Protein capture membrane and method of use thereof

In one aspect, the invention provides a protein capture membrane comprising a first side and a second side and a plurality of interstices extending contiguously from the first side to the second side, wherein the interstices are coated with a protein-reactive coating; and the porous substrate comprises nanoporous alumina or porous glass. In another aspect the invention provides a method of detecting a protein of interest in a plurality of proteins.

BACKGROUND OF THE INVENTION

The absence of a practical, cost effective, high-throughput technique for proteomics creates a bottleneck in a variety of areas of biotechnology, medicine and research in general. There is a need in the art for a practical method of identifying and quantifying a large number of proteins present in complex mixtures. The present invention addresses this need.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a protein capture membrane comprising a porous substrate comprising: a first side and a second side and a plurality of interstices extending contiguously from the first side to the second side, wherein the interstices are coated with a protein-reactive coating; and the porous substrate comprises nanoporous alumina or porous glass.

In various embodiments, the interstices have a diameter of about 500 nm or less than about 500 nm.

In various embodiments, the porous substrate has a thickness from the first side to the second side of about 50-100 μm.

In various embodiments, the porous substrate has a thickness from the first side to the second side of about 100 μm.

In various embodiments, the protein-reactive coating comprises a silane derivative.

In various embodiments, the silane derivative is covalently bound to the nanoporous alumina substrate.

In various embodiments, the protein-reactive coating is selected from the group consisting of:

In various embodiments, the protein-reactive coating is triethoxysilylundecanal

In various embodiments, the protein-reactive coating is selected from the group consisting of 3-thiocyanatopropyltriethoxysilane, triethoxysilylundecanal, trimethoxy[2-(7-oxabicyclo[4.1.0]hept-3-yl)ethyl]silane, (3-glycidyloxypropyl)triethoxysilane, 3-isocyanatopropyltriethoxysilane, N-[5-(trimethoxysilyl)-2-aza-1-oxopentyl]caprolactam, 11-(succinimidyloxy)undecyldimethylethoxysilane, 2-(4-chlorosulfonylphenyl)ethyltrimethoxysilane and triethoxysilylbutyraldehyde.

In various embodiments, the invention provides a method of transferring at least one protein of interest in a plurality of proteins to the protein capture membrane comprising: electrophoretically transferring the at least one protein of interest to the porous substrate.

In various embodiments, the invention provides a method of transferring at least one protein of interest to the protein capture membrane, wherein the porous substrate is comprises material with intrinsic protein covalent binding character.

In another aspect, the invention provides a method of detecting at least one protein of interest in a plurality of proteins, the method comprising: contacting the plurality of proteins with a porous substrate comprising: a first side and a second side, and a plurality of interstices extending contiguously from the first side to the second side, wherein the interstices are coated with a protein-reactive coating, thereby covalently binding at least a portion of the plurality of proteins to the protein-reactive coating, exposing the covalently bound plurality of proteins to a first molecule that binds the at least one protein of interest; and detecting the first molecule that binds at least one protein of interest, thereby detecting the at least one protein of interest; wherein contacting the plurality of proteins with the porous substrate comprises electrophoretic transfer of the proteins to the porous substrate.

In various embodiments, the porous substrate comprises nanoporous alumina or porous glass.

In various embodiments, the first molecule is selected from the group consisting of an antibody, an aptamer and a protein.

In various embodiments, contacting the plurality of proteins with the porous substrate comprises: separating the plurality of proteins using electrophoresis. In various embodiments, the plurality of proteins are separated using various biochemical techniques, including but not limited to polyacrylamide gel electrophoresis, and subsequently at least a portion of the separated proteins are transferred to the porous substrate.

In various embodiments, the method further comprises: stripping the first molecule that binds at least one protein of interest and exposing the covalently bound plurality of proteins to a second molecule that binds the same or a different protein of interest; and detecting the second molecule, thereby detecting the at least one protein of interest.

In various embodiments, the second molecule is selected from the group consisting of an antibody, an aptamer and a protein.

DETAILED DESCRIPTION

Definitions

As used herein, the term “stripping” is used to describe the removal of a detector molecule that specifically binds a protein of interest and may include steps of rinsing and/or preparing the sample for a second analysis. In the context of a Western blot, stripping refers to removal of an antibody, typically before probing with another antibody. As the term is defined herein, it can refer to the analogous operation with respect to any detector molecule.

Description

Protein Capture Membrane

In one aspect, the invention provides a protein capture membrane comprising a porous substrate comprising a first side and a second side and a plurality of interstices extending contiguously from the first side to the second side, wherein the interstices are coated with a protein-reactive coating. Porous, as applied to the substrate, refers to the interstices extending through the substrate from one side to another. The first and second sides refer to the opposite sides of the substrate, for example,FIG.4Adepicts a first side or a second side of an embodiment of the invention.FIG.4Bdepicts a cross-section showing the interstices extending contiguously from the first side to the second side.

The porous substrate may be composed of any material that can be engineered to hold the necessary shape and that may accommodate the protein-reactive coating. In various embodiments, the porous substrate comprises nanoporous alumina or porous glass. The porous substrate and the interstices may have any dimensions that can accommodate flow of proteins or a solution containing proteins from one side of the substrate to the other. The interstices may have a diameter of about 500 nm or less than about 500 nm. The porous substrate may have a thickness from the first side to the second side of about 100 μm or less than 100 μm, in some embodiments 50 μm or less than 50 μm. In various embodiments, the porous substrate may have a thickness from the first side to the second side of about 50-100 μm

The protein-reactive coating may be any substance that can coat the interstices and form a covalent bond to proteins that contact the protein-reactive coating. The protein reactive coating may form one or more covalent bonds to the peptide backbone or to side groups. In various embodiments, the protein reactive coating covalently binds proteins independent of the protein sequence. In various embodiments, the protein-reactive coating may be a silane derivative. In various embodiments, the protein-reactive coating may be covalently bonded to the porous substrate.FIG.2shows an embodiment of the invention comprising a silane derivative protein-reactive coating covalently bonded to a porous alumina substrate. In various embodiments the protein-reactive coating is:

In various embodiments, the protein-reactive coating is triethoxysilylundecanal

In various embodiments, the protein-reactive coating is selected from the group consisting of 3-thiocyanatopropyltriethoxysilane, triethoxysilylundecanal, trimethoxy[2-(7-oxabicyclo[4.1.0]hept-3-yl)ethyl]silane, (3-glycidyloxypropyl)triethoxysilane, 3-isocyanatopropyltriethoxysilane, N-[5-(trimethoxysilyl)-2-aza-1-oxopentyl]caprolactam, 11-(succinimidyloxy)undecyl dimethylethoxysilane, 2-(4-chlorosulfonylphenyl)ethyltrimethoxy-silane and triethoxysilylbutyraldehyde.

Method of Detecting a Protein of Interest

In another aspect, the invention provides a method of detecting at least one protein of interest in a plurality of proteins by contacting the plurality of proteins with a porous substrate having a first side and a second side, and a plurality of interstices extending contiguously from the first side to the second side, wherein the interstices are coated with a protein-reactive coating, thereby covalently binding at least a portion of the plurality of proteins to the protein-reactive coating, exposing the covalently bound plurality of proteins to a first molecule that binds the at least one protein of interest; and detecting the first molecule that binds at least one protein of interest, thereby detecting the at least one protein of interest. The methods of the invention allow the detection of one or more proteins in a complex mixture of proteins, by way of non-limiting example, crude cell lysate. The method may be performed using a small sample. In various embodiments, the plurality of proteins may have a volume of less than 10 μL, less than 5 μL, less than 3 μL or less than 1 μL.

Contacting the plurality of proteins covalently bound to the porous substrate with a first molecule that binds the at least one protein of interest may be achieved by passive diffusion. In other embodiments, this may be achieved by creating a flow through the interstices using pressure. In other embodiments, this may be achieved by electrophoresis. By way of non-limiting example,FIG.10depicts an embodiment in which flow is created by a pair of syringes. Various separation techniques may be employed as part of the contacting step in order to stratify a complex mixture of proteins. In various embodiments, contacting further comprises electrophoretic mobilization of the plurality of proteins. By way of non-limiting example, electrophoretic separation may be sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Contacting may also include electrophoretic transfer of the plurality of proteins to the porous substrate.FIG.6depicts an embodiment of the invention in which the plurality of proteins is electrophoretically transferred to the porous substrate following electrophoretic separation. This may allow multiple separate spatially resolved pluralities of proteins (by way of non-limiting example, various bands originating from the same or different wells of the gel) to be transferred and subsequently analyzed. As depicted inFIGS.13-15, this feature allows multiplexing which may vastly improve throughput.

Exposing the covalently bound plurality of proteins to molecules that bind the protein of interest may include generating a flow of solution containing the molecules through the interstices such that the molecules may bind the protein of interest. The flow may be created by diffusion or by using pressure or by using electrophoretic force. This may be the same as or different from the method used to contact the porous substrate with the plurality of proteins. Molecules that bind the protein of interest may be any molecule that specifically binds to the protein of interest and is detectable thereafter using any means known in the art, by way of non-limiting example, fluorescence. In various embodiments, the first molecule that binds the protein of interest may be an antibody, an aptamer or a protein.

The molecules that bind proteins of interest may be detected by any appropriate means known in the art. In various embodiments, detection of the molecule that binds the protein of interest may require the application of a second reagent, by way of non-limiting example a secondary antibody. In various embodiments, depending on the nature of the molecule that binds the protein of interest, one or more rinsing steps may be performed. Rinsing steps may be necessary or preferable in order to, by way of non-limiting example, lower background due to non-specific binding.

The covalent binding of the plurality of proteins to the protein-reactive coating allows repeated analysis of the same sample. In various embodiments the method further includes steps of stripping the first molecule that binds at least one protein of interest and exposing the covalently bound plurality of proteins to a second molecule that binds the same or a different protein of interest; and detecting the second molecule, thereby detecting the at least one protein of interest. Stripping and re-probing as described here may be repeated more than once. In various embodiments, molecules that bind proteins of interest present in the plurality of proteins may be stripped and new molecules that bind the same or different proteins of interest may be applied, three times, five times, ten times or more. In various embodiments, the second or subsequent molecule is an antibody, an aptamer or a protein. In various embodiments, depending on the nature of the molecule that binds the protein of interest, one or more rinsing steps may be performed during or after stripping and prior to applying a second molecule that binds the same or a different protein of interest.