Method and device for protein preparation

Abstract

The present invention provides a method of preparing a sample comprising one or more proteins of interest, the method comprising: providing a sample comprising a population of proteins of interest solubilised with a surfactant in a medium; exposing said sample to a mild precipitant to cause precipitation of said proteins; during or after the precipitation step, bringing said sample into contact with a matrix adapted to capture said precipitated proteins and prevent excessive aggregation of precipitated protein particles; and washing the matrix with captured precipitated proteins to remove the surfactant. A sample preparation device to carry out the same is also provided.

1 - 48 . (canceled) 
     
     
         49 . A method of preparing a sample comprising one or more proteins of interest, the method comprising the ordered steps of:
 providing a sample comprising a population of proteins of interest solubilised with a surfactant in a medium;   exposing said sample to a mild precipitant to cause precipitation of said proteins;   wherein the mild precipitant causes the protein solubilised with surfactant to precipitate to form a suspension of particles of protein, wherein said particles of protein are sensitive to protease digestion;   during or after the precipitation step, bringing said sample into contact with a matrix that is a depth filter adapted to capture said precipitated protein particles and prevent excessive aggregation of precipitated protein particles to thereby allow a protease to digest the protein particles in situ;   washing the matrix containing the captured precipitated protein particles to remove the surfactant; and   after washing the matrix, digesting the protein particles in situ.   
     
     
         50 . The method of  claim 49  wherein the matrix is porous or fibrous material which is able to be penetrated by the medium comprising the proteins and which permits the precipitated proteins to be reversibly captured by the matrix. 
     
     
         51 . The method of  claim 49  wherein the matrix comprises one or more of quartz, glass, polymer and cellulose materials. 
     
     
         52 . The method of  claim 49 , wherein the matrix: is adapted to capture and retain protein particles of 10 μm or smaller; is substantially inert with respect to the proteins in the sample such that undesirable reactions between the matrix and the sample are avoided; is able to reversibly capture proteins from the sample; and does not bind to, and therefore retain, the surfactant. 
     
     
         53 . The method of  claim 49  wherein the mild precipitant is an acid. 
     
     
         54 . The method of  claim 53  wherein the mild precipitant is phosphoric acid. 
     
     
         55 . The method of  claim 49  wherein the matrix is permeated with an aqueous methanolic solution, prior to introduction of the precipitated protein. 
     
     
         56 . The method of  claim 49 , further comprising exposing the matrix with protein particles captured thereon to a protease to generate fragments of the proteins. 
     
     
         57 . The method of  claim 56 , further comprising the step of eluting the proteins and/or fragments thereof from the matrix. 
     
     
         58 . The method of  claim 57 , wherein proteins or protein fragments are eluted from the matrix and then pass to a secondary matrix. 
     
     
         59 . The method of  claim 58 , comprising eluting the protein fragments from the secondary matrix using a suitable elution solution. 
     
     
         60 . The method of  claim 55 , wherein the protein solubilised with surfactant immediately forms a suspension of particles of protein after the addition of the solution comprising the mild precipitant and solubilised protein to the aqueous methanolic solution permeating the matrix. 
     
     
         61 . The method of  claim 49 , wherein the method does not comprise the use of urea. 
     
     
         62 . A method of preparing a sample comprising one or more proteins of interest, the method comprising the ordered steps of:
 providing a sample comprising a population of proteins of interest solubilised with a surfactant in a medium;   exposing said sample to a precipitant, wherein said precipitant solubilises the surfactant, causes precipitation of said proteins as a suspension of particles of protein, and does not render the precipitated proteins insensitive to protease digestion;   during or after the precipitation step,   bringing said sample into contact with a matrix that is a depth filter having a network of pore channels adapted to capture said precipitated protein particles throughout the depth filter and thereby prevent excessive aggregation of precipitated protein particles at a surface of initial contact between the precipitated protein particles and the depth filter, and   wherein further said matrix does not bind the solubilised surfactant; and   digesting the captured precipitated protein particles in situ using a protease.   
     
     
         63 . The method of  claim 62 , wherein the matrix is porous or fibrous material which is able to be penetrated by the medium comprising the proteins and which permits the precipitated proteins to be reversibly captured by the matrix. 
     
     
         64 . The method of  claim 62 , wherein the matrix comprises one or more of quartz, glass, polymer and cellulose materials. 
     
     
         65 . The method of  claim 62 , wherein the matrix: is adapted to capture and retain protein particles of 10 μm or smaller; is substantially inert with respect to the proteins in the sample such that undesirable reactions between the matrix and the sample are avoided; and is able to reversibly capture proteins from the sample. 
     
     
         66 . The method of  claim 62 , wherein the precipitant is an acid. 
     
     
         67 . The method of  claim 66 , wherein the acid is phosphoric acid. 
     
     
         68 . The method of  claim 62 , wherein the matrix is permeated with an aqueous methanolic solution prior to introduction of the precipitated protein. 
     
     
         69 . The method of  claim 62 , further comprising the step of eluting fragments of the digested protein particles from the matrix. 
     
     
         70 . The method of  claim 69 , wherein the fragments of the digested protein particles are eluted from the matrix and then pass to a secondary matrix. 
     
     
         71 . The method of  claim 70 , comprising eluting the protein fragments from the matrix using a suitable elution solution. 
     
     
         72 . The method of  claim 68 , wherein the protein solubilised with surfactant immediately forms a suspension of particles of protein after the addition of the solution comprising the precipitant and solubilised protein to the aqueous methanolic solution permeating the matrix. 
     
     
         73 . The method of  claim 62 , further comprising the step of washing the matrix with the captured precipitated protein particles to remove the surfactant, wherein the washing step is performed after the precipitation step and before the step of digesting the protein particles in situ. 
     
     
         74 . The method of  claim 62 , further comprising the step of washing the matrix with the captured precipitated protein particles to remove the surfactant, wherein the washing step is performed after the precipitation step and before the step of digesting the protein particles in situ, and wherein an aqueous methanolic solution is used in the washing step. 
     
     
         75 . The method of  claim 62 , wherein the precipitated protein particles are captured throughout the depth filter on the basis of particle size, and wherein the step of digesting the captured protein particles in situ comprises digesting the protein particles to fragments having sizes which are smaller than the network of pore channels in the depth filter matrix to facilitate elution of the digested protein particle fragments from the depth filter.