Multiplexed screening of enzyme activities using nanostructure-initiator mass spectrometry

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.