Biological sample component purification and differential display

Abstract

Provided are affinity support materials having intermediate binding affinity for biological samples. Among the materials provided by the present invention are hydrophilic solid supports composed of hydrophilic ligands coupled to hydrophilic matrixes which are compatible with biological samples, for example, a cell line, a biological fluid such as blood, or a tissue cell lysate. The ligands may include affinity property groups and hydrophilic groups pendent from a backbone, and be configured to at least partially resolve components of a biological sample. Affinity supports in accordance with the present invention may be used in a variety of techniques and apparatuses to achieve improved separations of complex biological samples and thereby enhance the results of biological sample component fractionations, enrichments, purifications, expression product determinations and comparisons, and other biological sample processing techniques. In addition, the affinity supports may be included in kits useful in processing biological samples.

1 . A method of processing a biological sample, comprising: 
 applying the biological sample to an affinity support comprising a ligand coupled to a biological sample-compatible hydrophilic matrix, said ligand comprising a backbone having a plurality of affinity property groups and hydrophilic groups pendent therefrom, and said ligand having a binding affinity characterized by a specificity for one or more components of the biological sample that is intermediate between charge-based and antibody-based ligands and being configured to at least partially resolve one or more components of said biological sample; and    fractionating biological sample components of said biological sample using the affinity support to thereby provide an enriched fraction.    
     
     
         2 . The method of  claim 1 , wherein the one or more biological sample components comprise proteins.  
     
     
         3 . The method of  claim 1 , wherein the one or more biological sample components comprise nucleotides.  
     
     
         4 . The method of  claim 1 , wherein said ligand comprises: 
 a peptoid backbone; and    a plurality of affinity property groups and hydrophilic groups being pendent from said peptoid backbone.    
     
     
         5 . The method of  claim 4 , wherein said hydrophilic groups are intercalated with said affinity property groups.  
     
     
         6 . The method of  claim 5 , wherein said hydrophilic groups alternate with said affinity property groups along said peptoid backbone.  
     
     
         7 . The method of  claim 6 , wherein said affinity property groups are selected from the group consisting of alkyl, (cycloalkyl)alkyl, (cycloheteroalkyl)alkyl, aralkyl, and heteroaralkyl, each substituted optionally from the group consisting of oxo, thia, halo, amino, hydroxy, cyano, nitro, thio, aminocarbonyl, carboxy, and imino.  
     
     
         8 . The method of  claim 7 , wherein said affinity property groups are selected from the group consisting of methyl, hydroxymethyl, prop-2-yl, 2-methylpropyl, pyrrolidylmethyl, methylthioethyl, 1-hydroxyethyl, thiomethyl, aminocarbonylmethyl, aminocarbonylethyl, carboxymethyl, carboxyethyl, 4-aminobutyl, and 3-guanidinopropyl, guanidinoaryl, hydroxyaryl, amidoalkyl, phosphonyl alkyl, phosphonyl aryl, oligoether, and polyhydroxyalkyl.  
     
     
         9 . The method of  claim 7 , wherein said affinity property groups are selected from the group consisting of optionally substituted aralkyl and heteroaralkyl.  
     
     
         10 . The method of  claim 9 , wherein said affinity property groups are selected from the group consisting of phenylmethyl, hydroxyphenylmethyl, imidazolylmethyl, purinylmethyl, pyrimidinylmethyl, and indolylmethyl.  
     
     
         11 . The method of  claim 7 , wherein said affinity groups are selected from the group consisting of optionally substituted amonioalkyl and trialkylamonioalkyl.  
     
     
         12 . The method of  claim 7 , wherein said affinity property groups are optionally substituted carboxylatoalkyl.  
     
     
         13 . The method of  claim 4 , wherein said hydrophilic groups are selected from the group consisting of alkyloxyalkylenyl, aminoalkyl, alkylaminoalkyl, quaternary ammoniumalkyl, hydroxyalkyl, thioalkyl, alkylthioalkylenyl, carboxyalkyl, alkyloxycarbonylalkyl, and aminocarbonylalkyl.  
     
     
         14 . The method of  claim 4 , wherein said hydrophilic group is alkyloxyalkyl.  
     
     
         15 . The method of  claim 4 , wherein said hydrophilic group is selected from the group consisting of methoxyethyl, hydroxyethyl, 1-hydroxyethyl-2-hydroxyethyl, and 2,3-dihydroxypropyl.  
     
     
         16 . The method of  claim 4 , wherein about 50% of said pendant groups are affinity property groups.  
     
     
         17 . The method of  claim 16 , wherein about 33% of said pendant affinity property groups have a common affinity property.  
     
     
         18 . The method of  claim 16 , wherein about 67% of said pendant affinity property groups have a common affinity property.  
     
     
         19 . The method of  claim 16 , wherein about 100% of said pendant affinity property groups have a common affinity property.  
     
     
         20 . The method of  claim 4 , wherein said affinity property groups and said hydrophilic groups are pendant from nitrogen atoms in the backbone.  
     
     
         21 . The method of  claim 4 , wherein said biological sample is derived from a homogeneous source.  
     
     
         22 . The method of  claim 21 , wherein said homogeneous source is a cell line.  
     
     
         23 . The method of  claim 4 , wherein said biological sample is derived from a heterogeneous source.  
     
     
         24 . The method of  claim 23 , wherein said heterogeneous source is one or more tissue samples.  
     
     
         25 . The method of  claim 23 , wherein said heterogeneous source is one or more blood samples.  
     
     
         26 . The method of  claim 1 , wherein the intermediate binding affinity is characterized by the ligand interacting with the components of the biological sample by a combination of non-specific molecular forces consisting essentially of ionic, van der Waal's and hydrogen bond interactions.  
     
     
         27 . The method of  claim 4 , wherein the ligand is selected from the group consisting of the following:  
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         28 . The method of  claim 1 , wherein said fractionation comprises chromatographically resolving the one or more components of the biological sample to provide thereby the enriched fraction.  
     
     
         29 . The method of  claim 28 , further comprising: 
 determining a biological sample component expression pattern for the biological sample using the enriched fraction in at least one of an electrophoretic and a mass spectroscopic technique; and    repeating the applying, resolving and determining steps for additional biological samples; and    comparing biological sample phenotypes by analyzing differences in biological sample component expression patterns among a plurality of the biological samples applied.    
     
     
         30 . The method of  claim 29 , wherein said biological sample components comprise proteins and said analyzing differences comprises determining the presence or absence of one or more of said proteins.  
     
     
         31 . The method of  claim 30 , wherein determining the presence or absence of one or more proteins further comprises determining the abundance of one or more proteins.  
     
     
         32 . The method of  claim 29 , further comprising applying the biological samples to a second peptoid, and analyzing the differences in biological sample component expression patterns determined using each peptoid.  
     
     
         33 . The method of  claim 29 , wherein one of said biological samples is derived from a healthy tissue sample and another of said biological samples is derived from a diseased tissue sample.  
     
     
         34 . The method of  claim 29 , wherein one of said biological samples is derived from a healthy tissue sample of a first type and another of said biological samples is derived from a diseased tissue sample of the same cell type.  
     
     
         35 . The method of  claim 29 , wherein one of said biological samples is derived from a diseased tissue sample of a first type and another of said biological samples is derived from a diseased tissue sample of a second type.  
     
     
         36 . The method of  claim 1 , further comprising selecting an affinity support for reducing the complexity of the biological sample, comprising: 
 applying a portion of the biological sample to each of a plurality of affinity supports, said plurality of affinity supports comprising an array of peptoids configured to at least partially resolve biological sample components, which peptoids are coupled to biological sample-compatible matrixes;    identifying differences in complexity reduction achieved by different the supports; and    selecting a support using said differences.    
     
     
         37 . The method of  claim 36 , wherein said plurality of affinity supports is provided in one or more multi-compartment containment structures.  
     
     
         38 . The method of  claim 37 , wherein said plurality of affinity supports is provided in a plurality of multi-compartment containment structures.  
     
     
         39 . The method of  claim 40 , further comprising at least one of identification and quantitation of the components of the biological sample.  
     
     
         40 . The method of  claim 41 , wherein said identification and/or quantitation includes mass spectral analysis.  
     
     
         41 . The method of  claim 38 , comprising: 
 applying said biological sample to a first multi-compartment containment structure wherein one or more compartments contains a plurality of affinity supports;    identifying differences in complexity reduction achieved by supports in different compartments of the first multi-compartment containment structure;    selecting a support-containing compartment from the first multi-compartment containment structure using said differences;    applying said biological sample component mixture sample to a second multi-compartment containment structure wherein separate compartments contain the one or more supports from said selected support-containing compartment from the first multi-compartment containment structure;    identifying differences in complexity reduction achieved by different supports of the selected support-containing compartment from the first multi-compartment containment structure; and    selecting a support from the array and represented in said second multi-compartment containment structure using said differences.    
     
     
         42 . The method of  claim 4 , where said hydrophilic peptoid coupled to a hydrophilic biological sample-compatible matrix, is prepared by a procedure comprising:  
       synthesizing a peptoid on a hydrophobic solid phase substrate, said peptoid comprising a peptoid backbone having affinity property and hydrophilic groups pendent therefrom wherein about 50% of said pendant groups are affinity property groups, said peptoid being configured to at least partially resolve biological sample components of a biological sample; 
 terminating said peptoid with a chemoselective ligation group;  
 cleaving said peptoid from said hydrophobic solid phase substrate; and  
 linking said peptoid to a hydrophilic solid phase substrate with the chemoselective ligation group.  
 
     
     
         43 . The method of  claim 42 , wherein about 33% of said pendant affinity property groups have a common affinity property.  
     
     
         44 . The method of  claim 42 , wherein about 67% of said pendant affinity property groups have a common affinity property.  
     
     
         45 . The method of  claim 42 , wherein about 100% of said pendant affinity property groups have a common affinity property.  
     
     
         46 . The method of  claim 4 , wherein said hydrophilic peptoid coupled to a hydrophilic biological sample-compatible matrix, is prepared by a procedure comprising: 
 synthesizing a peptoid on a hydrophobic solid phase substrate, said peptoid comprising a peptoid backbone having affinity property and hydrophilic groups pendent therefrom wherein about 50% of said pendant groups are affinity property groups, said peptoid being configured to at least partially resolve biological sample components of a biological sample; and    converting said hydrophobic substrate to a hydrophilic substrate.    
     
     
         47 . The method of  claim 46 , wherein about 33% of said pendant affinity property groups have a common affinity property.  
     
     
         48 . The method of  claim 46 , wherein about 67% of said pendant affinity property groups have a common affinity property.  
     
     
         49 . The method of  claim 46 , wherein about 100% of said pendant affinity property groups have a common affinity property.  
     
     
         50 . The method of  claim 46 , wherein said hydrophobic substrate is converted to a hydrophilic substrate by a deprotection reaction removing a hydrophobic protecting group.  
     
     
         51 . The method of  claim 1 , further comprising conducting said applying and fractionating steps with additional biological samples in parallel to thereby generate enriched fractions.  
     
     
         52 . The method of  claim 51 , wherein said enriched fractions comprise portions of each biological sample that do not bind to the affinity supports.  
     
     
         53 . The method of  claim 51 , wherein said enriched fractions comprise portions of each biological sample that initially bind to the affinity supports.  
     
     
         54 . The method of  claim 51 , wherein said enriched fractions comprise portions of each biological sample that are eluted from the affinity supports as a single elution fraction.  
     
     
         55 . The method of  claim 51 , wherein said enriched fractions comprise portions of each biological sample that are eluted from the affinity supports as a plurality of elution fractions.  
     
     
         56 . The method of  claim 1 , further comprising: 
 applying the enriched fraction to a second affinity support comprising a ligand coupled to a biological sample-compatible hydrophilic matrix, said ligand comprising a backbone having a plurality of affinity property groups and hydrophilic groups pendent therefrom, and said ligand having a binding affinity characterized by a specificity for one or more components of the biological sample that is intermediate between charge-based and antibody-based ligands and being configured to at least partially resolve one or more components of said biological sample according to a second affinity interaction with said biological sample; and    fractionating biological sample components of the enriched sample fraction using the second affinity support to provide a twice enriched fraction.    
     
     
         57 . The method of  claim 56 , further comprising one or more additional applications and fractionations of a previously enriched fraction using one or more additional affinity supports, each of said one or more additional supports comprising a ligand coupled to a biological sample-compatible hydrophilic matrix, said ligand comprising a backbone having a plurality of affinity property groups and hydrophilic groups pendent therefrom, and said ligand having a binding affinity characterized by a specificity for one or more components of the biological sample that is intermediate between charge-based and antibody-based ligands and being configured to at least partially resolve one or more components of said biological sample according to an additional affinity interaction with said previously enriched fraction.  
     
     
         58 . An affinity support, comprising: 
 a ligand coupled to a biological sample-compatible hydrophilic matrix, said ligand comprising, 
 a hydrophilic peptoid, comprising, 
 a peptoid backbone, and  
 a plurality of affinity property groups and hydrophilic groups being pendent from said peptoid backbone, and  
 said ligand having a binding affinity characterized by a specificity for one or more components of a biological sample that is intermediate between charge-based and antibody-based ligands,  
 wherein about 50% of said pendant groups are affinity property groups.  
 
   
     
     
         59 . The support of  claim 58 , wherein about 33% of said pendant affinity property groups have a common affinity property.  
     
     
         60 . The support of  claim 58 , wherein about 67% of said pendant affinity property groups have a common affinity property.  
     
     
         61 . The support of  claim 58 , wherein about 100% of said pendant affinity property groups have a common affinity property.  
     
     
         62 . The support of  claim 58 , wherein said hydrophilic groups are intercalated with said affinity property groups.  
     
     
         63 . The support of  claim 62 , wherein said hydrophilic groups alternate with said affinity property groups along said peptoid backbone.  
     
     
         64 . The support of  claim 58 , wherein said peptoid is coupled to a biological sample-compatible matrix using a chemoselective ligation group.  
     
     
         65 . The support of  claim 58 , wherein the support is combined with one or more additional such affinity supports and provided in separate compartments of a multi-compartment containment structure as part of a kit.  
     
     
         66 . The support of  claim 65 , wherein said multi-compartment containment structure comprises a well plate.  
     
     
         67 . The support of  claim 66 , wherein said well plate is a fritted well plate.  
     
     
         68 . The support of  claim 66 , wherein said multi-compartment containment structure comprises a patterned chip.  
     
     
         69 . The support of  claim 68 , wherein said chip comprises a substrate of a material selected from Si, Au, Al and glass.  
     
     
         70 . The support of  claim 58 , wherein the intermediate binding affinity is characterized by the ligand interacting with the components of the biological sample by a combination of non-specific molecular forces consisting essentially of ionic, van der Waal's and hydrogen bond interactions.  
     
     
         71 . The support of  claim 58 , wherein the ligand is selected from the group consisting of the following: