Multiplexed screening of enzyme activities using nanostructure-initiator mass spectrometry

Patent metadata

Field	Value
Publication number	US-10240180-B2
Application number	US-201213363695-A
Country	US
Kind code	B2
Filing date	Feb 1, 2012
Priority date	Feb 1, 2011
Publication date	Mar 26, 2019
Grant date	Mar 26, 2019

Abstract

Disclosed herein are methods, compositions and systems for analyzing and detecting enzyme activity. For examples, methods, compositions and systems for parallel detection and analysis of enzymatic activities of enzymes in complex biological mixtures are provided.

First claim

What is claimed is:

1. A method of detecting the activities of a plurality of enzymes in a multiplexed assay, comprising:
(a) providing an aqueous solution comprising substrates for a plurality of enzymes, wherein each of the substrates comprises a substrate head group linked to a hydrophobic fluorous tag and forms micelles in the aqueous solution, wherein the substrate head group is selected from the group consisting of a sugar head group, a lipid head group, and a polypeptide head group;
(b) incubating the aqueous solution comprising the substrates with a sample comprising the plurality of enzymes to carry out enzymatic reactions to form reaction products under aqueous conditions in a container, wherein the plurality of enzymes comprises enzymes having an activity that changes the mass of said substrates;
(c) applying the reaction products formed in (b) to a hydrophobic nanostructure-initiator mass spectrometry (NIMS) chip surface after incubating the sample with the aqueous solution comprising the substrates, wherein the hydrophobic NIMS chip surface comprises a perfluorinated coating configured to interact with the hydrophobic fluorous tags of the substrates;
(d) analyzing the reaction products and the substrates by mass spectrometry; and
(e) detecting a change in the mass of the substrate to identify the ratio of substrate-to-reaction product ions in a mass spectrum, wherein a change in the mass of the substrate is indicative of an activity of one or more of the plurality of enzymes in the sample.

2. The method of claim 1 , wherein the plurality of enzymes comprises an enzyme with plant cell wall degrading activity.

3. The method of claim 2 , wherein the enzyme with plant cell wall degrading activity reduces the chain length of a sugar head group.

4. The method of claim 3 , wherein the sugar comprises cellulose or hemicellulose.

5. The method of claim 2 , wherein the enzyme is a cellulase or a hemicellulase.

6. The method of claim 2 , wherein the enzyme with plant cell wall degrading activity degrades lignin.

7. The method of claim 6 , wherein the enzyme with plant cell wall degrading activity is a laccase.

8. The method of claim 1 , wherein the sample comprises isolated enzymes or is a crude environmental sample.

9. The method of claim 1 , wherein the sample is obtained by incubating a crude environmental sample with switchgrass or cellulose.

10. The method of claim 1 , wherein two or more of the reaction products are analyzed in parallel and wherein two or more of the reaction products are different in mass.

11. The method of claim 10 , wherein the reaction products comprise sugar molecules with identical mass and tags of different mass.

12. The method of claim 10 , wherein the reaction products comprise sugar molecules of different mass and tags of identical mass.

13. The method of claim 1 , wherein the plurality of enzymes comprises a hydrolase.

14. The method of claim 13 , wherein the hydrolase is a glucoside hydrolase.

15. The method of claim 1 , wherein the container is a tube or a microwell plate.

16. The method of claim 1 , further comprising quenching the enzymatic reactions before applying the reaction products to the hydrophobic NIMS chip surface.

17. The method of claim 1 , wherein the perfluorinated coating of the NIMS chip surface comprises bis(heptadecafluoro-1,1,2,2-tetrahydrodecyl)tetramethyl-disiloxane.

18. The method of claim 1 , wherein the plurality of enzymes comprises a lipase.

19. The method of claim 1 , wherein the plurality of enzymes comprises a protease.

20. The method of claim 1 , wherein the hydrophobic fluorous tag is a perfluorinated heptadecafluoro-1,1,2,2-tetrahydrodecyl (F17) tag, or bis(tridecafluoro-1,1,2,2-tetrahydrooctyldimethylsiloxy)-methylchloro-silane (F26) tag.

Assignees

• Northen Trent R
• Reindl Wolfgang E
• Deng Kai
• Singh Seema
• Singh Anup K
• Cheng Xiaoliang
• Univ California
• Nat Tech & Eng Solutions Sandia Llc

Inventors

• Northen Trent R
• Reindl Wolfgang E
• Deng Kai
• Singh Seema
• Singh Anup K
• Cheng Xiaoliang

Classifications

• G01N2560/00

  Chemical aspects of mass spectrometric analysis of biological material · CPC title

• C12Q1/00 Primary

  Measuring or testing processes involving enzymes, nucleic acids or microorganisms (measuring or testing apparatus with condition measuring or sensing means, e.g. colony counters, C12M1/34); Compositions therefor; Processes of preparing such compositions · CPC title

• B82Y30/00

  Nanotechnology for materials or surface science, e.g. nanocomposites · CPC title