Nanopatterned extracellular matrices enable cell-based assays with a mass spectrometric readout
The present disclosure provides methods in which adherent cells are treated with small molecules, cultured, lysed, and then analyzed by mass spectrometry to measure the activities of endogenous enzymes. The implementation of this method relies on the use of surfaces that are nanopatterned with cell adhesion ligands to mediate cell attachment and a peptide that is a substrate for the desired enzyme activity in the lysate.
1. A method of assaying activity of an intracellular enzyme, comprising:
(a) printing a surface with an array of immobilized cell adhesion ligands and immobilized substrates for the intracellular enzyme by
(i) coating a polymer pen lithography (PPL) tip array with a first monolayer reagent and printing the first monolayer reagent at selected positions on the surface to form an array of printed first monolayer reagent,
(ii) incubating the array of printed first monolayer reagent with a second monolayer reagent such that the second monolayer reagent is adsorbed onto unprinted portions of the surface,
wherein one of the first monolayer reagent and the second monolayer reagent comprises a monolayer reagent for adsorption of the cell adhesion ligand and the other comprises a monolayer reagent for chemical immobilization of the substrate for the intracellular enzyme,
(iii) contacting the resulting array of step (ii) with the substrate for the intracellular enzyme under conditions to immobilize the substrate to the surface at the portion of the surface comprising the monolayer reagent for chemical immobilization,
(iv) contacting the resulting array of step (ii) with the cell adhesion ligand under conditions to immobilize the cell adhesion ligand to the surface at the portion of the surface comprising the monolayer reagent for adsorption of the cell adhesion ligand;
wherein steps (iii) and (iv) can be performed in either order;
(b) contacting a cell and the surface of step (a), the contacting resulting in immobilization of the cell via interaction between the cell and the immobilized cell adhesion ligand;
(c) contacting the immobilized cell with a lysing solution to form a cell lysate and release the enzyme, thereby allowing contact between the enzyme and the immobilized substrate to transform the immobilized substrate to a product, the product having a different mass than the substrate; and
(d) measuring the amount of the product formed using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to assay the activity of the enzyme.
2. The method of claim 1 , wherein the surface comprises gold, silver, or copper.
3. The method of claim 1 , wherein the monolayer reagent for adsorption of the cell adhesion ligand comprises mercaptohexadecanoic acid (MHA), hexadecane thiol, or a C 8-20 hydroxyalkane.
4. The method of claim 1 , wherein the monolayer reagent for chemical immobilization of the substrate comprises an immobilizing moiety and an inert moiety.
5. The method of claim 4 , wherein the immobilizing moiety comprises a maleimide, a thiol, an alkyne, an azide, an amine, or a carboxyl group.
6. The method of claim 4 , wherein the inert moiety comprises mannitol or 3 to 6 ethylene glycol units.
7. The method of claim 1 , wherein (i) one of the monolayer reagent for chemical immobilization of the substrate and the substrate comprises a maleimide and the other an alkane thiol; (ii) one of the monolayer reagent for chemical immobilization of the substrate and the substrate comprises an alkyne and the other an azide; or (iii) one of the monolayer reagent for chemical immobilization of the substrate and the substrate comprises an amine and the other a carboxyl group, so as to form a chemical bond between the monolayer reagent for chemical immobilization and the substrate.
8. The method of claim 1 , wherein at least one of the immobilized substrate and the cell adhesion ligand comprises a peptide.
9. The method of claim 1 , wherein the cell adhesion ligand comprises a RGD peptide or an extracellular matrix (ECM) protein.
10. The method of claim 1 , wherein at least one of the cell adhesion ligand and the immobilized substrate is bound to the surface via a linker having a structure of formula I:
and Lig comprises the cell adhesion ligand or the immobilized substrate.
11. The method of claim 1 , wherein the enzyme is a deacetylase, acetyltransferase, esterase, phosphorylase/kinase, phosphatase, protease, methylase, demethylase, or a DNA or RNA modifying enzyme.
12. The method of claim 11 , wherein the immobilized substrate comprises an acylated peptide and the product comprises a deacylated peptide, or wherein the immobilized substrate comprises a deacylated peptide and the product comprises an acylated peptide.
13. The method of claim 11 , wherein the immobilized substrate comprises a phosphorylated peptide and the product comprises a dephosphorylated peptide, or wherein the immobilized substrate comprises a dephosphorylated peptide and the product comprises a phosphorylated peptide.
14. The method of claim 11 , wherein the immobilized substrate comprises a methylated peptide and the product comprises a demethylated peptide or wherein the immobilized substrate comprises a demethylated peptide and the product comprises a methylated peptide.
15. The method of claim 1 , further comprising washing the surface after immobilizing the cell on the surface and before lysing the cell to remove all cells not immobilized onto the surface.
16. The method of claim 1 , wherein the surface comprises a second immobilized substrate that associates with a second enzyme in the cell lysate to form a second product, the second product having a different mass than the second substrate.
17. The method of claim 1 , wherein the lysate further comprises a potential modulator of binding of the enzyme and the immobilized substrate; and the activity of the enzyme assayed indicates the potential modulator's effect on the binding of the enzyme and the immobilized substrate in the presence of the potential modulator.
18. The method of claim 17 , wherein the lysate comprises a second potential modulator of binding of the second enzyme and the second immobilized substrate; and the activity of the second enzyme assayed indicates the second potential modulator's effect on the binding of the second enzyme and the second immobilized substrate in the presence of the second potential modulator.
19. The method of claim 18 , wherein the potential modulator or the second potential modulator is an inhibitor of the enzyme and immobilized substrate binding, or wherein the potential modulator or the second potential modulator is an activator of the enzyme and immobilized substrate binding.
20. The method of claim 1 , wherein the PPL tip array comprises a compressible elastomeric polymer comprising a plurality of non-cantilevered tips each having a radius of curvature of less than 1 μm and a common substrate comprising a compressible elastomeric polymer, the tip array and the common substrate mounted onto a rigid support and the tip array, common substrate, and rigid support together being at least translucent.