Encoding substrates with mass tags to resolve stereospecific reactions using Nimzyme Kai Deng 1,2 , Kevin W. George 1 , Wolfgang Reindl 1,3 , Jay D. Keasling 1,4,5 , Paul D. Adams 1,5 , Taek Soon Lee 1 , Anup K. Singh 1,2 * and Trent R. Northen 1,3 * 1 Joint BioEnergy Institute, Physical Biosciences Division, Lawrence Berkeley National Laboratory, Emeryville, CA 94608, USA 2 Biotechnology and Bioengineering Department, Sandia National Laboratories, Livermore, CA 94551, USA 3 Department of Bioenergy/GTL & Structural Biology, Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94704, USA 4 Department of Chemical Engineering, University of California, Berkeley, CA 94720, USA 5 Department of Bioengineering, University of California, Berkeley, CA 94720, USA RATIONALE: The nanostructure-initiator mass spectrometry based enzyme assay (Nimzyme) provides a rapid method for screening glycan modifying reactions. However, this approach cannot resolve stereospecific reactions which are common in glycobiology and are typically assayed using lower-throughput methods (gas chromatography/mass spectrometry (GC/MS) or liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis) often in conjunction with stable isotopically labeled reactants. However, in many applications, library size necessitates the development of higher-throughput screening approaches of stereospecific reactions from crude sample preparations. Therefore, here we test the approach of utilizing Nimzyme linkers with unique masses to encode substrate identity such that this assay can resolve stereospecific reactions. METHODS: We utilize the nanostructure-initiator mass spectrometry (NIMS) enzyme assay in conjuction with an accurate mass tagging approach where each reactant is tagged with a unique perfluoronated tail. Mass spectrometric analysis was conducted using conventional MALDI-TOF instrumentation. RESULTS: Stereospecific reaction pathways of three stereoisomers (maltose, lactose and cellobiose) to afford the same product glucose were resolved simutaneously due to the presence of unique fluorous tags on both reactants and products. Not only purified enzymes, but also crude cell lysates can be used in this assay. CONCLUSIONS: The Nimzyme assay with accurate mass tagging provides a rapid method for screening for targeted stereospecific reactions using mass spectrometry and may be useful for high-throughput screening and functional annotation of a wide range of glycan-modifying enzymes. Copyright © 2012 John Wiley & Sons, Ltd. In recent years accurate mass tagging strategy has grown in popularity for the quantification of both small molecules [1] and biological molecules, particularly proteins. [2] For example, a mass tag is added to encode a peptide allowing multiple samples to be pooled and analyzed simultaneously to quantify relative expression of the peptide in various samples (e.g. iTRAQ [3] ). Previously, we reported that fluorous tags can be utilized to perform enzyme assays on perfluori- nated nanostructure-initiator mass spectrometry (NIMS) [4] surfaces to distinguish between products with different masses. [5] However, the highly degenerate mass of glycans (primarily hexoses and pentoses) and structural diversity due to glycosidic linkages, and range of branch points severely constrain application of Nimzyme to glycobiology. [6] Indeed, it is common to have two products with exactly the same mass but from different structures, for example, the beta-linked cellobiose vs. alpha-linked maltose. While tandem mass spectrometry (MS/MS) can be used to distinguish these substrates, [7] the products are identical and, hence, one cannot use glucose abundance to determine conversion of maltose vs. cellobiose. Clasically, stable isotopomers are used to differentiate stereoselective enzyme reactions [8] and enzyme inhibition. [9] More recently, isotope tagging of glycans has been applied for quantitative and comparative glycomis. For example, Bowman and Zaia [10] showed the generation of tags that vary only in their isotope content and the incorporation of these tags into glycans to study multiple samples using mass spectrometry. Withers and co-workers [11] developed an activity-based isotope-coded affinity tagging strategy for studying activities of glycosi- dases in biological systems. While similar approaches could be used to construct stable isotopically labeled Nimzyme substrates, a much more direct approach is to simply encode the idendity of the substrate using the unique mass of the fluorous linker. Here we describe the use of accurate mass tags to encode the identity of the substrate such that the mass spectrometry imaging (MSI) characterization for the product mixture can be used to simultaneously resolve the conversion of multiple glycoside hydrolase reactions resulting in the same product * Correspondence to: A. K. Singh or T. R. Northen, Joint BioEnergy Institute, Physical Biosciences Division, Lawrence Berkeley National Laboratory, Emeryville, CA 94608, USA. E-mail: aksingh@sandia.gov; trnorthen@lbl.gov Copyright © 2012 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2012, 26, 611–615 Research Article Received: 31 October 2011 Revised: 13 December 2011 Accepted: 18 December 2011 Published online in Wiley Online Library Rapid Commun. Mass Spectrom. 2012, 26, 611–615 (wileyonlinelibrary.com) DOI: 10.1002/rcm.6134 611