Device and method for the determination of protein domain boundaries

Abstract

A method and device to assist in the determination of protein domain boundaries is described. In particular the device is designed to provide a high throughput of proteolytic digestion of proteins to identify domains and their boundaries, for use in protein structure determination, in a manner that is amenable to automation. Proteases are immobilized in a convenient format such as a microtitre plate and preferably arranged in a matrix thereby allowing for simultaneous degradation of a protein by a number of proteases at a number of concentrations.

We claim:  
     
         1 . A method for the preparation of proteolytically digested fragments of a protein, free of protease, for the determination of domains and the boundaries of the domains in said protein, the method comprises: 
 (1) contacting a quantity of said protein with at least two concentrations of a protease under conditions which result in digested fragments of said protein, wherein each of said concentrations of protease is immobilized in a separate compartment; and    (2) separating said fragments from said immobilized protease.    
     
     
         2 . A method according to  claim 1  wherein said compartment is in an apparatus which comprises a plurality of compartments.  
     
     
         3 . A method according to  claim 2  wherein each compartment containing a quantity of said protease contains a concentration of said protease distinct from the concentration of said protease in every other compartment.  
     
     
         4 . A method according to  claim 3  wherein at least three concentrations of protease are each in one of at least three different said compartments.  
     
     
         5 . A method according to  claim 4  wherein said plurality of compartments forms a matrix in a linear format with at least three increasing concentrations of protease in said compartments.  
     
     
         6 . A method according to anyone of claims  1 - 5  wherein said matrix contains more than one protease.  
     
     
         7 . A method according to  claim 6  wherein said matrix is in a two-dimensional format wherein: (i) distinct concentrations of each protease are aligned in increasing concentration, along a first axis or concentration axis; and (ii) different proteases are aligned along a second axis that is perpendicular to the first axis.  
     
     
         8 . A method according to anyone of claims  1 - 7  wherein said protease is selected from the group consisting of aminopeptidase M; bromelain; carboxypeptidase A, B and Y; chymopapain; chymotrypsin; clostripain; collagenase; elastase; endoproteinase Arg-C, Glu-C and LysC; Factor Xa; ficin; Gelatinase; kallikrein; metalloendopeptinidase; papain; pepsin; plasmin; plasminogen; peptidase; pronase; proteinase A; proteinase K; subsilisin; thermolysin; thrombin; and trypsin.  
     
     
         9 . A method according to  claim 8  wherein the proteases are trypsin, chymotrypsin, proteinase K or papain.  
     
     
         10 . A method according to  claim 9  wherein the concentrations of each of the proteases along the first axis in each case is from about 50 μg/mL to about 0.5 μg/mL.  
     
     
         11 . A method according to  claim 10  wherein the concentration for each of the proteases is about 50 μg/mL in one container of a concentration axis; about 25 μg/mL in a second container of the concentration axis; about 5 μg/mL in a third container of the concentration axis; about 2.5 μg/mL in a fourth container of the concentration axis; about 0.5 μg/mL in a fifth container of the concentration axis; about 0 μg/mL in a sixth container of the concentration axis.  
     
     
         12 . A method according to any one of claims  1 - 11  wherein the surface upon which said protease is immobilized has been treated with a blocker of surface interaction.  
     
     
         13 . A method according to  claim 12  wherein the blocker is selected from the group consisting of BSA and beta-octyl glucoside.  
     
     
         14 . A method according to claim  1 - 13  wherein the apparatus may be prepared and stored prior to use.  
     
     
         15 . A method according to any one of  claims 1  to  13  wherein the surface to which said protease is immobilized is selected from the group consisting of an organic support of material selected from the group consisting of polyesters, polyamides, polyacrylates, polymethacrylates, polyacrylamides, poly(acrylic acid)m, poly(methacrylic acid); poly(galacturonic acid); poly(aspartic acid); ethylene-maleic anhydride copolymers; polyolefins; cellulose; cellulose derivatives; agarose gels; dextran gels and derivatives thereof, polysaccharides; polypeptides; collagen; and an inorganic support material selected from the group consisting of siliceous and nonsiliceous metal oxides.  
     
     
         16 . A method according to any one of  claims 1  to  14  wherein said apparatus is a microtitre plate.  
     
     
         17 . A method according to  claim 16  wherein one of the conditions is time for incubation and is between about 2 to 4 hours.  
     
     
         18 . A method according to  claim 17  wherein one of the conditions is temperature for a reaction and is room temperature.  
     
     
         19 . A method according to anyone of  claims 1  to  18  wherein only limited digestion of said protein occurs.  
     
     
         20 . A method for determining the boundaries of a proteolytically digested fragment of a protein which comprises the steps: (i) incubating a protein, according to the method of any one of claims  1 - 19  to yield protein fragments; (ii) isolatation of fragment(s) of interest; (iii) determining the mass of the isolated fragment(s); and (iv) matching the mass of the proteolytic fragment(s) to a protein amino acid sequence of the protein digested.  
     
     
         21 . A method according to  claim 20  wherein the determination of mass is by a method selected from the group consisting of nanospray time-of-flight mass spectrometry and matrix-assisted time-of-flight mass spectrometry.  
     
     
         22 . A method according to  claim 21  wherein the protein fragment isolation is carried out by a technique selected from the group consisting of HPLC and SDS PAGE.  
     
     
         23 . A method according to  claim 20 ,  21 , or  22  which is automated.  
     
     
         24 . An apparatus for degradation of a protein, said apparatus comprising a plurality of compartments, each compartment containing a quantity of a protease immobilized on a surface in each compartment wherein at least two compartments contain different concentrations of said protease.  
     
     
         25 . An apparatus according to  claim 24  wherein each compartment containing a quantity of said protease contains a concentration of said protease distinct from the concentration of said protease in every other compartment.  
     
     
         26 . An apparatus according to  claim 25  wherein at least three concentrations of protease are in said compartments.  
     
     
         27 . An apparatus according to  claim 26  wherein said plurality of compartments forms a matrix in a linear format with at least three increasing concentrations of protease in said compartments.  
     
     
         28 . An apparatus according to anyone of claims  24 - 27  wherein said matrix contains more than one protease.  
     
     
         29 . An apparatus according to  claim 28  wherein said matrix is in a two-dimensional format wherein: (i) distinct concentrations of each protease are aligned in increasing concentration, along a first axis or concentration axis; and (ii) different proteases are aligned along a second axis that is perpendicular to the first axis.  
     
     
         30 . An apparatus according to anyone of claims  24 - 29  wherein said protease is selected from the group consisting of aminopeptidase M; bromelain; carboxypeptidase A, B and Y; chymopapain; chymotrypsin; clostripain; collagenase; elastase; endoproteinase Arg-C, Glu-C and LysC; Factor Xa; ficin; Gelatinase; kallikrein; metalloendopeptinidase; papain; pepsin; plasmin; plasminogen; peptidase; pronase; proteinase A; proteinase K; subsilisin; thermolysin; thrombin; and trypsin.  
     
     
         31 . An apparatus according to  claim 30  wherein the proteases are trypsin, chymotrypsin, proteinase K or papain.  
     
     
         32 . An apparatus according to  claim 31  wherein the concentrations of each of the proteases along the first axis in each case is from about 50 μg/mL to about 0.5 μg/mL.  
     
     
         33 . An apparatus according to  claim 32  wherein the concentration for each of the proteases is about 50 μg/mL in one container of a concentration axis; about 25 μg/mL in a second container of the concentration axis; about 5 μg/mL in a third container of the concentration axis; about 2.5 μg/mL in a fourth container of the concentration axis; about 0.5 μg/mL in a fifth container of the concentration axis; about 0 μg/mL in a sixth container of the concentration axis.  
     
     
         34 . An apparatus according to any one of claims  24 - 33  wherein the surface upon which said protease is immobilized has been treated with a blocker of surface interaction.  
     
     
         35 . An apparatus according to  claim 34  wherein the blocker is selected from the group consisting of BSA and beta-octyl glucoside.  
     
     
         36 . An apparatus according to any one of  claims 24  to  33  wherein the surface to which said protease is immobilized is selected from the group consisting of an organic support of material selected from the group consisting of polyesters, polyamides, polyacrylates, polymethacrylates, polyacrylamides, poly(acrylic acid)m, poly(methacrylic acid); poly(galacturonic acid); poly(aspartic acid); ethylene-maleic anhydride copolymers; polyolefins; cellulose; cellulose derivatives; agarose gels; dextran gels and derivatives thereof, polysaccharides; polypeptides; collagen; and an inorganic support material selected from the group consisting of siliceous and nonsiliceous metal oxides.  
     
     
         37 . An apparatus according to claim  24 - 36  wherein the apparatus may be prepared and stored prior to use.  
     
     
         38 . An apparatus according to any one of  claims 24  to  37  wherein said apparatus is a microtitre plate.