Selective chemical oxidation and depolymerization of (Panicum virgatum L.) xylan with switchgrass oligosaccharide product analysis by mass spectrometry † Michael J. Bowman 1 * , Bruce S. Dien 1 , Patricia J. O’Bryan 1 , Gautam Sarath 2 and Michael A. Cotta 1 1 USDA, Agricultural Research Service, National Center for Agricultural Utilization Research, Bioenergy Research Unit, 1815 North University Street, Peoria, IL 61604, USA 2 Grain, Forage, and Bioenergy Research Unit, Agricultural Research Service, U.S. Department of Agriculture, 314 Biochemistry Hall, UN‐L East Campus, Lincoln, NE 68583‐0737, USA Xylan is a barrier to enzymatic hydrolysis of plant cell walls. It is well accepted that the xylan layer needs to be removed to efficiently hydrolyze cellulose; consequently, pretreatment conditions are (in part) optimized for maximal xylan depolymerization or displacement. Xylan consists of a long chain of β‐1,4‐linked xylose units substituted with arabinose (typically α‐1,3‐linked in grasses) and glucuronic acid (α‐1,2‐linked). Xylan has been proposed to have a structural function in plants and therefore may play a role in determining biomass reactivity to pretreatment. It has been proposed that substitutions along xylan chains are not random and, based upon studies of pericarp xylan, are organized in domains that have specific structural functions. Analysis of intact xylan is problematic because of its chain length (> degree of polymerization (d.p.) 100) and heterogeneous side groups. Traditionally, enzymatic end‐point products have been characterized due to the limited products generated. Analysis of resultant arabino‐xylo‐oligosaccharides by mass spectrometry is complicated by the isobaric pentose sugars that primarily compose xylan. In this report, the variation in pentose ring structures was exploited for selective oxidation of the arabinofuranose primary alcohols followed by acid depolymerization to provide oligosaccharides with modified arabinose branches intact. Switchgrass samples were analyzed by hydrophilic interaction chromatography (HILIC)‐liquid chromatography (LC)‐mass spectrometry/mass spectrometry (MSMS) and off‐line nanospray MS to demonstrate the utility of this chemistry for determination of primary hydroxyl groups on oligosaccharide structures, with potential applications for determining the sequence of arabino‐xylo‐oligosaccharides present in plant cell wall material. Published in 2011 by John Wiley & Sons, Ltd. As focus on the conversion of plant biomass to liquid transportation fuels becomes more intense due to limited petroleum resources, an increased understanding of constit- uent cell wall polysaccharides from herbaceous energy crops will provide benefits for the efficient conversion to mono- saccharide components. Typical grasses are composed of approximately: cellulose (20–30 %), xylan (20–40 %), lignin (minor), and pectin (5 %) in the primary cell wall; and cellulose (35–45 %), xylan (40–50 %), lignin (20 %), and pectin (0.1 %) in the secondary cell wall. [1] Xylan is a heterogeneous biopolymer, consisting of repeating 1,4‐β‐linked xylose residues with potential acetyl or arabinose substitutions at either the 2‐O or 3‐O positions or both. Further complexity of the chains comes from additional substitutions (hexose, hexuronic acids, and/or phenolics). [2,3] It has been proposed that substitutions along xylan chains are not random and, based upon studies of wheat, [4,5] barley, [5] and rye [6] pericarps, are organized in domains that have specific structural functions. In contrast, the view that the xylan structure occurs in repeating units due to the enzyme complexes responsible for biosynthesis has also been expressed. [2,7,8] Xylan plays a role in determining the reactivity of switchgrass (SG) to pretreatments and final sugar yields. For example, when switchgrass is pretreated with dilute acid, the xylan component displays bimodal kinetics where the slow reacting component (20–35 %) largely determines the severity of the reaction conditions applied. [9] Determining structural features present in xylan from biofuel feedstocks will allow for targeted chemical and/or enzymatic approaches for effective saccharification. In addition to the potential benefits of xylan structural knowledge on biomass conversion, many other aspects of the grass life‐cycle including biomass production yield, plant development, pathogen resistance, and forage digestibility are likely influenced by xylan structure. The size and heterogeneity of cell wall polysaccharides make intact sequence analysis infeasible, if not impossible, * Correspondence to: M. J. Bowman, USDA, Agricultural Research Service, National Center for Agricultural Utilization Research, Bioenergy Research Unit, 1815 North University Street, Peoria, IL 61604, USA. E-mail: michael.bowman@ars.usda.gov † This article is a U.S. Government work and is in the public domain in the U.S.A. Published in 2011 by John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2011, 25, 941–950 Research Article Received: 12 November 2010 Revised: 20 January 2011 Accepted: 20 January 2011 Published online in Wiley Online Library Rapid Commun. Mass Spectrom. 2011, 25, 941–950 (wileyonlinelibrary.com) DOI: 10.1002/rcm.4949 941