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(12) Patent Application Publication (10) Pub. No.: US 2012/0225797 A1 Northen Et Al 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 ENZYME (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 enzymes in complex bio (21) Appl. No.: 13/363,695 logical mixtures are provided. b ii. Sif cleanup gyCohydrolase S activity Patent Application Publication Sep. 6, 2012 Sheet 1 of 14 US 2012/0225797 A1 glycohydrolase activity & & sugar units periests if hexoses C cleaved sugas sits assifioritates: tsg SS Figure I Patent Application Publication Sep. 6, 2012 Sheet 2 of 14 US 2012/0225797 A1 Replacement Sheet Figure 2 Patent Application Publication Sep. 6, 2012 Sheet 3 of 14 US 2012/0225797 A1 Replacement Sheet Mass Figure 3 Patent Application Publication Sep. 6, 2012 Sheet 4 of 14 US 2012/0225797 A1 Replacement Sheet a),t 100% -- Glucose b) 100% : -- Glucose w 3 xylose k 8 xylose 88% 30 & As ar : : -" s : S. 60% : s S. : g : : g 40% : Six 40% : 20% : 20% : : : & s : E. 0, ..........y......sos.syr..........:s: - - - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - - - O. : 2 3 4 5 8 Temperature I Corris C) 100% d) 8% --8 XyloseGlucose rxYx t 5 86% N. 80% s f E ses, 60% 9. s 3s 48% 5 40% s s 3 s. 2 - E. xxxas&assa-saraway 8% oxy www. N- ww. Y 2 88: SC 3. 25 s Title Tin -e- Concunlabeled cellobiose M -- Figure 4 Patent Application Publication Sep. 6, 2012 Sheet 5 of 14 US 2012/0225797 A1 Replacement Sheet 090|,000|096 n 006 006 Patent Application Publication Sep. 6, 2012 Sheet 6 of 14 US 2012/0225797 A1 Replacement Sheet is Glucose {: Xylose 8& Glucosef... Xylose formation 4& 3:. E3 Patent Application Publication Sep. 6, 2012 Sheet 7 of 14 US 2012/0225797 A1 Replacement Sheet ? 04.080209, 8 9208000$900$99089c080008| 6HdIH0GHd£Hd O008000€0.08020900080208, Patent Application Publication Sep. 6, 2012 Sheet 8 of 14 US 2012/0225797 A1 Replacement Sheet 100% y is St. S. a Glucose 38%. , cellobiose i & Cellotriose sts Celotetraose aw S $ss ------ ------------ -- a Vsw--&sw VXXSk NS50010 UBG Cs GH5-1 CsCH5-2 ProgH5 Cel5A Cel9A Figure 8 Patent Application Publication Sep. 6, 2012 Sheet 9 of 14 US 2012/0225797 A1 & s:s 3. SS f5 p e E 3. e t E 25 3. sai 830 22O 8: 8 Figure 9 Patent Application Publication Sep. 6, 2012 Sheet 10 of 14 US 2012/0225797 A1 iss Figure 10 Patent Application Publication Sep. 6, 2012 Sheet 11 of 14 US 2012/0225797 A1 www. es g (i. g is r wers (c. & an grow s S 's Yew 9 st N. na. ed x gar t y R . is ( C & c. O & c. ) & S C n. S. is ef: N wr ver A:Sueu% Patent Application Publication Sep. 6, 2012 Sheet 12 of 14 US 2012/0225797 A1 AS 8 9, Patent Application Publication Sep. 6, 2012 Sheet 13 of 14 US 2012/0225797 A1 s aereeeeeeeeeeeeeeeeeesw s f (C. w wr c N. saar r ar s t Ev S. has ass' (D asses co a. | st s r k C & E. is y { C. a. E. s. cy kNE san rger AS jeu 9, Patent Application Publication Sep. 6, 2012 Sheet 14 of 14 US 2012/0225797 A1 ÁSue 9. US 2012/0225797 A1 Sep. 6, 2012 MULTIPLEXED SCREENING OF ENZYME head group. In some embodiments, the Sugar comprises cel ACTIVITIES USING lulose, hemicellulose, 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 cellulose, and a hemicellulase. In some embodiments, APPLICATIONS the enzyme with plant cell wall degrading activity degrades 0001. This application claims priority under 35 U.S.C. lignin. 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 hydrolases and transferases 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.
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