Cellular assays with a molecular endpoint measured by samdi mass spectrometry

Abstract

The disclosure provides a cell-based, label-free assay compatible with high-throughput screening (HTS) that can report quantitatively on enzyme activities by measuring mass changes of substrates with MALDI-mass spectrometry.

What is claimed is: 
     
         1 . A method of assaying activity of an intracellular enzyme, comprising:
 (a) contacting a cell and a surface, the surface comprising an immobilized cell adhesion ligand and an immobilized substrate for the enzyme, the contacting resulting in immobilization of the cell via interaction between the cell and the immobilized cell adhesion ligand;   (b) contacting the 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   (c) 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 a multi-well plate. 
     
     
         3 . The method of  claim 1  or  2 , wherein the surface comprises gold, silver, or copper. 
     
     
         4 . The method of any one of  claims 1 - 3 , wherein more than one cell is applied to the monolayer. 
     
     
         5 . The method of  claim 4 , wherein 2, 5, 10, 20, 50, or 100 cells are applied to the monolayer. 
     
     
         6 . The method of any one of  claims 1 - 5 , wherein at least one of the immobilized substrate and the cell adhesion ligand comprises a peptide. 
     
     
         7 . The method of  claim 6 , wherein the peptide is bound to the surface via a cysteine residue. 
     
     
         8 . The method of any one of  claims 1 - 7 , wherein the cell adhesion ligand comprises a RGD peptide. 
     
     
         9 . The method of any one of  claims 1 - 8 , wherein at least one of the cell adhesion ligand and the immobilized substrate is bound to the surface via a linker. 
     
     
         10 . The method of  claim 9 , wherein the linker has a structure of formula I: 
       
         
           
           
               
               
           
         
       
       and Lig comprises the cell adhesion ligand or the immobilized substrate. 
     
     
         11 . The method of  claim 9 , wherein the surface comprises a monolayer. 
     
     
         12 . The method of  claim 11 , wherein the monolayer comprises (i) the linker and (ii) an ethylene glycol and a C 2-20 alkylene moiety. 
     
     
         13 . The method of  claim 11  or  12 , wherein the monolayer is attached to the surface via a thiol bond. 
     
     
         14 . The method of any one of  claims 1 - 13 , wherein the enzyme is a deacetylase, acetyltransferase, esterase, phosphorylase/kinase, phosphatase, protease, methylase, demethylase, or a DNA or RNA modifying enzyme. 
     
     
         15 . The method of  claim 14 , wherein the deacetylase is KDAC8. 
     
     
         16 . The method of  claim 14 , wherein the esterase is cutinase or acetylcholine esterase. 
     
     
         17 . The method of  claim 14 , wherein the protease is TEV. 
     
     
         18 . The method of any one of  claims 14 - 17 , wherein the immobilized substrate comprises an acylated peptide and the product comprises a deacylated peptide. 
     
     
         19 . The method of any one of  claims 14 - 17 , wherein the immobilized substrate comprises a deacylated peptide and the product comprises an acylated peptide. 
     
     
         20 . The method of  claim 14 , wherein the immobilized substrate comprises a phosphorylated peptide and the product comprises a dephosphorylated peptide. 
     
     
         21 . The method of  claim 14 , wherein the immobilized substrate comprises a dephosphorylated peptide and the product comprises a phosphorylated peptide. 
     
     
         22 . The method of  claim 14 , wherein the immobilized substrate comprises a methylated peptide and the product comprises a demethylated peptide. 
     
     
         23 . The method of  claim 14 , wherein the immobilized substrate comprises a demethylated peptide and the product comprises a methylated peptide. 
     
     
         24 . The method of any one of  claims 1 - 23 , 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. 
     
     
         25 . The method of any one of  claims 1 - 24 , 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. 
     
     
         26 . The method of any one of  claims 1 - 25 , wherein the lysate 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. 
     
     
         27 . The method of any one of  claims 1 - 26 , 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. 
     
     
         28 . The method of  claim 26  or  27 , wherein the potential modulator or the second potential modulator is an inhibitor of the enzyme and immobilized substrate binding. 
     
     
         29 . The method of  claim 26  or  27 , wherein the potential modulator or the second potential modulator is an activator of the enzyme and immobilized substrate binding.