JOURNAL OF MASS SPECTROMETRY J. Mass Spectrom. 2007; 42: 428–439 Published online 3 January 2007 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/jms.1169 Differential fragmentation patterns of pectin oligogalacturonides observed by nanoelectrospray quadrupole ion-trap mass spectrometry using automated spectra interpretation Kudzai E. Mutenda, Rune Matthiesen and Peter Roepstorff * Department of Biochemistry and Molecular Biology, University of Southern Denmark Campusvej 55, DK-5230, Odense M, Denmark Received 3 February 2006; Accepted 26 November 2006 Oligogalacturonides of different degrees of polymerization (DP) and methyl esterification (DE) were structurally analyzed by nanoESI quadrupole ion-trap mass spectrometry. The fragmentation patterns of the oligogalacturonides were compared using the program ‘Virtual Expert Mass Spectrometrist’ (VEMS) for structural annotation. In the analyzed oligogalacturonides of lower DP, the generation of C/Y ions, i.e. ions retaining the glycosidic oxygen, was higher than that of B/Z ions. In general, with oligogalacturonides of higher DP, the B/Z ions were generated more abundantly. Oligogalacturonides with free carboxylic acid groups underwent higher water loss compared to fully methyl-esterified oligogalacturonides under the same fragmentation conditions. Cross-ring cleavage, in which fragmentation occurs across the ring system of the galacturonate residue and signified by unique mass losses, was observed to be higher in fully methyl-esterified oligogalacturonides than in non-methyl-esterified ones. This study demonstrates the different fragmentation patterns of oligogalacturonides as influenced by the presence or absence of methyl ester groups. For a detailed analysis of unknown oligogalacturonides, cross-ring fragmentation gives more structural information than glycosidic bond cleavage. One implication of this is that more structural information is obtained when analyzing methyl-esterified oligogalacturonides than non-methyl- esterified ones in an ion-trap instrument. This is of particular importance in pectin chemistry, where mass spectrometry has become the technique of choice for structural determination. Although this study was not designed to explain the mechanisms of oligogalacturonide fragmentation, possible explanations for why non-methyl-esterified oligogalacturonides undergo more water loss than methyl-esterified ones will be postulated. In addition, the VEMS program was extended to automatically interpret and assign the fragment ions peaks generated in this study. Copyright  2007 John Wiley & Sons, Ltd. KEYWORDS: ion-trap mass spectrometry; nanoelectrospray ionization; oligogalacturonides; pectin; VEMS INTRODUCTION Oligogalacturonides are oligomers of galacturonic acid residues. They are formed via a condensation reaction that links the anomeric hydroxy group of one galacturonic acid molecule to the hydroxy group of another. Oligogalactur- onides, as components of pectins (see below), are regularly analyzed by mass spectrometry. 1–5 Although other tech- niques are used for the characterization of oligogalactur- onides, MS has become the preferred technique of analysis. Pectins are anionic polysaccharides consisting of a core chain of ˛-(1,4)-linked D-galacturonic acid (GalA) residues. The carboxy groups on the GalA residues can either be methyl-esterified or free. The GalA residues can also be acetylated on O-2 and/or O-3. Branching side chains, L Correspondence to: Peter Roepstorff, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230, Odense M, Denmark. E-mail: roe@bmb.sdu.dk generally referred to as ‘hairy’ regions as opposed to the ‘smooth’ region, are present, and these consist of neutral sugars like L-rhamnose, and D-galacturonic acid. 6 The GalA backbone is synthesized in the cis Golgi, methyl-esterified in the medial Golgi, modified with side chains in the trans Golgi and exported to the cell wall. The most abundant pectins are homogalacturonans (HG), rhamnogalacturonan I (RG I) and RGII. Pectins also associate with cellulose to form the structural network in plant cell walls. Different chains of the polygalacturonate form a pectin network via Ca 2C bridges between GalA residues, as pro- posed in the ‘egg box’ model. 7 Esterification of the carboxy- late groups prevents cross-linking by calcium. This is an important phenomenon in pectin function. Esterification in pectins affects their mechanical properties. The degree of esterification (DE) in pectins determines their ability to form gels. The cell wall pH is also affected by the DE of the GalA residues in a pectin. Pectins of a high DE form flexible cell walls, as in elongating cells. Pectins of a low DE form stiffer Copyright  2007 John Wiley & Sons, Ltd.