US 20120225797A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2012/0225797 A1 Northen et al. (43) Pub. Date: Sep. 6, 2012

(54) MULTIPLEXED SCREENING OF (22) Filed: Feb. 1, 2012 ACTIVITIES USING NANOSTRUCTURE-NITIATORMASS Related U.S. Application Data SPECTROMETRY (60) Provisional application No. 61/438,586, filed on Feb. 1, 2011. (75) Inventors: Trent R. Northen, Walnut Creek, CA (US); Wolfgang E. Reindl, Publication Classification Berkeley, CA (US); Kai Deng, (51) Int. C. Albany, CA (US); Seema Singh, C40B 30/08 (2006.01) Fremont, CA (US); Anup K. Singh, C40B 40/04 (2006.01) Danville, CA (US) (52) U.S. Cl...... 506/11:506/15 (73) Assignees: Sandia Corporation, Livermore, (57) ABSTRACT CA (US); The Regents of the Disclosed herein are methods, compositions and systems for University of California, Oakland, analyzing and detecting enzyme activity. For examples, CA (US) methods, compositions and systems for parallel detection and analysis of enzymatic activities of in complex bio (21) Appl. No.: 13/363,695 logical mixtures are provided.

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MULTIPLEXED SCREENING OF ENZYME head group. In some embodiments, the Sugar comprises cel ACTIVITIES USING lulose, , Xylose, cellobiose, cellotetraose, or NANOSTRUCTURE-NITIATORMASS xylobiose. SPECTROMETRY 0009. In some embodiments, the enzyme is selected from an endoglucanase, an exoglucanase, a glucosidase, a pecti CROSS-REFERENCE TO RELATED nase, a , and a hemicellulase. In some embodiments, APPLICATIONS the enzyme with plant degrading activity degrades 0001. This application claims priority under 35 U.S.C. . In some embodiments, the enzyme is a laccase or S119(e) to U.S. Provisional Patent Application No. 61/438, peroxidase. 586 filed Feb. 1, 2011. The content of this related application 0010. In some embodiments, analyzing the reaction prod is hereby expressly incorporated by reference in its entirety. uct by nanostructure-initiator mass spectrometry (NIMS) comprises applying the reaction product to a hydrophobic STATEMENT OF GOVERNMENT RIGHTS NIMS chip surface. In some embodiments, the hydrophobic NIMS chip surface comprises a perfluorinated coating. 0002 This invention was made with government support under Contract No. DE-AC02-05CH11231 awarded by the 0011. In some embodiments, the substrate comprises a U.S. Department of Energy. The government has certain Substrate head group linked to a perfluorinated tag that forms rights in the invention. This work is a collaboration between micelles under aqueous conditions. inventors at Lawrence Berkeley National Laboratory and 0012. In some embodiments, the substrate interacts with Sandia National Laboratories for the Joint BioEnergy Insti the NIMS chip surface via fluorous-phase-interactions. tute. 0013. In some embodiments, the sample is an isolated enzyme or a crude environmental sample. In some embodi FIELD OF THE INVENTION ments, the sample is obtained by incubating the crude envi ronmental sample with Switchgrass or cellulose. 0003. The present application relates to the fields of 0014. In some embodiments, the reaction products are microbiology, molecular biology, biofuel technology, and analyzed in parallel and wherein the reaction products are biomedicine. More specifically, the present application different in mass. In some embodiments, the reaction prod relates to methods, compositions and systems for analyzing ucts comprise Sugar molecules with identical mass and per and detecting enzymatic activities. fluorinated tags of different mass. In some embodiments, the reaction products comprise Sugar molecules of different mass BACKGROUND and perfluorinated tags of identical mass. 0004 Analysis of enzyme activity and identification of 0015. Some embodiments disclosed herein provide a mul Sources of enzyme activity are important in life Science tech tiplexed system for the activities of a plurality of enzymes, nologies. For instance, glycoside and comprising: (i) perfluorinated tagged Substrates for the plu (glycohydrolases and glycotransferases) play important roles rality of enzymes; and (ii) a nanostructure-initiator mass in a multitude of biological processes, both in eukaryotes spectrometry (NIMS) chip having a surface comprising a (e.g., processing of glycans in glycoproteins) and prokaryotes perfluorinated coat adapted to interact with the substrates via (e.g., utilization of Sugar polymers as carbon Source). How fluorous-phase-interactions. ever, current technology to identify and characterize sources 0016. In some embodiments, the substrates comprise a of these enzymes is limited in terms of the ability to resolve cellulose Substrate, a hemicellulase Substrate, aligninase Sub complex samples and evaluate large numbers of enzymatic strate, a lipase Substrate, a protease substrate, or a combina reaction products in parallel. There is a need for efficient tion thereof. In some embodiments, the Substrates are tagged methods capable of parallel detection and analysis of enzy with a heptadecafluoro-1,1,2,2-tetrahydrodecyl (F17) tag. matic activities of enzymes in complex biological mixtures. 0017. In some embodiments, the NIMS chip surface is coated with bis(heptadecafluoro-1,1,2,2-tetrahydrodecyl)tet SUMMARY ramethyl-disiloxane. 0005. The present application relates to methods, compo sitions, and systems for analyzing and detecting enzymatic BRIEF DESCRIPTION OF THE DRAWINGS activities using Substrate analogs and nanostructure-initiator mass spectrometry. 0018 FIG. 1 is a schematic illustration showing one 0006. Some embodiments disclosed herein provide a embodiment of a method for detecting enzymatic activity method of detecting the activities of a plurality of enzymes in wherein glycohydrolase activity in a sample is identified by a a multiplexed assay, where the method comprises: incubating multiplex glycan array. FIG. 1a is a schematic illustration a sample with substrates for the plurality of enzymes to obtain showing enzymatic reactions performed in a multiwall plate reaction products; analyzing the reaction products by nano in which a reaction well contains a Substrate including a Sugar structure-initiator mass spectrometry (NIMS); and detecting head group linked to a hydrophobic perfluorinated tag that activities of the plurality of enzymes in the sample by iden forms micelles under aqueous reaction conditions. FIG. 1b is tifying the ratio of Substrate-to-reaction product ions in a a schematic illustration of samples spotted onto a nanostruc mass spectrum. ture-initiator mass spectrometry (NIMS) chip after enzymatic 0007. In some embodiments, the plurality of enzymes cleavage. FIG. 1c is a schematic illustration showing vapor comprises an enzyme with plant cell wall degrading activity, ization of the initiator by laser irradiation, thereby transfer a lipase, or a protease. ring the applied samples into the gas phase. FIG. 1d is a 0008. In some embodiments, the enzyme with plant cell schematic showing ions of reaction products and uncleaved wall degrading activity reduces the chain length of a Sugar Substrates detected by mass spectrometry. US 2012/0225797 A1 Sep. 6, 2012

0019 FIG. 2 is an illustration of various exemplary sub tubes or microtiter plates and the reaction product is spotted strates that contain a Sugarhead group coupled to a perfluori onto a nanostructured, liquid-coated mass spectrometry chip nated heptadecafluoro-1,1,2,2-tetrahydrodecyl (F17) tag. Surface, and analyzed by Nanostructure-Initiator mass spec 0020 FIG. 3a is a small-angle neutron scattering curve trometry (NIMS) in a standard MALDI-TOF mass spectrom showing colloid formation of cellobiose (CB-F17). FIG.3b is eter. a graph showing the size distribution of the hydrodynamic 0033. The methods, compositions and systems described radius for cellobiose (CB-F17). herein can be used in diverse fields such as microbiology, 0021 FIG. 4a is a line graph showing the temperature development of biofuels, conversion of , and bio optima for B-glucosidase and B-Xylosidase. FIG. 4b is a line medical applications. For example, the methods, systems, graph showing the pH optima for B-glucosidase and B-Xylosi and compositions described herein can provide simultaneous dase. FIG. 4c is a line graph showing activity of B-glucosidase identification and characterization of the enzymatic activities and B-Xylosidase over time. FIG. 4d is a bar graph showing of multiple enzymes (e.g., glycoside hydrolases, lipases and competition between CB-F17 and unlabeled cellobiose for proteases) directly from crude environmental samples. conversion by B-glucosidase. Because enzymatic of into fer 0022 FIG. 5 is a mass spectrum showing signals for mentable Sugars by glycoside hydrolases is an important step xylose (FIG.5a), glucose (FIG.5b), xylobiose (FIG.5c), and in the conversion of biomass to lignocellulosic biofuels, the cellobiose (FIG. 5d). methods, systems and compositions described herein are use 0023 FIG. 6 is a bar graph showing B-glucosidase and ful for development of biofuels. B-Xylosidase activities in eight environmental samples. 0024 FIG. 7a is a bar graph showing activity Enzymes of Interest present in a Jepson Prairie compost sample under a range of pH condition. FIG.7b is a bar graph showing Xylanase activ 0034. Any enzyme having an activity that changes the ity present in a Jepson Prairie compost sample under a range mass of a Substrate can serve as an enzyme of interest in the of ionic liquid (IL) conditions. FIG. 7c is a bar graph showing embodiments described herein. Non-limiting examples of activity present in a Jepson Prairie compost sample enzymes of interest include transferases, hydrolases, , under a range of pH conditions. FIG. 7d is a bar graph show and . For example, the transferases can include glyco ing cellulase activity present in a Jepson Prairie compost Syltransferases, methyltransferase, acyltransferases Sul sample under a range of ionic liquid (IL) conditions. furtransferase, and Sulfotransferase. Examples of 0025 FIG. 8 is a bar graph showing activities of control include, but are not limited to, lipases, phosphatases, glyco enzymes on glucose, cellobiose, cellotriose and cellotetraose. side hydrolases, and proteases. 0026 FIG. 9 is a mass spectrum of cellotetraose-F17. 0035. In some embodiments, the enzyme of interest is 0027 FIG. 10 is a bar graph showing the influence of a involved in Sugar modification. For example, the enzyme of carbon Source on enzyme expression. interest can have an activity related to changing the chain 0028 FIGS.11a and 11b are mass spectra showing signals length of a Sugar head group. For example, enzymes of inter for lipase substrate F17008-Gly-Cys(palmitoyl)-Met-Gly est include enzymes, that cleave off one or more Sugar mono Leu-Pro-Art-OH and product F17008-Gly-Cys-Met-Gly mers (glycohydrolases) or enzymes that extend the Sugar Leu-Pro-Art-OH. head group by attaching one or more Sugar units (glycotrans 0029 FIGS.12a and 12b are mass spectra showing signals ferases). Examples of enzyme of interest include, but are not for protease substrate F17008-Ala-Pro-Arg-Thr-Pro-Gly limited to, glycohydrolases, glycotransferases, endogluca Gly-Arg-Arg-OH and product F17008-Ala-Pro. nases, exoglucanases, and hemicellulases. 0036. In some embodiments, the enzyme of interest is involved in degrading Sugar. Non-limiting examples of Sugar DETAILED DESCRIPTION degrading enzymes of interest include C-; B-amy 0030 The description that follows illustrates various lase; glucan 1,4-O-glucosidase; cellulose; endo-1.3(4)-3-glu embodiments of the subject matter disclosed herein. Those of canase; ; endo-1,4-B-Xylanase, oligo-1,6-glucosi skill in the art will recognize that there are numerous varia dase; ; ; ; ; tions and modifications of the subject matter provided herein exo-O-Sialidase; C-glucosidase; B-glucosidase; C-galactosi that are encompassed by its scope. Accordingly, the descrip dase; B-galactosidase; C-; 3-mannosidase; tion of certain embodiments should not be deemed to limit the B-fructofuranosidase; C-; 3-glucuronidase; Xylan Scope of the present application. endo-1,3-B-Xylosidase; amylo-1,6-glucosidase; hyalurono 0031. The present application relates to methods of detect glucosaminidase; ; Xylan 1,4-B-Xy ing enzyme activity in a sample. Activities of a plurality of losidase; B-D-; glucan endo-1,3-B-D-glucosidase; enzymes of interest in a sample can be detected by incubating B-L-rhamnosidase; ; GDP-glucosidase; B-L- a sample to be tested for enzymatic activity with substrates for rhamnosidase; ; ; galactosyl the plurality of enzymes of interest to obtain reaction prod ceramidase; galactosylgalactosylglucosylceramidase; ucts, and analyzing the reaction products by nanostructure Sucrose B-glucosidase; Cl-N-acetylgalactosaminidase; Cl-N- initiator mass spectrometry (NIMS). The ratio of substrate acetylglucosaminidase; C-L-fucosidase; B-L-N-acetylhex to-reaction productions in the mass spectrum can be analyzed osaminidase; B-N-acetylgalactosaminidase; cyclomaltodext to determine the activity of one or more enzymes of the rinase; Cl-N-arabinofuranosidase; glucuronosyl plurality of enzymes in the sample, and thus determine the disulfoglucosamine glucuronidase; isopululanase; glucan presence of one or more enzymes of interest in the sample. 1.3-3-glucosidase; glucan endo-1,3-O-glucosidase; glucan 0032. In some embodiments, glycan Substrate analogs are 1,4-O-maltotetraohydrolase; ; glycosylcera used for the analysis of glycohydrolase or glycotransferase midase; 1.2-O-L-fucosidase; 2,6-B-fructan 6-levanbiohydro activities. In various aspects, enzymatic reactions using lase; ; quercitrinase; galacturan 1,4-O-galactur amphiphilic Substrates can be carried out, for example, intest onidase; ; glucan 1,6-O-glucosidase; glucan endo US 2012/0225797 A1 Sep. 6, 2012

1.2-f-glucosidase; Xylan 1.3-3-xylosidase; licheninase; enzymes known as hemicellulases. Lignin gets broken down glucan 1.4-3-glucosidase; glucan endo-1,6-B-glucosidase; by “ligninases, e.g. laccases or lignin peroxidases. Some L-; mannan 1.2-(1,3)-O-mannosidase; mannan embodiments disclosed herein relate to detection of these endo-1,4-B-mannosidase; fructan B-fructosidase; : enzymes involved in degrading or breaking down plant cell exo-poly-C-galacturonosidase: K-carrageenase; glucan 1,3- wall. f3-glucosidase; 6-phospho-?3-galactosidase; 6-phospho-B- 0038. In several embodiments, the enzyme(s) of interest glucosidase; capsular- endo-1,3-O-galactosi include cellulase, which includes but is not limited to endo dase; f-L-arabinosidase; arabinogalactan endo-1,4-B- (endocellulases) for example, endo-1,4-beta-glu galactosidase; cellulose 1,4-B-cellobiosidase; peptidoglycan canase, carboxymethyl cellulase (CMCase), endo-1,4-beta f-N-acetylmuramidase; O-phosphotrehalase; glucan 1,6-O- D-, beta-1,4-glucanase, beta-1,4-endoglucan isomaltosidase; dextran 1,6-O-isomaltotriosidase; mannosyl hydrolase, and celludextrinase; exoglucanases (exocellu glycoprotein endo-B-N-acetylglucosaminidase; glycopep lases); and beta-. In several embodiments, the tide Cl-N-acetylgalactosaminidase; glucan 1,4-O- enzymes of interest include cellulase enzymes identified by maltohexaosidase; arabinan 1.5-O-L-arabinosidase; mannan the EC 3.2.1.4, which is herein 1,4-mannobiosidase; mannan endo-1,6-O-mannosidase; fully incorporated by reference in its entirety for examples of blood-group-substance endo-1,4-B-galactosidase; keratan enzymes contemplated herein. Sulfate endo-1,4-?3-galactosidase; steryl-?3-glucosidase; stric 0039. In some embodiments, the enzyme(s) of interest tosidine f-glucosidase; mannosyl- glucosi include ligninase, which includes but is not limited to lignin dase; protein-glucosylgalactosylhydroxylysine glucosidase; peroxidase, manganese peroxidase, laccase, and cellobiose ; endogalactosaminidase; mucinaminylserine muci dehydrogenase. In some embodiments, the enzymes of inter naminidase; 1.3-O-L-fucosidase; 2-deoxyglucosidase; man est include ligninase enzymes identified by the Enzyme Com nosyl-oligosaccharide 1.2-O-mannosidase; mannosyl-oli mission number EC 1.14.99, which is herein fully incorpo gosaccharide 1,3-1,6-O-mannosidase; branched-dextran exo rated by reference in its entirety for examples of enzymes 1.2-O-glucosidase; glucan 1.4-O-maltotriohydrolase; contemplated herein. amygdalin f3-glucosidase; prunasin B-glucosidase; vicianin 10040. In some embodiments, the enzyme of interest is f3-glucosidase; oligoxyloglucan f3-glycosidase; polymannur involved in catalyzing the formation or hydrolysis of lipids. onate hydrolase; maltose-6'-phosphate glucosidase; endogly Examples of lipase include, but are not limited to, Lipase cosylceramidase; 3-deoxy-2-octulosonidase; raucaffricine AP4, Lipase AP6, Lipase F-AP15, Lipase OF, Lipase AP12, f-glucosidase; coniferin B-glucosidase; 1,6-O-L-fucosidase; Lipase M-AP5, Lipase M-AP10, Lipase M-AP20, Lipase glycyrrhizinate f3-glucuronidase; endo-C.-sialidase; glyco Saiken, Lipase PS, Lipase MY, and Lipase B. protein endo-C-1.2-mannosidase, Xylan C-1,2-glucuronosi I0041. In some embodiments, the enzyme of interest is dase; ; glucan 1.4-O-maltohydrolase; difructose involved in conducting proteolysis. For example, the enzyme anhydride synthase; ; glucuronoarabinoxylan of interest can be a protease, such as serine protease, a threo endo-1,4-B-Xylanase; mannan exo-1,2-1,6-B-mannosidase; nine protease, cysteine protease, an aspartate protease, a met C-glucuronidase; lacto-N-biosidase: 4-O-D-(1->4)-C-D- alloprotease, and a glutamic acid protease. glucano trehalose trehalohydrolase; dextrinase; poly(ADP ribose) glycohydrolase; 3-deoxyoctulosonase; galactan 1,3- Enzyme Substrates and Substrate Analogs B-galactosidase; B-galactofuranosidase; thioglucosidase; 0042. In some embodiments, enzyme substrates are linked f-primeverosidase; oligoxyloglucan reducing-end-specific to tags that interact with a surface of a NIMS chip. As used cellobiohydrolase; Xyloglucan-specific endo-?3-1,4-gluca herein, the terms "substrate(s)" and “substrate analog(s) are nase; mannosylglycoprotein endo-B-mannosidase; fructan used interchangeably and generally refer to a substrate head f-(2,1)-fructosidase; fructan B-(2,6)-fructosidase; oligosac group linked to a tag. In some embodiments, substrates or charide reducing-end xylanase; and the like. Substrate analogs include a sugar head group linked to a 0037. Some embodiments are related to detecting activity hydrophobic tag, which is capable of interaction with a of an enzyme involved in degrading plant cell wall material. hydrophobic NIMS chip surface. For example, substrates can For example, glycoside hydrolases are important for the include a sugar head group linked to a perfluorinated tag that development of biofuels from lignocellulosic biomass: long interacts with a NIMS chip surface having a perfluorinated chain polysaccharides from plant cell walls are enzymatically coat. Head groups can be sugar monomers, oligomers, or hydrolyzed and the resulting Sugar monomers are fermented (branched) multimers. In other words, head groups can into ethanol or advanced biofuels. Three major components include , , polysaccharides, forming plant cell walls that are deconstructed include the and . Examples of monosaccharides, disac polysaccharides cellulose and hemicellulose, and the highly charides, polysaccharides, and oligosaccharides that can be phenolic macromolecule lignin. Cellulose is comprised of used as head groups in substrate analogs are known in the art. linear chains off-1.4-linked D-glucose units, while hemicel In several embodiments, substrates or substrate analogs can lulose consists mainly of mixtures of pentoses with D-xylose be phenolic substrates degradable by ligninases. Further and D-arabinose being the most abundant. Cellulose is hydro more, a wide range of initiators including but not limited to lyzed into glucose through the concerted action of at least lauric acid, polysiloxanes, chlorosilanes, methoxy and ethy three known classes of enzymes collectively referred to as oxy silanes, fluorous siloxanes, and silanes can be used for cellulase: endoglucanases, exoglucanases, and f-glucosi NIMS chip surface coating. dases. Without being bound by theory, endoglucanases ran 0043 Sugars modified by any of the aforementioned domly produce free ends from cellulose fibrils that are further enzymes can be linked to a hydrophobic tag and used as degraded by exoglucanases that release cellobiose, which in Substrate analogs in various embodiments of the enzyme turn is hydrolyzed by f6-glucosidases into glucose. Hemicel activity detection methods described herein. In some embodi luloses are degraded by a complex class of multi-domain ments, substrate analogs are based on hexoses, such as cello US 2012/0225797 A1 Sep. 6, 2012 biose and cellotetraose, or pentoses, such as Xylobiose. Other fore, the enzymatic reactions can be performed using the Sugars that can be used as Substrate analogs include, without amphiphilic Substrate analogs in standard reaction tubes or limitation, glucose, fructose, galactose, mannose, maltose, plates where all reaction conditions can easily be controlled. Sucrose, lactose, arabinose, Xylose, and rhamnose. 0044. In some embodiments, substrates can include a lipid Enzymatic Reactions head group linked to a hydrophobic tag, which is capable of 0049. Enzymatic reactions can be carried out in solution interaction with a hydrophobic NIMS chip surface. For using test tubes or microwell microtiter plates (96-well, 384 example, Substrates can include a lipid head group linked to a well). Enzymatic reactions can be performed using the perfluorinated tag that interacts with a NIMS chip surface amphiphilic Substrate analogs in standard reaction tubes or having a perfluorinated coat. Head groups can, for example, plates where all reaction conditions can easily be controlled. include lipidic groups or fatty acidic groups. Non-limiting Various embodiments described herein involve a solution examples of lipidic group group include oleoyl group, based assay system that can be applied to all kinds of standard Stearoyl group, palmitoyl group, lauroyl group, myristoyl reaction tubes and microtiter plates. Furthermore, all liquid group, arachidoyl group, behenoyl group, lignoceoyl group. handling and sample spotting can be interfaced with existing and the like. In some embodiments, the head group comprises pipetting robots and liquid handling systems, so that several a lipidic group. In some embodiments, the head group com described embodiments are highly suitable for high-through prises a fatty acidic group. In some embodiments, the head put applications. group comprises a palmitoyl group. 0050. After completing an enzymatic reaction under the 0045. In some embodiments, substrates can include a desired conditions, reactions can be quenched, e.g., with polypeptide head group linked to a hydrophobic tag, which is methanol which denatures all enzymes. For analysis of the capable of interaction with a hydrophobic NIMS chip surface. occurring enzymatic reactions, Small samples Volumes (one For example, Substrates can include a polypeptide head group microliter and below) can be spotted onto the mass spectrom linked to a perfluorinated tag that interacts with a NIMS chip etry chip surface. The nanostructured chip can be coated with Surface having a perfluorinated coat. Head groups can be, for ultrathin liquid layers of perfluorinated (di)siloxanes. The example, a polypeptide. The length of the polypeptide can fluorous tails of the used amphiphilic Substrates can interact vary. For example, the polypeptide can have about 1, about 2, with this Surface via fluorous-phase-interactions, so that Sub about 3, about 4, about 5, about 6, about 7, about 8, about 9, strates are driven downto the chip surface when the sample is about 10, about 15, about 20, or more amino acids. In some spotted. In a "chromatographic' step, all other components embodiments, the head group has about 9 amino acids. In of the reaction sample do not interact with the surface and can Some embodiments, the head group is a polypeptide compris be washed away, or simply be pipetted off again, while the ing one or more Arginine. Substrates Stick to the Surface. Analysis of enzymatic activi 0046. A fluorous tag of a substrate analog can be formed ties in the mass spectrometer can be performed based on the by several perfluorinated chemical structures, e.g., aliphatic ratios of Substrate-to-product ions, which is advantageous carbon chains, phenyl rings, etc. For example, fluorinated because it is independent from variations of total intensities aliphatic molecules. Such as (heptadecafluoro-1,1,2,2-tet on various sites on the chip. rahydrodecyl)-dimethylchlorosilane (“F17) and bis(tride cafluoro-1,1,2,2-tetrahydrooctyldimethylsiloxy)-methyl Samples chloro-silane (“F26’), can be used in synthesis of a fluorous tagged Substrate. In some embodiments, Substrate analogs are 0051 Methods described herein are not limited to detect tagged with a heptadecafluoro-1,1,2,2-tetrahydrodecyl (F17) ing activity of purified enzymes, but also include detecting tag. enzyme activity in crude samples like cells, cellular lysates, 0047. The fluorous tag of a substrate analog can be sepa cellular extracts, and environmental samples. For example, rated from the substrate head group by a linker. Without being microbial communities (e.g., fungi or ) capable of bound by any particular theory, it is believed that the linker growing on lignocellulose have gained increasing attention as can facilitate the accessibility of the Substrate head group. Sources for discovering glycoside hydrolases. As such, sev The length of the linker can vary. For example, the linker can eral embodiments relate to detecting enzyme activities in be a 2-carbon, 3-carbon, 4-carbon, 5-carbon, 6-carbon, 7-car samples Suspected of containing Such microbial communi bon, 8-carbon, 9-carbon, 10-carbon, 15-carbon or 20 carbon ties. Non-limiting examples of samples that can be assayed in group. In some embodiments, the linker comprises a five several embodiments described herein include plant matter, carbon group. Such as 5 methylene group. In some embodi wood, leaves, paper waste, soil, compost, agriculture waste ments, the linker comprises an ionization enhancer. As used (e.g. livestock waste), mulch, dirt, clay, and garbage. herein, the term "ionization enhancer” refers to a chemical 0052. In some embodiments, samples are cultivated and group that can enhance ionization of the Substrate analog extracted for analysis of enzymatic activity using standard during analysis of the enzyme(s) of interest. Non-limiting techniques available in the field. For example, environmental examples of the ionization enhancer include arginine and samples can be inoculated and grown in liquid cultures con dimethyl-arginine. taining a biomass feedstock Such as Switchgrass. The Super 0048. In some embodiments, substrate analogs are natants of the liquid cultures can be collected for analysis and amphiphilic and thus soluble in water or aqueous buffers. detection of enzyme activity as described herein. Without being bound by any particular theory, dissolving the Substrate analogs is possible because they form Supramolecu Nanostructure-Initiator Mass Spectrometry lar amphiphilic assemblies (e.g., micelles, liposomes, 0053. In some embodiments, the mass of the reaction vesicles, colloids, etc.), where the hydrophobic fluorous tail is product generated by incubating a sample or enzyme with a 'shielded from the aqueous solution, whereas Sugar head Substrate can be determined by nanostructure-initiator mass groups remain accessible for the enzymes’ active sites. There spectrometry (NIMS). NIMS is described in T. R. Northen, O. US 2012/0225797 A1 Sep. 6, 2012

Yanes, M. T. Northen, D. Marrinucci, W. Uritboonthai, J. enzyme negatively correlates with the ratio of Substrate-to Apon, S. L. Golledge, A. Nordstrom, G. Siuzdak, Nature reaction product ions. As described above, NIMS is 2007, 449, 1033-1036: T. R. Northen, J. C. Lee, L. Hoang, J. described, for example, in US Patent Publication Raymond, D. R. Hwang, S. M. Yannone, C. H. Wong, G. 2008.0128608, which is hereby expressly incorporated by Siuzdak, Proc. Natl. Acad. Sci. USA 2008, 105, 3678-3683; reference. and U.S. Patent Application Publication No. 2008/0128608, 0059. In some embodiments, applying the reaction prod which are hereinfully incorporated by reference. Production uct can be applied to a hydrophobic NIMS chip surface for of NIMS chips is described in detail in H. K. Woo, T. R. analyzing the reaction product. The hydrophobic NIMS chip Northen, O. Yanes, G. Siuzdak, Nat. Protoc. 2008, 3, 1341 Surface can, for example, include a perfluorinated coating. In 1349, which is herein fully incorporated by reference. The Some embodiments, the Substrate comprises a Substrate head ratio of Substrate-to-reaction productions in the mass spec group linked to a perfluorinated tag that forms micelles under trum can be analyzed to determine the presence of the enzyme aqueous conditions. In some embodiments, the Substrate of interest in the sample. interacts with the NIMS chip surface via fluorous-phase 0054) A variety of apparatuses can be used in NIMS to interactions. measure the mass-to-charge ratio of the ionized target. For 0060. The methods disclosed hereincan, in some embodi example, in several embodiments a time-of-flight mass ana ments, analyze the reaction products in parallel. In some lyZer is used for measuring the desorbed and ionized target. embodiments, the reaction products are different in mass. In However, other non-limiting examples of mass analyzers that Some embodiments, the reaction produces comprise Sugar can be used include magnetic ion cyclotron resonance instru molecules with identical mass and perfluorinated tags of dif ments, deflection instruments, and quadrupole mass analyZ ferent mass. In some embodiments, the reaction products CS. comprise Sugar molecules of different mass and perfluori 0055 Thus, the methods and apparatuses described herein nated tags of identical mass. In some embodiments, the reac permit parallel detection of large numbers of reaction prod tion products comprise amino acid molecules with identical ucts based on the Substrates and reaction products differing in mass and perfluorinated tags of different mass. In some mass. Some embodiments disclosed herein relate to enzyme embodiments, the reaction products comprise amino acid activity detection involving parallel use of pentose and hex molecules of different mass and perfluorinated tags of iden ose-based substrates. Substrate analogs including head tical mass. groups with identical masses can be used by linking them to tags of different mass. For example, polysaccharides with Systems for Detecting Plant Cell Wall Degrading Enzymatic identical masses (e.g., cellobiose and maltose) can be ana Activity lyzed in parallel by using different chemical structures in the 0061 Some embodiments are drawn to systems for detect linker or tags of varying length. Accordingly, some embodi ing plant cell wall degrading enzymatic activity. A system for ments described herein allow simultaneous testing of a plu detecting plant cell wall degrading enzymatic activity can rality of Samples and enzymatic reactions without analytical include any of the above-described substrates and NIMS interference. chips. For example, a system for detecting plant cell wall 0056. The above description discloses several methods degrading enzyme activity can include a tagged Xylobiose, and materials of the present invention. This invention is sus cellobiose, or cellotetraose Substrate, or combinations ceptible to modifications that will become apparent to those thereof, and a NIMS chip having a surface which includes a skilled in the art from a consideration of this disclosure or tag capable of interacting with the Substrate. In some embodi practice of the invention disclosed herein. Consequently, it is ments, the substrate(s) have a fluorous tag and the NIMS chip not intended that this invention be limited to the specific surface is coated with a fluorous initiator that is capable of embodiments disclosed herein, but that it cover all modifica interacting with the tag. For example, a system for detecting tions and alternatives coming within the true scope and spirit plant cell wall degrading activity can include a Substrate. Such of the invention. All references cited herein are incorporated as but not limited to xylobiose, cellobiose, cellotetraose, or by reference in their entirety and are hereby made a part of combinations thereof tagged with a heptadecafluoro-1.1.2.2- this specification. tetrahydrodecyl (F17) tag and a NIMS chip having a surface coated with bis(heptadecafluoro-1,1,2,2-tetrahydrodecyl)tet Methods for Detecting Enzymatic Activity ramethyl-disiloxane. In some embodiments, systems for 0057. Some embodiments disclosed herein provide a detecting plant cell wall degrading enzymatic activity further method of detecting the activities of a plurality of enzymes in include a sample such as a crude environmental sample Sus a multiplexed assay. The method, in some embodiments, pected of having the enzyme activity of interest. allows simultaneous detection of the activities of multiple enzymes. For example, the activities of two, three, four, or EXAMPLES more enzymes may be detected simultaneously by the 0062 Having generally described embodiments of the method. present application, a further understanding can be obtained 0.058. In some embodiments, the method includes incubat by reference to certain specific examples which are provided ing a sample with Substrates for the plurality of enzymes to herein for purposes of illustration only, and are not intended obtain reaction products, analyzing the reaction products by to be limiting. nanostructure-initiator mass spectrometry (NIMS); and detecting activities of the plurality of enzymes in the sample Example 1 by identifying the ratio of Substrate-to-reaction productions in a mass spectrum. The activity of the enzyme, in some 0063. Self-assembled colloids and NIMS for the multi embodiments, correlate with the ratio of substrate-to-reaction plexed identification and characterization of glycoside hydro product ions. In some embodiments, the activity of the lase activities are described in this Example. US 2012/0225797 A1 Sep. 6, 2012

0.064 FIG. 1 shows a schematic illustration of one emission patterns that can be resolved separately. In contrast, embodiment of a method for detecting enzymatic activity, mass spectrometry can resolve thousands of ions and there wherein glycohydrolase activity in a sample is identified by a fore we have discovered that it is well-suited for the parallel multiplex glycan array. As illustrated in FIG. 1a, enzymatic detection of large numbers of reaction products where the reactions can be performed in a multiwall plate in which a Substrates and occurring products differ in mass. This is the reaction well contains a Substrate including a Sugar head case, for example, when using pentose and hexose-based group linked to a hydrophobic perfluorinated tag that forms Substrates at the same time. The hydrolysis of polysaccha micelles under aqueous reaction conditions. The samples can rides with identical masses (e.g. cellobiose and maltose) can then be spotted onto a nanostructure-initiator mass spectrom even be analyzed in the same run by using different chemical etry (NIMS) chip after enzymatic cleavage (FIG. 1b). As structures in the linker or perfluorinated tags of varying shown in FIG. 1c, the initiator can be vaporized by laser length. Thus, potentially dozens of enzymatic conversions irradiation, thereby transferring the applied samples into the can be tested simultaneously using this method. Here both gas phase. And then, ions of reaction products and uncleaved B-glucosidase and B-Xylosidase activities were detected substrates can be detected by mass spectrometry (FIG. 1d). simultaneously utilizing a Substrate cocktail comprised of 0065 Assays were performed with amphiphilic substrate equal amounts of CB-F17 and XB-F17. In the non-enzyme analogs comprising a polar Sugar head group and a highly control only signals for cellobiose and xylobiose were detect hydrophobic perfluorinated tail (F17) (FIG. 2). The sugar able (FIG.5a). Incubation of the two substrates with a B-glu groups used in this study were based on hexoses, such as canase/Xylanase enzyme cocktail lead to complete cleavage cellobiose (CB) and cellotetraose (CT), or pentoses, such as of both (FIG. 5b). After incubation with B-glucosidase, cel xylobiose (XB). Due to their amphiphilic character, sub lobiose was almost completely converted to glucose, while strates spontaneously formed colloids, as identified for CB there was only a slight increase in the Xylose signal, probably F17 by small-angle neutron scattering (SANS) and dynamic due to side reactions of the enzyme (FIG. 5c). For B-xylosi light scattering (DLS). As shown in FIG. 3a-b, both SANS dase there was conversion of Xylobiose to Xylose and no and DLS techniques showed a consistent radius for the enzymatic cleavage of cellobiose (FIG. 5d). These data dem detected main particles: 27.5 A (polydispersity 14%) for onstrate the applicability of this method for the analysis of SANS and 30.0 A (polydispersity 12%) for DLS. Addition several activities in parallel. ally, SANS measurements revealed a spherical shape for the particles (FIG.3a). With an estimated molecule size of -24. A Enzyme Activity Assays for CB-F17, the recorded diameters of -55-60 A and the spherical shape Suggest the formation of micelles (FIG. (0068. The B-glucosidase (NS50010) and the B-glucanase/ 3a-b). Thus, despite containing a large hydrophobic moiety, Xylanase mixture (NS22002) were part of the Biomass Kit Substrates could be dissolved in aqueous solutions, allowing from Novozymes (Davis, Calif.). 1,4-B-D-xylosidase from enzymatic reactions to be performed in standard buffers and Bacillus pumilus was purchased from MegaZyme (Wicklow, labware. After enzymatic cleavage, Samples could be spotted Ireland). Two different expression versions of the exogluca directly onto a NIMS surface without any further sample nase Cs GH5 from Caldicellulosiruptor saccharolyticus, the preparation steps, even if they contained crude environmental endoglucanases Pr GH5 from Prevotella ruminicola, Cel5A extracts with visible particles. This is due to the fact that the from Thermotoga maritima, and Cel9A from Alicyclobacil substrates selectively bind to the surface via fluorous-phase lus acidocaldarius were kindly provided by Joshua Park and interactions, while other sample components (i.e. enzymes, Supratim Datta (Joint BioEnergy Institute, Emeryville, cleaved off Sugar units, salts) can be washed away in situ, Calif., USA). An additional undisclosed B-glucosidase which enhances signal intensities. Therefore, the assay sys (UBG) was also included. tem comprises the benefits of sample purification, while requiring only a minimal fraction of time in comparison to Separate Analysis of B-Glucosidase and B-Xylosidase Activ standard chromatography techniques. Once spotted onto the ity NIMS surface, samples were stable for several weeks. 0069. The separate characterization of B-glucosidase and 0066. The next step was the analysis by NIMS in a B-Xylosidase activity was carried out in reaction Volumes of MALDI mass spectrometer, where laser irradiation leads to 20-50 uL, by mixing 0.1 mM cellobiose with 8 ug of vaporization of the initiator liquid on the chip Surface and NS50010 in 50 mM sodium acetate buffer (pH 4.8), or 0.1 Subsequent transfer of applied samples into the gas phase. mM xylobiose with 0.13 Lug (in 20 mL) or 0.33 Lug (in 50 uL) Acquired spectra show pairs of Substrate and product signals, of 1,4-B-D-xylosidase in 50 mM potassium phosphate buffer Such that enzymatic activities can be measured as product-to (pH 7.5) with 1 mg/mL BSA. For the competition studies with substrate ratios, which is completely independent from total cellobiose, samples additionally contained 10, 25, or 50 mM signal intensities. This method was validated by analyzing of unlabeled cellobiose (Sigma-Aldrich: St. Louis, Mo.). commercially available B-glucosidase and B-Xylosidase Other buffers used for the determination of the pH optima activities using cellobiose (CB-F17) or xylobiose (XB-F17) were McIlvaine's citrate/phosphate buffer (pH 2.6 and 6.0), as Substrates in separate setups (FIG. 4): determined tempera 50 mM Tris buffer (pH 9.0) for NS50010, and 50 mM potas ture and pH optima for both enzymes match the known reac sium phosphate buffer (pH 6.0), 50 mM Tris buffer (pH 9.0), tion optima for these enzymes as specified by the manufac 50 mM sodium borate buffer (pH 11.0), all with 1 mg/mL turers; additionally, kinetic and competition studies could be BSA for the B-xylosidase. Samples were incubated for 1 h at performed. 35° C. (B-xylosidase) or 50° C. (NS50010), for determination 0067. It can be difficult to discriminate between several oftemperature optima Samples were additionally incubated at products in simultaneous reactions using conventional spec the given temperatures. Reactions were quenched by adding troscopic methods since these reactions typically yield the one sample Volume of ice-cold methanol. For monitoring the same outputs and there are only a few unique absorption or enzymatic reactions over time, Small aliquots were taken out US 2012/0225797 A1 Sep. 6, 2012

ofa bigger reaction Volume at the given time points, quenched clear Substrate conversion for both targeted enzymatic activi by adding one aliquot Volume of methanol, and kept on ice ties (FIG. 6). The Jepson Prairie (JP) compost sample was the until further analysis. most effective in terms of overall liberation of monomeric sugars with 87.1%+2.1% cellobiose and 92.3%+0.4% xylo Multiplexed f-Glucosidase/B-Xylosidase Assay biose conversion. 0070 For the simultaneous analysis of B-glucosidase and (0073. For a detailed multiplexed characterization of the JP B-xylosidase activity a mixture of 0.1 mM cellobiose and 0.1 secretome, the extract was incubated with a mixture of XB mM xylobiose was incubated with 8 ug NS50010 in 50 mM F17 and CT-F17 for 30 min at 50° C. or 80° C. at pH 3, 5, 7, sodium acetate buffer (pH 4.8), with 6.5ug B-xylosidase in 50 and 9, and in the presence of 0%, 10%, 20%, and 30% ionic mM potassium phosphate buffer (pH 7.5) with 1 mg/mL liquid (IL; ethyl-3-methyl imidazolium (EMIM) acetate). BSA, or with 2.4 mg NS22002 in 50 mM sodium acetate The ionic liquid reagent is a molten salt used for pretreatment buffer (pH 6.0) in a total reaction volume of 20 uL each. of biomass, and IL-tolerance is highly desirable for enzymes Samples were incubated for 1 h at 35°C. (B-xylosidase) or and microbes involved in biomass degradation. The B-Xylosi 50° C. (NS50010 and NS22002). Reactions were quenched dase activity showed a clear preference for higher tempera by adding one sample Volume of ice-cold methanol. tures under every reaction condition tested, suggesting that the B-xylosidase(s) present in the JP secretome are thermo Characterization off-Glucosidase, Exoglucanase, and Endo philic enzymes (FIGS. 7a and 7b). Essentially no f-xylosi glucanase Activity dase activity was detected at pH 3, while XB-F17 was con 0071. To assess the spectrum of potentially catalyzed reac verted at pH 5, 7, and 9. At 80° C. more than 90% of XB-F17 tions by the three different groups of cellulose degrading was degraded at pH 5, 7, and 9. The 50° C. data show a pH enzymes, two B-glucosidases, two exoglucanases, and three optimum at around pH 7 (FIG. 7a). The IL profile of B-xy endoglucanases were incubated for 15 min with 0.02 mM losidase activity revealed that these enzymes have a Surpris cellotetraose in a total reaction volume of 25 uL. The used ingly strong IL-tolerance. Even at 30% IL 32.3%+4.2% of enzymes, buffers and temperatures are shown in Table 1. All XB-F17 was hydrolyzed at 80° C. (FIG.7b). enzymes were used, at their optimal reaction temperatures. 0074 The cellulase activity in the secretome was also Reactions were quenched by adding one sample Volume of highest at 80° C. (FIG. 7c), showing that the cellulase ice-cold methanol. enzymes are thermophilic, as well. Strong cellobiose and

TABLE 1 Overview of enzymes and reaction conditions Enzyme Enzyme Enzyme l8le type amount Buffer Temp NSSOO10 3-glucosidase 1.2 mg 10 mM sodium acetate (pH 4.8) 50° C. UBG 3-glucosidase 2 Jug McIlvaine's citrate/phosphate buffer (pH 7.0) 50° C. Cs GH5-1 exoglucanase 2.9 g McIlvaine's citrate phosphate buffer (pH 6.0) 80° C. Cs GH5-2 exoglucanase 3.2 g McIlvaine's citrate phosphate buffer (pH 6.0) 80° C. Pr GHS endoglucanase 3.8 Lig McIlvaine's citrate/phosphate buffer (pH 6.0) 37o C. Cel5A endoglucanase 1.9 g McIlvaine's citrate/phosphate buffer (pH 6.0) 80° C. Cel9A endoglucanase 12.5 Lig McIlvaine's citrate phosphate buffer (pH 6.0) 70° C.

Example 2 glucose signals suggest that the JP Secretome is composed rimarily of exo- and endoglucanases, as it showed an activi 0072 The described nanostructure-initiator mass spec- p y 9. s ty trometry (NIMS)-based method was capable of identifying pattern comparable to exo- and endoglucanase control and characterizing multiple glycoside hydrolase activities enzymes (FIG. 8). However, as no triose formation was directly from crude environmental samples. The multiplexed observed for the control exo- and endoglucanases, but for assay system was used for screening for potential glycoside control B-glucosidases (FIG. 8) and a significant amount of hydrolase activities in environmental samples. The following triose was detected for the JP secretome (FIG. 7c), this indi workflow was applied for obtaining these samples: Soil cates that the secretome also contains 3-glucosidases. The pH samples were collected at various sites with different soil optimum for cellulase activity was determined to be at pH 5 characteristics; a small amount of each sample was used to (FIG. 7c). In contrast to the B-xylosidases, the were inoculate minimal growth medium with Switchgrass as sole much less IL-tolerant. At 50° C., there was hardly any detect carbon source; after one week of incubation the remaining able cellulase activity at 30% IL, with 89.6%+5.1% of the soil particles were removed by centrifugation and the result CT-F17 substrate remaining (FIG. 7d). Surprisingly, the lim ing Supernatants containing secreted enzymes (secretome) ited tolerance was greater at 50° C. than 80°C. Without being from the present microbial community were used for further bound by theory, we hypothesize that the particular reaction analysis. The secretomes of the environmental samples were leading to the inactivation of the affected glycoside hydrolase incubated with a mixture of equal amounts of CB-F17 and (s) is kinetically favored at higher temperatures. XB-F17 for 1 hour at 50° C. and analyzed for conversion of 0075 Thus, this approach was found to screen major these substrates. This approach proved to be very effective, classes of lignocellulolytic enzymes (B-glucosidases, exo-f and at least one of the targeted activities was detected in the endoglucanases, and B-Xylosidases) from a range of relevant majority of samples tested, while several samples showed sample types for the development of sustainable biofuels. For US 2012/0225797 A1 Sep. 6, 2012 a more comprehensive analysis of plant cell wall degradation, I0081 Screening for B-Glucosidase/B-Xylosidase Activ several more substrates can be used for the analysis of addi ity. tional enzymatic activities. Longer B-1.4-linked glucose I0082 For the detection of glycoside hydrolase activity a chains can be used to study endoglucanases in more detail, mixture of 0.02 mM cellobiose and 0.02 mMxylobiose were longer Xylose chains or also other Sugars, such as arabinose or mixed with 75% (v/v) per environmental sample secretome in mannose, can be used to aid in the identification and charac 50 mM sodium acetate buffer (pH 5.0). The total reaction terization of hemicellulases, and phenolic Substrates can be volume was 30 uL. Samples were incubated for 1 h at 50° C. used to study lignin degradation. and reactions quenched by adding four reaction Volumes of ice-cold methanol. I0083. Enzymatic Profiling of the JP MC Environmental Analysis of Environmental Samples Sample. Environmental Samples. I0084. For analysis of the activity profile of cellulose-de 0076 grading glucoside hydrolase(s) and 13-Xylosidase(s) of the JP 0077. Two commercially available compost samples and MC sample a mixture of 0.006 mM cellotetraose and 0.01 four soil samples collected at various sites in Berkeley, Calif. mM xylobiose were mixed with 42% (v/v) of the JP MC or Walnut Creek, Calif, were used: cow (COM) and chicken secretome. For determination of the pH profile reactions were (CHM) manure, soil mixed with leafs from under an oak tree carried out in McIlvaine's citrate/phosphate buffer (pH 3.0, (OAK), clay-rich soil (CS), mixed organic compost (MOC). 5.0, 7.0) or 10 mM Tris buffer (pH 9.0). For determination of and a sample from light woodland (WS). These samples were the ionic liquid (IL) tolerance 0%, 10%, 20%, or 30% (v/v) manually fragmented with a hammer. Additionally two ethyl-3-methyl imidazolium (EMIM) acetate (Sigma-Ald samples collected from two different green waste compost rich; St. Louis, Mo.) were added to the sample in McIlvaine's sites in northern California were included: Zamora (Z) and citrate/phosphate buffer (pH 5.0). All samples were incubated Jepson Prairie (JP). The latter compost sample was collected for 30 min at 50° C. and 80°C. Reactions were quenched by at the Jepson Prairie Organics facility (Vacaville, Calif.). This adding one sample Volume of ice-cold methanol. facility processes municipal green waste in turned and watered windrows. Compost was collected from 7.30, and 60 Example 3 day windrows. The 7 day windrow was in the mesophilic I0085 Measuring Colloid Formation. stage of composting and slightly warm to the touch. The 30 I0086. In FIG. 3a, Small-angle neutron scattering (SANS) and 60 day windrows were in the thermophilic composting curves were acquired for 0.35% (w/w) and 0.97% (w/w) stage, hot to the touch, and steaming. The top 12 inches of cellobiose (CB-F17) in D.O. Scattering intensity was propor each windrow was removed with a spade and the exposed tional to Solution concentration. The upturn in the low q range biomass underneath was packed into 50 ml tubes, stored at of 0.004

0089. Dynamic Light Scattering (DLS). 0090 DLS experiments were performed at 25°C. with a 0.35% (w/w) solution of CB-F17 in DO on a DynaPro plate N N 1 HCI reader (Wyatt Technology; Santa Barbara, Calif.) with a wavelength of 832.6 nm and a detection angle of 158. The NH + size distribution was obtained by analyzing the auto correla tion function using regularization analysis. HO O Example 4 1 0091 Method Validation with Known Enzymes. O EtN, 0092. In FIG. 4a, the determined temperature optima for CF3(CF).7CH2CH2O OS DMF the used enzymes were 50-60° C. for B-glucosidase (black) N He and 35°C. for B-xylosidase (gray). In FIG.4b, the determined pH optima were around pH 4.8 for B-glucosidase (black) and O around pH 7.5 for B-xylosidase (gray). FIG. 4c shows moni O toring B-glucosidase (black) and B-Xylosidase (gray) activity over time. Kinetic studies were performed by analyzing ali 2 quots of each reaction sample at the given time points. In FIG. N N 1. 4d. product inhibition was studied by analyzing B-glucosi dase activity in the presence of untagged cellobiose. As HN 1. N 1n 1%. OCH2CH2(CF)7CF2-12Q-2Jiu-3 expected, there was competition for conversion with increas ing amounts of non-perfluorinated cellobiose with an ICso of O about 15-20 mM at the used assay conditions. All activities HO O were corrected by the values of negative control samples 3 without enzyme. Error bars represent standard deviation of three independent experiments. N N 1 HCI Example 5 NH 0093 Assessment of Activities of Control Enzymes. HN r 2 + 0094. In FIG. 8, for an analysis of the polysaccharide HO O degrading capabilities of cellulases, pure B-glucosidases 1 (NS50010, UBG), exoglucanases (Cs GH5-1, Cs GH5-2), O and endoglucanases (Pr GH5, Cel5A, Cel9A) were incu Et3N, bated with perfluorinated cellotetraose (CTF17) as substrate. DMF All activities were corrected by the values of negative control O-N -e- samples without enzyme. Error bars represent standard devia F3C(FC)7 O -( tion of three independent experiments. 0095 Bars 1-2: Both f3-glucosidases were able to stepwise \ ( ) / O O degrade CT-F17 from the tetraose to cellotriose, cellobiose, and glucose. The preferred reaction of B-glucosidases is the 4 conversion of cellobiose to glucose, but the production of NN1 (CF2)7CF Sugar monomers from longer is also catalyzed, presumably at a slower reaction speed. The most abundant al 1N1. lo signal for both B-glucosidases was cellotriose, followed by HN N I. O cellobiose and glucose. HO O 0096 Bars 3-7: The main reaction for exo- and endoglu canases with cellotetraose is hydrolysis into cellobiose units. 5 Accordingly, both groups of enzymes showed cellobiose as the most significant signal present. However, in some cases (0099 Compound 3. significant signals for glucose were detectable, showing that 0100 Triethylamine (0.18 mL, 1.31 mmol) was added to a these enzymes are also capable of cleaving off cellotriose stirred solution of asymmetric dimethyl-arginine HCl salt units directly. This effect was more significant for the endo (Compound 1: 120 mg, 0.436 mmol) and Compound 2 (308 glucanases. Exo- and endoglucanases basically showed no mg, 0.523 mmol) in DMF (11.0 mL) under nitrogen at 0°C. triose signal, indicating that in general these two groups of Subsequently, the ice-bath was removed and the reaction enzymes cannot cleave off Sugar monomers. mixture was stirred at room temperature overnight. The Sol vent was removed under reduced pressure and the residue was Example 6 purified by column chromatography (MeOH/EtOAc/HO=5/ 3/0.1 to 5/3/1) to provide the desired Compound 3 (112 mg, 0097. Synthesis of Perfluorinated Tags. 37% yield) as a white viscous solid substance. "H NMR (600 0098 Perflurinated tags (Compound 3 and Compound 5) MHz, CDOD) 8 (ppm) 4.36 (t, J=6.0 Hz, 2H), 4.08 (t, J–7.2 were obtained by coupling dimethyl-arginine (Compound 1) HZ, 1H), 3.32-3.27 (m, 2H), 3.05 (s, 6H), 2.65-2.52 (m, 2H), to perfluorinated (F17) molecules of varying length by amide 1.92-1.82 (m. 1H), 1.76-1.65 (m, 3H). LRMS (NIMS) m/z bond formation (Compound 2 and Compound 4). calculated for CHF.N.O., 692.13, found 692.74. US 2012/0225797 A1 Sep. 6, 2012

0101 Compound 5. 3.27 (m, 2H), 3.04 (s, 6H), 2.95-2.89 (m, 2H), 2.52-2.40 (m, 0102 Triethylamine (0.19 mL, 1.35 mmol) was added to a 2H), 1.91-1.84 (m. 1H), 1.75-1.63 (m, 3H). stirred solution of asymmetric dimethyl-arginine HCl salt (Compound 1: 125 mg, 0.450 mmol) and Compound 4 (2.5- Example 7 dioxopyrrolidin-1-yl-(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10 heptadecafluorodecyl)benzyl car-bonate; 380 mg. 0.550 (0103) Synthesis of xylobiose-F17. A (CH)-linker (Com mmol) in DMF (11.0 mL) under nitrogen at 0° C. Subse pound 7) was coupled to the reducing end of a Xylobiose quently, the ice-bath was removed and the reaction mixture molecule (Compound 6) using Schmidtimidate chemistry to was stirred at room temperature overnight. The solvent was yield Compound 8. After removing the acetyl protection removed under reduced pressure and the residue was purified groups from the hydroxyl groups of the Sugar molecule by column chromatography (MeOH/EtOAc/H2O=5/3/0.1 to (Compound 9), hydrogenation by Pd/C removed the Car 5/3/1) to provide the desired Compound 5 (120 mg, 34% bobenzyloxy (CbZ) protection group to give a primary amine yield) as a white solid substance. "H NMR (600 MHz, (Compound 10). A peptide coupling reaction with a fluorous CDOD) 8 (ppm) 7.33 (d. J–7.8 Hz, 2H), 7.27 (d. J=7.8 Hz, tag (Compound 3) yielded the desired perfluorinated enzyme 2H), 5.06 (AB, J=12.6 Hz, 2H), 4.07 (t, J=6.0 Hz, 1H), 3.32 substrate (Compound 11).

O AcOXco O O AcO OAc 1N1 S-1a TMSOTf, CH2Cl2 AcO s - HO NHCbz -- 7 NH 6 O AcO 1. NaOMe MeOH Xco AcOO O O 1N1N1N NHCbz -e- OAc 2. DoweX 50 OAc

8 O

HQ, OHO O O 1N1N1 a NHCbz -e-Po?C OH MeOH:THF = 1:1 OH 9

O HO HO OHO O O 1N1N1N NH2 3 e OH HOBT, DIC, DMF OH 10

N1

O H HO O HO OHO O O 1nn-s-DC 1\1\ N O OH H OH 11 US 2012/0225797 A1 Sep. 6, 2012

0104 Compound 8. for 14 h. The mixture was filtered and the filtrate was concen 0105 Compound 6 (114 mg., 0.18 mmol), and Compound trated by solvent evaporization under reduced pressure. The 7 (51 mg, 0.21 mmol) were mixed in 4.0 mL of anhydrous resulting residue containing Compound 10 (117 mg, 98% methylene chloride under nitrogen. Then 3 A molecular yield) was used in the next step without further purification. sieves were added. After the resulting mixture was stirred at "H NMR (600 MHz, CDOD) 8 (ppm)4.32 (d. J=7.8 Hz, 1H), room temperature for 0.5h, it was cooled to -20°C., followed 4.22 (d. J=7.8 Hz, 1H), 4.00 (dd, J=11.4, 5.2 Hz, 1H), 3.91 by injection of TMSOTf (5uL, 0.027 mmol). After the reac (dd, J=11.4, 5.2 Hz, 1H), 3.82 (m. 1H), 3.64 (m. 1H), 3.58 tion mixture was stirred at this temperature for 1 h, triethy 3.48 (m, 2H), 3.45 (t, J=8.8 Hz, 1H), 3.38-3.27 (m, 2H), lamine was added to quench the reaction. The solvent was 3.27-3.18 (m,3H), 3.13 (t, J=6.8 Hz, 2H), 1.67-1.59 (m, 2H), removed under reduced pressure and the Substance was puri 1.58-148 (m, 2H), 1.47-1.36 (m, 2H). ''C NMR (150 MHz, fied by flash column chromatography to give 49 mg of Com CDOD) (ppm) 104.85, 103.98, 78.23, 77.61, 75.93, 74.72, pound 8 with 37% yield. "H NMR (400 MHz, CDC1) & 74.27, 71.03, 70.54, 67.07, 64.51, 42.00, 31.85, 30.36, 24.24. (ppm) 7.36-7.27 (m, 5H), 5.14-5.03 (m, 4H), 4.91-4.75 (m, 0110 Compound 11. 4H), 4.52 (d. J=6.0 Hz, 1H), 4.37 (d. J=7.6 Hz, 1H), 4.07 (dd. 0111 N,N'-Diisopropylcarbodiimide (11 uL, 0.071 J=12.0, 4.8 Hz, 1H), 3.96 (dd, J=11.6, 5.2 Hz, 1H), 3.85-3.72 mmol) and 1-hydroxybenzotriazole (11 mg, 0.071 mmol) (m. 2H), 3.49-3.40 (m. 1H), 3.37 (dd, J=12.0, 7.6 Hz, 1H), were added to a stirred solution of Compound 10 (26 mg, 3.27 (dd, J=11.6, 10.0 Hz, 1H), 3.16 (q, J=6.8 Hz, 2H), 2.04 0.071 mmol) and Compound 3 (36 mg, 0.052 mmol) in DMF (s.3H), 2.03 (s.3H), 2.02 (s.3H), 2.01 (s.3H), 2.00 (s.3H), (2.0 mL) at room temperature, and the reaction was kept 1.62-1.51 (m, 2H), 1.51-1.42 (m, 2H), 1.38-1.28 (m, 2H). stirring overnight. The mixture was concentrated under 0106 Compound 9. reduced pressure, and the residue was purified by flash col 0107 NaOMe in MeOH (3.4 uL:25% w/w) were added to umn chromatography to provide Compound 11 (40 mg, 74% Compound 8 (43 mg, 0.06 mmol) in 2 mL of methanol. After yield). "H NMR (600 MHz. CDOD) 8 (ppm) 4.43-4.34 (m, the resulting mixture was stirred at room temperature for 4 h, 2H), 4.32 (d. J–7.8 Hz, 1H), 4.21 (d. J–7.8 Hz, 1H), 4.11-4.05 prewashed DOWEX 50WX2-200 (H+) was added. The mix (m. 1H), 4.02-3.96 (m. 1H), 3.89 (dd, J=10.8, 4.8 Hz, 1H), ture was stirred for 0.5 h and filtered. The solvent was evapo 3.87-3.74 (m, 2H), 3.65 (m, 1H), 3.66-3.59 (m, 1H), 3.58-3. rated under reduced pressure and the remaining residue was 46 (m, 2H), 3.46 (t, J=8.4 Hz, 1H), 3.36-3.14 (m, 7H), 3.04 (s, purified by flash column chromatography using 30% MeOH 6H), 2.93 (t, J=6.0 Hz, 2H), 2.69-2.53 (m, 2H), 1.85 (m. 1H), in chloroform to give Compound 9 (30 mg) in quantitative 1.78-1.60 (m, 5H), 1.60- 1.47 (m, 2H), 1.47-1.36 (m, 2H). yield. "H NMR (400 MHz. CDOD) 8 (ppm) 7.40-728 (m, 5H), 5.07 (s. 2H), 4.31 (d. J=5.6 Hz, 1H), 4.22 (d. J=7.8 Hz, Example 8 1H), 4.01 (dd, J=11.6, 5.2 Hz, 1H), 3.91 (dd, J=11.2, 5.2 Hz, (O112 Synthesis of cellobiose-F17. 1H), 3.82 (m, 1H), 3.64 (m, 1H), 3.58-3.48 (m, 2H), 3.45 (t, 0113 A (CH)s-linker (Compound 7) was coupled to the J=8.8 Hz, 1H), 3.38-3.27 (m, 2H), 3.27-3.18 (m,3H), 3.13 (t, reducing end of a cellobiose molecule (Compound 12) using J=6.8 Hz, 2H), 1.67-1.59 (m, 2H), 1.58-1.48 (m, 2H), 1.47 Schmidt imidate chemistry to yield Compound 13. After 1.36 (m, 2H). removing the acetyl protection groups from the hydroxyl 0108 Compound 10. groups of the Sugar molecule (Compound 14), hydrogenation 0109 Compound 9 (163 mg, 0.325 mmol) was dissolved by Pd/C removed, the carbobenzyloxy (Cbz) protection in a mixture of methanol (2.0 mL) and THF (2.0 mL). Then group to give a primary amine (Compound 15). A peptide 10% Pd/C (34 mg., 0.016 mmol) was added and the reaction coupling reaction with a fluorous tag (Compound 3) yielded mixture was stirred under H2 (g; 1 atm) at room temperature the desired perfluorinated enzyme substrate (Compound 16).

AcO AcO O AcO AcO O O AcO OAc TMSOTf, CH2Cl2 AcO O CCl3 + Ho1N1,N1NNick, Hip r 7 NH 12

AcO AcO O AcO 1. NaOMe AcO O O MeOH AcO o1N1 N1). Nick, -- OAc 2. DoweX 50 OAc 13 US 2012/0225797 A1 Sep. 6, 2012 12

-continued HO HO O

HQ, OHO O O 1N1N1\ NHCbz -e-Po?C OH MeOH:THF = 1:1 OH 14

N1

HN 1. N 1N1 4, 8. OCH2CH2(CF2)7CF2-12Q-2Jiu-3 HO H HO O HO O O HO O HO OHO O 1N1-1N NH2 3 OH HOBT, DIC, DMF OH 15

N1

HO HO HN 1-1%. OCH2CH2(CF2)7CF2-12Q-2Jiu-3 O H HO HO OHO O O 1n 11a N O O OH H OH 16

0114 Compound 13. evaporated under reduced pressure and the remaining residue 0115 Compound 12(330 mg, 0.42 mmol) and Compound was purified by flash column chromatography using 30% 7 (150 mg. 0.55 mmol) were mixed in 8 mL of anhydrous MeOH in chloroform to give Compound 14 (128 mg, 90% methylene chloride under nitrogen. Then 3 A molecular yield). "H NMR (600 MHz. CDOD) 8 (ppm) 7.40-7.28 (m, sieves were added. After the resulting mixture was stirred at 5H), 5.06 (s. 2H), 4.40 (d. J=7.8 Hz, 1H), 4.27 (d. J=7.8 Hz, 1H), 3.90-3.82 (m, 4H), 3.66 (dd, J=11.4, 5.4 Hz, 1H), 3.58 room temperature for 0.5 h, it was cooled down to -20°C., 3.48 (m, 3H), 3.41-3.28 (m, 4H), 3.26-3.20 (m, 2H), 3.11 (t, followed by injection of TMSOTf (14 uL, 0.077 mmol). After J=6.8 Hz, 2H), 1.67-1.58 (m, 2H), 1.55-1.46 (m, 2H), 1.45 the reaction mixture was stirred at this temperature for 1 h, 1.35 (m, 2H). triethylamine was added to quench the reaction. The solvent 0118 Compound 15. was removed under reduced pressure and the residue was 0119 Compound 14 (89 mg, 0.16 mmol) was dissolved in purified by flash column chromatography to give 140 mg of a mixture of methanol (1.5 mL) and THF (1.5 mL). Then 10% Compound 13 in 39% yield. "H NMR (400 MHz, CDC1) & Pd/C (16.8 mg, 0.008 mmol) was added and the reaction (ppm) 7.40-7.28 (m, 5H), 5.19-5.08 (m, 5H), 4.95-4.79 (m, mixture was stirred under H (g; 1 atm) at room temperature 3H), 4.60-4.50 (m, 2H), 4.45 (d. J=8.0 Hz, 1H), 4.39 (dd. for 14 h. The mixture was filtered and the filtrate was concen J=12.8, 4.4 Hz, 1H), 4.18-4.00 (m, 2H), 3.88-3.75 (m, 2H), trated by solvent evaporization under reduced pressure. The 3.69 (m. 1H), 3.61 (m. 1H), 3.48 (m. 1H), 3.20 (q, J=6.8 Hz, resulting residue containing Compound 15 (65 mg., 96% 2H), 2.10 (s.3H), 2.07 (s.3H), 2.01 (s.3H), 2.00 (s, 6H), 1.99 yield) was used in the next step without further purification. (s, 3H), 1.96 (s.3H), 1.67-1.56 (m, 2H), 1.56-1.47 (m, 2H), "H NMR (600 MHz, CDOD) 8 (ppm)4.41 (d. J=7.8 Hz, 1H), 1.47-1.32 (m, 2H). 4.28 (d. J=7.8 Hz, 1H), 3.94-3.83 (m, 4H), 3.65 (dd, J=12.0, 0116 Compound 14. 5.4 Hz, 1H), 3.59-3.53 (m, 2H), 3.51 (t, J=9.0 Hz, 1H), 3.42 0117 NaOMe in MeOH (20 uL; 25% w/w) were added to 3.32 (m, 4H), 3.32-3.29 (m, 2H), 3.27-3.20 (m, 2H), 2.72 (t, Compound 13 (217 mg, 0.25 mmol) in 2.5 mL of methanol. J–7.2 Hz, 2H), 1.68-1.60 (m, 2H), 1.60- 1.51 (m, 2H), 1.50 After the resulting mixture was stirred at room temperature 1.41 (m, 2H). CNMR (150 MHz, CDOD) 8 (ppm) 104.9, for 4 h, prewashed DOWEX 50WX2-200 (H) was added. 104.5, 81.1, 78.4, 78.2, 76.8, 76.7, 75.2, 75.1, 71.7, 70.9, 62.7, The mixture was stirred for 0.5 hand filtered. The solvent was 62.2, 42.2, 32.2, 30.6, 24.5. US 2012/0225797 A1 Sep. 6, 2012

0120 Compound 16. Example 9 0121 N,N'-Diisopropylcarbodiimide (8.1 uL, 0.052 mmol) and 1-hydroxybenzotriazole (8.0 mg, 0.052 mmol) 0.122 Synthesis of cellotetraose-F17. A fully acetylated were added to a stirred solution of Compound 15 (22 mg, 0.052 mmol) and Compound 3 (30 mg, 0.043 mmol) in DMF cellotetraose molecule (Compound 17) was first converted to (1.5 mL) at room temperature, and the reaction was kept a trichloro-imidate (Compound 18), and a (CH)s-linker stirring overnight. The mixture was concentrated under (Compound 7) was Subsequently coupled to the reducing end reduced pressure, and the residue was purified by flash col of the cellotetraose molecule (Compound 18) using Schmidt umn chromatography to provide Compound 16 (35 mg, 74% imidate chemistry to yield Compound 19. After removing the yield). "H NMR (600 MHz. CDOD) 8 (ppm) 4.41 (d. J–7.8 acetyl protection groups from the hydroxyl groups of the HZ, 1H), 4.40-4.32 (m, 2H), 4.28 (d. J–7.8 Hz, 1H), 4.10-4.04 (m. 1H), 3.94-3.83 (m, 4H), 3.66 (dd, J=12.0, 5.4 Hz, 1H), sugar molecule (Compound 20), hydrogenation by Pd/C 3.61-3.47 (m, 3H), 3.44-3.25 (m, 4H), 3.25-3.16 (m, 4H), removed the Carbobenzyloxy (Cbz) protection group to give 3.04 (s, 6H), 2.96-2.91 (t, J–7.2 Hz, 2H), 2.65-2.53 (m, 2H), a primary amine (Compound 21). A peptide coupling reaction 1.88-1.79 (m, 1H), 1.73-1.59 (m, 5H), 1.58-1.48 (m, 2H), with a fluorous tag (Compound 5) yielded the desired perflu 1.47-1.39 (m, 2H) orinated enzyme Substrate (Compound 22).

US 2012/0225797 A1 Sep. 6, 2012 15

ponunuoo US 2012/0225797 A1 Sep. 6, 2012

(0123 Compound 18. acetic acid (1.0 mL). Then 10% Pd/C (18 mg, 0.008 mmol) 0.124 Benzylamine (52 uL, 0.47 mmol) was added to a was added and the reaction mixture was stirred under H (g: 1 stirred solution of D-(+)-cellotetraose tetradecaacetate atm) at room temperature for 14 hours. The mixture was (Compound 17:391 mg, 0.31 mmol) in THF (13 mL) at room filtered and the filtrate was concentrated by solvent evapora temperature every 12 h for 60 h. Subsequently, the solvent tion under reduced pressure. The resulting residue (14 mg. was removed and the resulting residue was purified by flash Compound 21) was used in the next step without further column chromatography to give Compound 17 (intermedi purification. "H NMR (600 MHz, DO) 8 (ppm) 4.51-4.47 ate)(365 mg, 97% yield). CCICN (0.30 mL, 2.97 mmol) was (m. 2H), 4.46 (d. J–7.8 Hz, 1H), 4.43 (d. J–8.4 Hz, 1H), added to a stirred solution of Compound 17 (intermediate) 3.94-3.81 (m, 5H), 3.80-3.71 (m, 3H), 3.70-3.49 (m, 9H), (360 mg, 0.30 mmol) in methylene chloride (10.0 mL) at 0° 3.48-3.39 (m, 2H), 3.40-3.33 (m. 1H), 3.31 (t, J=8.4 Hz, 2H), C., followed by the addition of DBU (9 uL, 0.06 mmol). The 3.28-3.21 (m, 2H), 2.95 (t, J–7.2 Hz, 2H), 1.67-1.56 (m, 4H), resulting mixture was stirred for 2 h and the solvent was 1.43-1.35 (m, 2H). evaporated under reduced pressure. The residue was sub I0131 Compound 22. jected to flash column chromatography purification to give (0132 N,N'-Diisopropylcarbodiimide (1.0 uL, 0.0063 Compound 18 (341 mg, 84% yield). H NMR (400 MHz, mmol) and 1-hydroxybenzo-triazole (1.0 mg, 0.0063 mmol) CDC1) & (ppm) 8.65 (s, 1H), 6.47 (s, 1H), 5.69 (br. 1H), 5.50 were added to a stirred solution of Compound 21 (4.0 mg. (t, J=9.6 Hz, 1H), 5.16-5.00 (m, 6H), 4.90 (t, J=8.0 Hz, 1H), 0.0053 mmol) and Compound 5 (4.1 mg, 0.0053 mmol) in 4.89 (q, J=8.8 Hz, 2H), 4.55-4.38 (m, 7H), 4.34 (dd, J=12.8, DMF (0.4 mL) at room temperature, and the reaction was kept 4.4 Hz, 1H), 4.15-4.05 (m,3H), 4.34 (dd, J=12.4, 2.0 Hz, 1H), stirring overnight. The resulting mixture containing Com 3.85-3.68 (m, 3H), 3.66-3.51 (m, 3H), 2.14 (s, 6H), 2.11 (s, pound 22 was purified by flash column chromatography as 3H), 2.08 (s.3H), 2.034 (s.3H), 2.031 (s.3H), 2.02 (s, 3H), before, however, in this case it was not possible to purify 2.01 (s.3H), 2.00 (s.3H), 1.998 (s.3H), 1.99 (s.3H), 1.98 (s, sufficiently enough for NMR. Nanostructure-initiator mass 3H), 1.96 (s.3H). spectrometry was used to confirm the Success of the coupling 0125 Compound 19. reaction. The calculated exact mass for Css HENO is 0126 Compound 18 (112 mg, 0.0825 mmol) and Com 1515.48, which was correctly identified at 1516.45 M+H" pound 7 (25 mg 0.107 mmol) were mixed in 5 mL of anhy (Figure S8). drous methylenechloride under nitrogen. Then3A molecular sieves were added. After the resulting mixture was stirred at Example 10 room temperature for 0.5 h, it was cooled down to -20°C., I0133) Mass spectrum of cellotetraose-F17 (FIG.9). After followed by injection of TMSOTf (14 uL, 0.077 mmol). After synthesis, cellotetraose-F17 (CT-F17) was purified by flash the reaction mixture was stirred at this temperature for 2 h, column chromatography. Purity was controlled by nanostruc triethylamine was added to quench this reaction. The solvent ture-initiator mass spectrometry. was removed under reduced pressure and the residue was purified by flash column chromatography to give Compound Example 11 19 (35 mg,30% yield). "HNMR (600 MHz. CDOD) 8 (ppm) I0134. The influence of carbon source on enzyme expres 737-7.27 (m, 5H), 5.16-5.00 (m, 7H), 492-4.78 (m, 5H), sion was determined (FIG.10). A Jepson Prairie environmen 4.51 (dd, J=12.0, 1.8 Hz, 1H), 4.47-4.36 (m, 5H), 4.34 (dd. tal sample (JP) was incubated in minimal medium containing J=12.6, 4.2 Hz, 1H), 4.13-402 (m, 3H), 4.01 (dd, J=12.6, 2.4 either switchgrass (SG) or microcrystalline cellulose (MC) as HZ, 1H), 3.83-3.67 (m, 4H), 3.65-3.59 (m, 1H), 3.57-3.50 (m, sole carbon Source. The resulting secretomes were used for 3H), 3.47-3.39 (m, 1H), 3.15 (q, J=6.8 Hz, 2H), 2.13 (s.3H), glycohydrolase activity assays using perfluorinated cellotet 2.12 (s.3H), 2.10 (s.3H), 2.07 (s.3H), 2.02 (s, 3H), 2.01 (s, raose as Substrate. Conversion of cellotetraose into Smaller 3H), 1.999 (s.3H), 1996 (s.3H), 1992 (s.3H), 1.98 (s.3H), Sugar chains was monitored over time with sampling time 1.97 (s, 3H), 1.96 (s, 3H), 1.94 (s, 3H), 1.60- 1.51 (m, 2H), points after 5 min, 15 min, and 30 min. At 5 min an additional 1.51-1.42 (m, 2H), 1.40-1.27 (m, 2H). JPMC sample with only half the amount of secretome was 0127. Compound 20. included. 0128 NaOMe in MeOH (30 uL; 25% w/w) was added to Compound 19 (32 mg, 0.022 mmol) in a mixture of methanol Example 12 (1.5 mL) and methylene chloride (1.5 mL). After the resulting mixture was stirred at room temperature for 14 h, prewashed Nanostructure-Initiator Mass Spectrometry (NIMS) DOWEX 50WX2-200 (H) was added. The mixture was I0135 Fabrication of NIMS chips. A 4" silicon wafer stirred for 0.5 h and filtered. The solvent was evaporated (single-sided polished P/Boron, orientation <1-O-0>, resis under reduced pressure to give a white solid substance (18 tivity 0.01-0.02 S2cm, thickness 525+25um) obtained from mg; Compound 20), which was used in the next step without Silicon Quest International (Santa Clara, Calif.) was cut into further purification. H NMR (600 MHz, DO) 8 (ppm)7.41 a 70x70mm square and cleaned thoroughly with methanol, 7.28 (m, 5H), 5.06 (s. 2H), 4.49 (d. J–7.8 Hz, 1H), 4.48 (d. followed by anodic etching with 25% hydrofluoric acid in J=7.8 Hz, 1H),447 (d. J=7.8 Hz, 1H), 4.41 (d.J=7.8 Hz, 1H), ethanol in a custom made Teflon etching chamber using 3.97-3.90 (m, 3H), 3.90-3.82 (m, 2H), 3.81-3.72 (m, 3H), extreme caution. A current of 2.4A was applied for 15 min 3.69 (dd, J=12.6, 6.0 Hz, 1H), 3.68-3.55 (m, 9H), 3.53 (m, utes. After etching, chips were coated by adding 400 uL of the 1H), 3.49-3.42 (m, 2H), 3.39-3.22 (m, 5H), 3.08 (t, J=6.0 Hz, initiator liquid bis(heptadecafluoro-1,1,2,2-tetrahydrodecyl) 2H), 1.61-1.53 (m, 2H), 1.50-1.43 (m, 2H), 1.35-1.28 (m, tetramethyl-disiloxane (Gelest; Morrisville, Pa.) for 20 min 2H). utes. Excess initiator was blown off with nitrogen. 0129. Compound 21. 0.136 Mass Spectrometry. 0130 Compound 20 (18 mg, 0.020 mmol) was dissolved 0.137 In each case 0.5-1 LIL per quenched reaction sample in a mixture of methanol (1.5 mL), ethyl acetate (1.5 mL), and was spotted onto the NIMS surface and removed after an US 2012/0225797 A1 Sep. 6, 2012

incubation of -30s. A grid drawn manually on the NIMS chip 0143 Protease substrate F17008-Ala-Pro-Ay-Thr-Pro using a diamond-tip scribe helped with spotting and identifi Gly-Gly-Arg-Arg-OH cation of sample spots in the spectrometer. Chips were loaded 0144. The mixture was incubated for 15 minutes at 37°C. using a modified standard MALDI plate. NIMS was per and reaction were quenched by adding 3 ul of ice-cold metha formed on a 4800 MALDI TOF/TOF mass analyzer from nol (analytical grade) and kept on ice for NIMS-based analy Applied BioSystems (Foster City, Calif.). In each case signal sis. The resulting mass spectra from the NEVIS-analysis are intensities were identified for the ions of the used substrates shown in FIG.12a-b. FIG.12a shows the signals for protease and occurring products. Enzyme activities were determined substrate F17008-Ala-Pro-Ay-Thr-Pro-Gly-Gly-Arg-Arg by forming product-to-substrate ratios. OH and FIG.12b shows the signals for the protease substrate and the reaction product F17008-Ala-Pro. 0145 This example demonstrates that the method dis Example 13 closed herein can be used for analysis of protease activity in a sample. Detection of Lipase Activity What is claimed is: 0138 2 ul lipase substrate F17008-Gly-Cys(palmitoyl)- 1. A method of detecting the activities of a plurality of Met-Gly-Leu-Pro-Art-OH was mixed with a lipase sample (2 enzymes in a multiplexed assay, wherein the method com ul solution of purified Lipase from Candida rugosa (Sigma, prises: incubating a sample with substrates for the plurality of St. Louis) at 0.06 units/ul) in 5ul phosphate buffer (pH7.5) in enzymes to obtain reaction products; a microwell plate. analyzing the reaction products by nanostructure-initiator mass spectrometry (NIMS); and detecting activities of the plurality of enzymes in the sample by identifying the ratio of Substrate-to-reaction productions in a mass spectrum. 2. The method of claim 1, wherein the plurality of enzymes comprises an enzyme with plant cell wall degrading activity, a lipase, or a protease. 3. The method of claim 2, wherein the enzyme with plant cell wall degrading activity reduces the chain length of a Sugar Gly-Cys(palmitoyl)-Met-Gly-Leu-Pro-Art-OH head group. 4. The method of claim 3, wherein the sugar comprises 0139 Lipase substrate F17008-Gly-Cys(palmitoyl)-Met cellulose, hemicellulose, Xylose, cellobiose, cellotetraose, or Gly-Leu-Pro-Art-OH xylobiose. 0140. The mixture was incubated for 15 minutes at 37° C. 5. The method of claim 2, wherein the enzyme is selected and reaction were quenched by adding 3 ul of ice-cold metha from the group consisting of an endoglucanase, an exogluca nol (analytical grade) and kept on ice for NIMS-based analy nase, a glucosidase, a pectinase, a cellulose, and a hemicel sis. The resulting mass spectra from the NIMS-analysis are lulase. shown in FIG. 11a-b. FIG. 11a shows the signals for lipase 6. The method of claim 2, wherein the enzyme with plant substrate F17008-Gly-Cys(palmitoyl)-Met-Gly-Leu-Pro cell wall degrading activity degrades lignin. Art-OH and FIG. 11b shows the signals for the lipase sub 7. The method of claim 6, wherein the enzyme is a laccase strate and the reaction product F17008-Gly-Cys-Met-Gly or peroxidase. Leu-Pro-Art-OH. 8. The method of claim 1, wherein analyzing the reaction 0141. This example demonstrates that the method dis product by nanostructure-initiator mass spectrometry closed herein can be used for analysis of lipase activity in a (NIMS) comprises applying the reaction product to a hydro sample. phobic NIMS chip surface. 9. The method of claim 8, wherein the hydrophobic NIMS Example 14 chip Surface comprises a perfluorinated coating. 10. The method of claim8, wherein the substrate comprises Detection of Protease Activity a Substrate head group linked to a perfluorinated tag that forms micelles under aqueous conditions. 0142. 2 ul protease substrate F17008-Ala-Pro-Ay-Thr 11. The method of claim 10, wherein the substrate interacts Pro-Gly-Gly-Arg-Arg-OH was mixed with a protease sample with the NIMS chip surface via fluorous-phase-interactions. (2 ul solution of purified trypsin (Sigma, St. Louis) at 0.08 12. The method of claim 1, wherein the sample is an iso units/ul) in 5ul phosphate buffer (pH7.5) in a microwell plate. lated enzyme or a crude environmental sample. 13. The method of claim 12, wherein the sample is obtained by incubating the crude environmental sample with Switch grass or cellulose. 14. The method of claim 1, wherein the reaction products are analyzed in parallel and wherein the reaction products are different in mass. 15. The method of claim 14, wherein the reaction products comprise Sugar molecules with identical mass and perfluori nated tags of different mass. Ala-Pro-Arg-Thr-Pro-Gly-Gly-Arg-Arg-OH 16. The method of claim 14, wherein the reaction products comprise Sugar molecules of different mass and perfluori nated tags of identical mass. US 2012/0225797 A1 Sep. 6, 2012

17. A multiplexed system for the activities of a plurality of nase Substrate, a lipase Substrate, a protease substrate, or a enzymes, comprising: combination thereof. (i) perfluorinated tagged substrates for the plurality of 19. The system of claim 18, wherein the substrates are enzymes; and tagged with a heptadecafluoro-1,1,2,2-tetrahydrodecyl (F17) (ii) a nanostructure-initiator mass spectrometry (NIMS) tag. chip having a Surface comprising a perfluorinated coat 20. The system of claim 18, wherein the NIMS chip surface adapted to interact with the substrates via fluorous is coated with bis(heptadecafluoro-1,1,2,2- phase-interactions. tetrahydrodecyl)tetramethyl-disiloxane. 18. The system of claim 17, wherein the substrates com prise a cellulose Substrate, a hemicellulase Substrate, aligni c c c c c