+177. So why is this important? Obviously, in the first instance, the wrong sugar composition can be purported to exist on a protein. Secondly, even if the correct monosaccharide composition is determined, the 3D structure of the oligosaccharide that is presented on the protein will vary considerably depending on the way the sugars are linked together. As an example, two important blood group determinants have the same mass composition (Hex1HexNAc1dHex1), but they differ in their component monosacJournal of Proteome Research • Vol. 5, No. 6, 2006 1291

editorial

charides, sequence, and linkages. Blood group A is [GalNAca13(Fuca1-2)Galb1-], and blood group Lewis a is [Galb1-3(Fuca14)GlcNAcb1-]. More importantly, in the glycobiological context, they differ in the epitopes they present and in the function of the molecules to which they are attached. Too many publications, and conclusions, include complete sugar structures that have been assigned from the parent ion mass alone, on the basis of either what has been seen before or a best guess. Supposedly new and unusual structural compositions deduced solely from molecular mass data have been published, when the masses could have been explained equally well by other compositions and structures. As in protein anal-

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ysis, when mass fragmentation is used, current instruments have the capacity to identify sugar sequences and, in some cases, the monosaccharide linkage positions, and this type of data should be an essential prerequisite of any published oligosaccharide structural assignments. Whilst the fragmentation data will not always allow the full sequence of oligosaccharides to be deduced, it will, at the least, support the assigned mass composition. This will often be enough initial information for the determination of differentially expressed oligosaccharides in glycomics applications. Nicolle H. Packer and Niclas G. ­K arlsson Proteome Systems Ltd., Sydney, ­Australia