U.S. patent application number 11/591747 was filed with the patent office on 2007-03-01 for microarrays on mirrored substrates for performing proteomic analyses.
This patent application is currently assigned to Novartis Vaccines and Diagnostics, Inc.. Invention is credited to Eric Beausoleil, Deborah Charych, Ronald N. Zuckermann.
Application Number | 20070048806 11/591747 |
Document ID | / |
Family ID | 30114049 |
Filed Date | 2007-03-01 |
United States Patent
Application |
20070048806 |
Kind Code |
A1 |
Charych; Deborah ; et
al. |
March 1, 2007 |
Microarrays on mirrored substrates for performing proteomic
analyses
Abstract
Provided are peptidomimetic protein-binding arrays, their
manufacture, use, and application. The protein-binding array
elements of the invention include a peptidomimetic segment linked
to a solid support via a stable anchor. The invention contemplates
peptidomimetic array element library synthesis, distribution, and
spotting of array elements onto solid planar substrates, labeling
of complex protein mixtures, and the analysis of differential
protein binding to the array. The invention also enables the
enrichment or purification, and subsequent sequencing or structural
analysis of proteins that are identified as differential by the
array screen. Kits including proteomic microarrays in accordance
with the present invention are also provided.
Inventors: |
Charych; Deborah; (Albany,
CA) ; Beausoleil; Eric; (San Francisco, CA) ;
Zuckermann; Ronald N.; (El Cerrito, CA) |
Correspondence
Address: |
NOVARTIS VACCINES AND DIAGNOSTICS INC.
CORPORATE INTELLECTUAL PROPERTY R338
P.O. BOX 8097
Emeryville
CA
94662-8097
US
|
Assignee: |
Novartis Vaccines and Diagnostics,
Inc.
|
Family ID: |
30114049 |
Appl. No.: |
11/591747 |
Filed: |
October 31, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10190308 |
Jul 3, 2002 |
7153682 |
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11591747 |
Oct 31, 2006 |
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09874091 |
Jun 4, 2001 |
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10190308 |
Jul 3, 2002 |
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60209711 |
Jun 5, 2000 |
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Current U.S.
Class: |
435/7.2 |
Current CPC
Class: |
B82Y 15/00 20130101;
G01N 2500/00 20130101; G01N 33/6845 20130101; G01N 33/545 20130101;
C07K 17/14 20130101; C40B 30/04 20130101; G01N 33/543 20130101;
B82Y 30/00 20130101; B82Y 40/00 20130101; G01N 33/6842
20130101 |
Class at
Publication: |
435/007.2 |
International
Class: |
G01N 33/567 20060101
G01N033/567; G01N 33/53 20060101 G01N033/53 |
Claims
1. A method of performing a differential binding assay, comprising:
labeling proteins in a protein-containing biological sample
solution; contacting an aliquot of said labeled protein-containing
biological sample solution with an array of protein-binding agents
stably attached to the surface of a solid support, said array
comprising, a solid substrate having a substantially planar surface
comprising an organic chemically-modified dielectric-coated
reflective metal; a plurality of protein-binding agents bound to
said substrate, each of said protein-binding agents comprising, an
anchoring segment stably bound to said substrate surface, a
peptidomimetic protein-binding segment, and a linker segment
connecting and separating the anchoring and peptidomimetic
segments; and a di-thiol modified polyethylene glycol non-protein
chemical blocking agent and a protein blocking agent bound to the
substrate surface not occupied by protein-binding agent array
elements bound to the substrate surface; and analyzing the array to
determine differential binding of proteins in the sample to
protein-binding agents of the array.
2. The method of claim 1, wherein in the array the metal is
aluminum, the dielectric is SiO.sub.2 and said modifying organic
chemical is an aminosilane.
3. The method of claim 2, wherein the aminosilane is functionalized
with a maleimide.
4. The method of claim 3, wherein in the array the peptidomimetic
segment is a peptoid.
5. The method of claim 4, wherein the protein blocking agent is
selected from the group consisting of casein, non-fat milk and
BSA.
6. The method of claim 5, wherein the chemical blocking agent is
casein.
7. The method of claim 6, wherein the protein labels comprise
fluorescent dyes.
8. The method of claim 7, wherein the fluorescent dyes comprise
amine-reactive cyanine dyes.
9. The method of claim 8, wherein the amine-reactive cyanine dyes
comprise Cyanine 3 and Cyanine 5 dyes.
10. The method of claim 9, wherein in the array the SiO.sub.2
coating is about 800 angstroms thick.
11. The method of claim 10, wherein in the array the aluminum is
disposed on a glass slide.
12. The method of claim 11, wherein the assay is used to compare
the binding profiles of a first biological sample untreated with a
particular chemical agent and a second biological sample treated
with the particular chemical agent.
13. The method of claim 12, wherein the biological samples are cell
lines.
14. The method of claim 13, wherein proteins in the lyastes of the
cell lines are labeled.
15. The method of claim 14, wherein proteins in the lyastes are in
their native states.
16. The method of claim 14, wherein proteins in the lyastes are in
their denatured states.
17. The method of claim 14, wherein the assay is part of a
screening process to identify drug candidates.
18. The method of claim 1, wherein the protein blocking agent is
selected from the group consisting of casein, non-fat milk and
BSA.
19. The method of claim 18, wherein the chemical blocking agent is
casein.
20. The method of claim 1, wherein in the array the SiO.sub.2
coating is about 800 angstroms thick.
21. The method of claim 1, wherein the protein labels comprise
fluorescent dyes.
22. The method of claim 1, wherein the assay is used to compare the
binding profiles of a first biological sample untreated with a
particular chemical agent and a second biological sample treated
with the particular chemical agent.
23. The method of claim 22, wherein the biological samples are cell
lines.
24. The method of claim 23, wherein proteins in the lyastes of the
cell lines are labeled.
25. The method of claim 24, wherein proteins in the lyastes are in
their native states.
26. The method of claim 24, wherein proteins in the lyastes are in
their denatured states.
27. The method of claim 1, wherein the assay is part of a screening
process to identify drug candidates.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 10/190,308 titled MICROARRAYS ON MIRRORED SUBSTRATES FOR
PERFORMING PROTEOMIC ANALYSES, filed Jul. 3, 2002; which is a
continuation-in-part of U.S. patent application Ser. No. 09/874,091
titled MICROARRAYS FOR PERFORMING PROTEOMIC ANALYSES, filed Jun. 4,
2001; which claims priority from U.S. Provisional Application No.
60/209,711, entitled MICROARRAYS FOR PERFORMING PROTEOMIC ANALYSES,
filed Jun. 5, 2000; the disclosure of each of which is incorporated
by reference herein in its entirety for all purposes.
BACKGROUND OF THE INVENTION
[0002] The present invention relates to cell product analysis and
materials. In one embodiment, the invention is directed to
proteomic microarrays and methods of using them to conduct
proteomic analyses.
[0003] In recent years, microarray technology has developed from a
specialized sub-field into an important tool for basic and applied
studies in molecular biology, microbiology, pharmaceutics,
agriculture, and many other biotechnologies. DNA microarray
technology attempts to link the genome of an organism or cell to an
expressed phenotype or protein function.
[0004] The overwhelming publication and patent literature on
microarray technology describes arrays of DNA (or other forms of
nucleic acid, such as cDNA or RNA), displayed on a solid surface
such as a glass slide (often referred to as a "chip"). The arrayed
DNA is typically in the form of short oligonucleotides (e.g., about
8 to 25 bases) or longer clones or PCR products (about 500 to 2000
bases). The former are typically synthesized on the solid support,
whereas the latter are robotically "spotted" onto a solid support
into an array format.
[0005] While there are reports of peptide and protein arrays on
solid surfaces, these have received considerably less attention in
comparison to DNA arrays. This is likely due to the inherent
instability of these materials at interfaces, and in the presence
of complex biological matrices. For example, it is well known, that
many proteins denature upon contact with solid surfaces. Peptides,
as well as proteins, are also subject to hydrolysis by any
proteases that may be present in the biological sample being
analyzed. In addition, peptide arrays are typically synthesized
in-situ on solid surfaces using photolithographic methods. These
techniques require the use of expensive custom-made masks that must
be designed and manufactured for each chip. Furthermore, chemical
characterization of surface-synthesized peptides is nearly
impossible to perform due to the tiny amount of peptide
generated.
[0006] Currently, the most common way of analyzing the proteome of
biological samples employs two-dimensional ("2-D") gel
electrophoresis. This method is problematic because the results are
very sensitive to the experimental protocol (for example,
development time of the gel as well as other parameters).
Therefore, it is very difficult to get reproducible data from 2-D
gels. Also, the sensitivity of the silver stain used in these gels
is limited, and is less than that of the fluorescent labels used in
microarray technologies.
[0007] Thus, there is an overwhelming need to develop effective
microarray technology that is useful in a protein context. In many
cases, functional pathways cannot be directly linked to a
particular gene. Proteins often undergo a variety of
post-translational modifications, interactions, or degradations
that ultimately determine function. Even the seemingly simple
evaluation of a protein's abundance cannot be directly correlated
with the level of corresponding mRNA. The only solution is to
evaluate the state of the cell, tissue or organism at the protein
level. Therefore, a high throughput format that allows rapid
display of protein differentials in complex mixtures such as cells,
tissues, serum, etc., would provide a powerful counterpart and
complement to DNA microarray technology.
SUMMARY OF THE INVENTION
[0008] To achieve the foregoing, the present invention provides
peptidomimetic protein-binding arrays, their manufacture, use, and
application. The protein-binding array elements of the invention
include a peptidomimetic segment, an anchor segment and a linker
segment connecting the peptidomimetic and anchor segment. The
invention contemplates peptidomimetic array element library
synthesis, distribution, and spotting of array elements onto solid
planar substrates, labeling of complex protein mixtures, and the
analysis of differential protein binding to the array. The
invention also enables the enrichment or purification, and
subsequent sequencing or structural analysis of proteins that are
identified as differential by the array screen. Kits including
proteomic microarrays in accordance with the present invention are
also provided.
[0009] In one aspect, the invention pertains to an array of
protein-binding agents stably attached to the mirrored surface of a
solid support. The array includes a solid substrate having a
substantially planar surface including an organic
chemically-modified dielectric-coated reflective metal, and a
plurality of different protein-binding agents bound to the
substrate. Each of the protein-binding agents includes an anchoring
segment stably bound to the substrate surface, a peptidomimetic
protein-binding segment, and a linker segment connecting and
separating the anchoring and peptidomimetic segments. The array may
also include chemical blocking agents to prevent non-specific
binding of proteins in samples run on the array.
[0010] In another aspect, the invention pertains to a method of
making an array comprising a plurality of different protein-binding
agents stably associated with the surface of a mirrored solid
support. The method involves preparing for bonding a solid
substrate having a substantially planar surface including an
organic chemically-modified dielectric-coated reflective metal, and
contacting a plurality of different protein-binding agents with the
substrate under conditions sufficient for the protein-binding
agents to become bound to the substrate surface. Each of the
protein-binding agents includes an anchoring segment stably bound
to the substrate surface, a peptidomimetic protein-binding segment,
and a linker segment connecting and separating the anchoring and
peptidomimetic segments. A chemical blocking agent designed to
prevent non-specific binding of proteins in samples run on the
array, may also be applied to the array.
[0011] These and other features and advantages of the present
invention will be presented in more detail in the following
specification of the invention and the accompanying figures which
illustrate by way of example the principles of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIGS. 1A and 1B schematically depict the structure of a
protein-binding agent array element and array portion,
respectively, in accordance with one embodiment of the present
invention.
[0013] FIG. 1C depicts the molecular structure a protein-binding
agent linking segment composed of ethylene oxides in accordance
with one embodiment of the present invention.
[0014] FIG. 2 schematically depicts a process of making an array
having a plurality of different protein-binding agents stably
associated with the surface of a solid support in accordance with
one embodiment of the present invention.
[0015] FIG. 3A schematically depicts the molecular structures of
six 12-mer peptoid array elements in accordance with one embodiment
of the present invention.
[0016] FIG. 3B illustrates a peptoid-based chemical blocking agent
in accordance with the present invention designed to inhibit
non-specific binding of proteins.
[0017] FIGS. 4A and 4B schematically depict alternative modes of
binding a protein-binding agent to a solid support in accordance
with one embodiment of the present invention.
[0018] FIG. 5 schematically depicts a process for conducting a
differential protein binding assay in accordance with one
embodiment of the present invention.
[0019] FIG. 6 schematically depicts a various processes for
identifying and characterizing proteins identified in accordance
with embodiments of the present invention.
[0020] FIG. 7A depicts the chemical formula for a NHS-LC-LC-biotin
molecule used in a solution to coat aluminum slides to be used as a
substrate in accordance with one embodiment of the present
invention.
[0021] FIG. 7B depicts a representation of an avidin-derviatized
aluminum slide spotted with a biotinylated protein-binding agent in
accordance with one embodiment of the present invention.
[0022] FIG. 8 depicts a graph of results of size exclusion
chromatography conducted to separate labeled protein from the
unreacted dye prior to application of the labeled protein sample to
a microarray in accordance with embodiments of the present
invention.
[0023] FIGS. 9A-9C depict scans of microarray chips bearing a
library of 1,000 peptoid-based protein-binding agents used to prove
the concept of the present invention.
[0024] FIG. 10 depicts biotinylated hexameric peptides (A through
E) and the corresponding signal intensity obtained when the
biotinylated peptides were spotted onto avidin-treated slides,
blocked with casein, and probed with Cy5-labelled antiglu
antibodies.
[0025] FIG. 11 depicts the dependence of signal strength on the
mode of surface attachment of a peptide for two modes of surface
attachment in accordance with the present invention.
[0026] FIG. 12 depicts the difference in signal from glass vs.
mirrored substrates in a comparative experiment to illustrate the
advantages of mirrored substrate arrays in accordance with the
present invention.
DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
[0027] The materials and associated techniques and apparatuses of
the present invention will now be described with reference to
several embodiments. Important properties and characteristics of
the described embodiments are illustrated in the structures in the
text and in the accompanying drawings. While the invention will be
described in conjunction with these embodiments, it should be
understood that the invention it is not intended to be limited to
these embodiments. On the contrary, it is intended to cover
alternatives, modifications, and equivalents as may be included
within the spirit and scope of the invention. In the following
description, numerous specific details are set forth in order to
provide a thorough understanding of the present invention. The
present invention may be practiced without some or all of these
specific details. In other instances, well known process operations
have not been described in detail in order not to unnecessarily
obscure the present invention.
[0028] In this specification and the appended claims, the singular
forms "a," "an," and "the" include plural reference unless the
context clearly dictates otherwise. Unless defined otherwise, all
technical and scientific terms used herein have the same meaning as
commonly understood to one of ordinary skill in the art to which
this invention belongs.
[0029] Introduction
[0030] The present invention provides peptidomimetic
protein-binding arrays, in addition to methods for their
manufacture, use, and application. The protein-binding array
elements of the invention include a peptidomimetic segment (an
example of which is a peptoid), linked to a solid support using a
stable anchor. In one embodiment, the invention provides
peptidomimetic array element library synthesis, distribution, and
spotting of array elements onto solid, planar substrates, labeling
of complex protein mixtures, and the analysis of single protein
binding or differential protein binding by contacting labeled pure
protein or complex protein mixtures to the peptidomimetic array.
Such analysis may lead to the identification of novel therapeutic
targets involved in diseases such as cancer, HIV or diabetes. These
novel targets can then be utilized in high throughput screening
assays to find new drug candidates. The invention also enables the
enrichment or purification, and subsequent sequencing or structural
analysis, of proteins that are identified as differential by the
array screen. The arrays can also be used to identify new synthetic
ligands for proteins of interest (for protein purification or
development of high throughput assays) or synthetic antigens for
antibodies of interest. They may also be used to find new enzyme
inhibitors or other high affinity ligands for drug targets, or as
initial scaffolds for new therapeutics. Kits including proteomic
microarrays in accordance with the present invention are also
provided.
1. Protein-Binding Agent Arrays
[0031] Peptidomimetic arrays in accordance with the present
invention are composed of a number of different array elements
comprising protein-binding agents attached to the surface of a
solid support. The different protein-binding agent array elements
each include an anchoring segment attached to the substrate
surface, a peptidomimetic protein-binding segment, and a linker
segment connecting and separating the anchoring and peptidomimetic
segments. A "peptidomimetic" as used herein refers to nonpeptide
synthetic polymers or oligomers that detectably interact with
proteins or receptors in a manner analogous to protein-protein or
protein-peptide physical and/or chemical interactions under assay
conditions. The anchoring segment is typically attached to the
substrate surface such that the association between the anchoring
group and the substrate surface, and thus the concentration and
density of the protein-binding agent on the substrate surface, is
maintained during the processing to which the microarray is
subjected under its normal operating conditions, for example, as
described herein. The number of different chemical species of
protein-binding agent present on the surface of the array is at
least 2, or at least 10, or at least 100, and can be much higher,
generally being at least about 1,000, or at least about 5,000 to
about 50,000, for example, between about 5,000 and about 10,000, as
described further below.
[0032] FIGS. 1A and 1B provide a representation of one such array
element and an array in accordance with the invention. In FIG. 1A,
a protein-binding array element 100 includes three segments: a
peptidomimetic segment 102, an anchor segment 104, and a linker
segment 106. The array element is attached to a solid support or
substrate 108 by a bond between its anchor segment 104 and the
substrate surface 110. In some cases, the substrate may be composed
of a plurality of layers 112a, 112b, 122c forming a laminate. In
FIG. 1B, a protein-binding array includes a plurality of different
array elements 100, such as illustrated in FIG. 1A, attached to a
planar substrate 108. Each of these components of the array is
described in greater detail separately below.
[0033] A. Substrate
[0034] The solid support (FIGS. 1A and 1B, element 108) employed in
arrays in accordance with the present invention may vary greatly
depending on the intended use of the product to be produced. The
solid support may be any suitable material for binding the
protein-binding agent that is also compatible with any analytical
methods with which the array is to be used. Compatibility can be
determined using methods and materials known to those having skill
in the surface or materials chemistry arts. In one embodiment, the
solid support comprises an impermeable, rigid material. Suitable
materials include plastics, such as polymers, e.g.
polyvinylchloride, polyethylene, polystyrenes, polyacrylates,
polycarbonate and copolymers thereof, e.g., vinyl
chloride/propylene polymer, vinyl chloride/vinyl acetate polymer,
styrenic copolymers, and the like. Suitable materials also include
glasses, such as those formed from quartz, or silicon; and metals
(including alloys), e.g., gold, platinum, silver, copper, aluminum,
titanium, chromium and the like.
[0035] As noted above, in many embodiments of interest, the support
or substrate 108 will be a composite of two or more different
layers of material, where the composition includes at least a base
material, e.g., as represented by element 112a in FIG. 1A, and one
or more surface coating materials, as represented by elements 112b
and 112c in FIG. 1A. For example, one embodiment of interest
includes a base material 112a, such as a glass, which is coated
with a metallic layer 112b, e.g., gold or aluminum overcoated with
silicon dioxide, or titanium overcoated with silicon dioxide, and a
functionalized organic, e.g., amino-modified thiol or
amino-modified silane, layer 112c. The planar solid support may be
in the dimensions of a standard 3''.times.1'' microscope slide or
in the shape of a 3'' or 5'' diameter circular wafer, for example.
Other configurations will be apparent to those having skill in the
surface or materials chemistry arts.
[0036] The solid support material or substrate may have a variety
of different configurations, depending on the intended use of the
material. Thus, in the broadest sense the support or base material
may be in the form of a plate, sheet, cone, tube, well, bead,
nanoparticle (e.g., about 5-100 nm), wafer, etc. In some
embodiments, the base support material is one that has at least one
substantially planar surface, e.g., as found on a plate, slide,
sheet, disc, etc. In these embodiments, supports having an overall
slide or plate configuration, such as a rectangular or disc
configuration are employed.
[0037] In the planar, rectangular embodiments of the
above-described slides, the length of the support will generally be
at least about 1 cm and may be as great as about 40 cm or more, but
will usually not exceed about 30 cm and may often not exceed about
20 cm. The width of support will generally be at least about 1 cm
and may be as great as about 40 cm, but will usually not exceed
about 30 cm and will often not exceed about 20 cm. The height of
the support will generally range from about 0.01 mm to about 10 mm,
depending at least in part on the material from which the rigid
substrate is fabricated and the thickness of the material required
to provide the requisite rigidity. Of particular interest in many
embodiments are supports having the dimensions of a standard
microscope slide. One typical substrate size is about 2.54 cm
.times.7.62 cm and about 1-2 mm thick. However, any suitable
dimensions can be employed.
[0038] Where the support is a bead, nanoparticle or microparticle,
the diameter of the support typically ranges from about 5 nm to
about 1000.mu., particularly from about 10 nm to about 500.mu..
[0039] The protein-binding agents can either be attached directly
to the inorganic solid surface of layers 112a or 112b (as
illustrated and described in more detail below with reference to
FIG. 4A) or attached using the functionalized organic layer of 112c
(as illustrated and described in more detail below with reference
to FIG. 4B). In the latter case, the non-substrate-bound termini of
the organic molecules in the layer are functionalized with a
reactive group that can attach to the anchoring group of a
subsequently bound protein-binding agent. Suitable terminal
reactive groups may be, for example, maleimide, hydrazide,
aminooxy, an activated ester such as N-hydroxysuccinimide,
anhydride, aldehyde, disulfide, thiol, azide, phosphine or avidin,
streptavidin, neutravidin or other altered forms of the protein
avidin that bind biotin, depending upon the anchor's functional
group.
[0040] In those embodiments in which the surface of the base
support material, such as glass, is coated with a thin layer of a
metal, such as aluminum, gold, or titanium, the thickness of the
metal layer will generally range from about 300 .ANG. to about
10,000 .ANG., more particularly from about 750 .ANG. to about 2,000
.ANG., and still more particularly from about 1,000 .ANG. to about
1,500 .ANG.. The metal layer may be deposited on the substrate
surface using any suitable protocol, including e-beam deposition,
vapor deposition, sputtering and the like, as are known to those of
skill in the art. An adhesion metal layer may be present between
the metal layer and the substrate, where adhesion metals of
interest include titanium, chromium, and the like. When present,
the adhesion metal layer will typically range in thickness between
about 5 .ANG. and about 100 .ANG., usually between about 25 .ANG.
and about 75 .ANG. and in many embodiments will be about 50 .ANG..
In some embodiments, the above-described adhesion layer can be a
molecular adhesion layer. Examples of materials suitable for
forming molecular adhesion layers in accordance with the present
invention include mercaptopropyltriethyoxysilane, and other
mercaptoalkoxysilanes, such as mercaptopropyltrimethoxysilane,
mercaptopropyltrichlorosilane, or other chain lengths such as
mrcaptohexyltriethoxysilane and other mercaptohexylalkoxysilanes,
as are known in the art. Where the adhesion layer is a molecular
adhesion layer, the thickness of the adhesion layer typically
ranges from about 5 .ANG. to about 50 .ANG..
[0041] The anchoring group of a protein-binding agent may be used
to directly bond the protein-binding agent to an inorganic, e.g.,
metal or glass, substrate surface. For example, a thiol anchoring
group may be used to bond directly to metals such as gold silver,
copper or platinum without an intervening functionalized layer
(e.g., maleimide/amine/thiol). Or, if the anchor group is an
activated silane (i.e., modified to be reactive with other organic
species, for example, hydrolyzable silane or modified silane that
can condense with hydroxyls or other silanes to form siloxane
bonds), e.g., chlorosilane or an alkoxysilane, the protein-binding
agent can be directly bonded to glass or other oxide surfaces, such
as titanium oxide, silicon oxide or aluminum oxide. Other activated
silanes for this purpose include triethoxysilane, trichlorosilane,
trimethoxysilane or other trialkoxysilanes. Within each of the
classes, chain lengths may be from three atoms up to about eighteen
atoms, for example.
[0042] As will be described in more detail below, in the case of
bonding of the protein-binding agent directly to a substrate's
inorganic surface, the protein-binding agent's linking segment
(between the peptidomimetic and the anchor) should be long enough
to keep the peptidomimetic sufficiently far from the hard substrate
surface that the surface does not interfere with protein binding
occurring (subsequently) at the peptidomimetic. The length can be
from about 2 atoms to about 200 atoms, or about 2 Angstroms to
about 300 Angstroms, for example. A typical linking segment may
have a backbone of between about 2 to about 30 atoms, preferably
about 6 to about 12 atoms. The linking segment may be composed of,
for example, suitably derivatized aliphatic chains (e.g.,
aminoalkanoic acids, such as aminohexanoic acid), ethylene oxides
(e.g., such as shown in FIG. 1C), sulfoxides, or "non-binding"
(orthogonal) short peptoid or peptide elements that remain constant
for each element of the array (e.g., a glycine heptamer), or some
combination of these components.
[0043] As noted above, the protein-binding agents may also be
attached using a functionalized organic layer (FIG. 1A, element
112c), such as an amino-modified thiol (aminothiol) (for use with
some metal substrate surfaces) or an amino-modified silane
(aminosilane) (for use with glass, metallic or other oxide
substrate surfaces). Where such an organic layer is used, the
termini of the organic molecules of the layer are functionalized
with a reactive group that can stably attach to the substrate
surface at on end, and to the anchor segment of a
subsequently-bound protein-binding agent in accordance with the
present invention at another end. Suitable terminal groups include,
for example, maleimide which can form a covalent bond upon reaction
with a thiol presented in the anchor. A further advantage of such
synthetic anchoring systems is their stability, conferring long
shelf life. Other possible terminal groups on the substrate include
hydrazide, which can react to form covalent bonds with aldehyde or
ketone moieties in the anchor; aminooxy, which can react to form
covalent bonds with ketone moieties in the anchor, aldehyde,
disulfide, thiol, azide, phosphine.
[0044] In another implementation, proteins such as avidin,
streptavidin, or other analogs may be used as a coating for the
solid support. In this case, biotin is used as the anchoring moiety
of the protein-binding agent to form a stable, non-covalent bonding
complex with avidin on the surface. Without wishing to be bound by
any particular theory of action, it is believed that this format
displays the protein-binding agent in a particularly biocompatible
environment with desirable distances between display molecules and
their neighbors, and desirable distances between display molecules
and the solid surface. Other protein-coatings can also be employed,
provided they sufficiently bind a small molecule anchoring group.
These may include anti-digoxigenin/digoxigenin,
anti-dinitrophenol/dinitrophenol, or many other protein/small
molecule pairs known in the art. Avidin/biotin is of particular
value because of it's extremely stable binding interaction. In
addition, substantial signal increases have been observed for
biotin/avidin immobilization compared to other suitable
immobilization techniques in accordance with the present invention,
for example 1000.times. higher than thiol/maleimide. Suitable
synthetic macromolecules may also be used as mimics of such protein
spacers. These may include high molecular weight polymers such as
polyethyleneimines, dendrimers, polyacrylic acids, polylysines and
the like.
[0045] Of course, other suitable binding combinations of this
character (namely, as noted above, sufficiently stable to maintain
the bond during the processing to which the microarray is subjected
under its normal operating conditions) are also possible. In
addition, those having skill in the surface chemistry arts will
understand that the above-listed anchoring groups on the peptoid
can function as reactive groups on the surface, and the reactive
surface groups can also function as a peptidomimetic anchoring
groups.
[0046] In one embodiment of the present invention, aluminum slides
may be coated with a layer of silicon dioxide or silicon monoxide
having a thickness of between about 500 .ANG. to about 2,000 .ANG..
The thickness is chosen to roughly correspond to 1/4 the wavelength
of the emission or excitation light. A layer of an aminoalkyl
trialkoxysilane, such as aminopropyl triethoxysilane (APS),
trichlorosilane, trimethoxysilane, and any other trialkoxysilane is
coated on the surface of the oxide. In addition, other
amino-silanes could also be used, for example, compounds having
longer alkyl groups, such as octyl, decyl, hexadecyl, etc., that
may form more ordered silane layers as will be appreciated by those
having skill in the surface chemistry arts. The thickness of this
silane layer may be from about 3 .ANG. to about 100 .ANG., more
preferably about 5 .ANG. to about 50 .ANG., even more preferably
about 7 .ANG. to about 20 .ANG.. One suitable example is an APS
layer that is about 7 .ANG. thick. The amino-modified Al surfaces
may be functionalized with a reactive group that will bind to the
anchor functional group on a protein-binding agent. In one
embodiment, the functional group may be maleimide. In another
embodiment, the functional group may be a whole protein such as
avidin. An implementation of an avidin-presenting substrate is
described with reference to FIGS. 7A and 7B in Example 3,
below.
[0047] In another embodiment, a gold-surfaced substrate slide (or a
slide surfaced with any metal capable of forming metal-thiol bonds,
such as Ag, Pt, or Cu) may be coated with an aminothiol layer that
is functionalized with a group that will bind to the anchor
functional group on a protein-binding agent, such as maleimide.
However, the anchor functional group and the surface-bound reactive
group should be chosen to have orthogonal reactivities. As noted
above, these anchor and substrate-bound groups can be interchanged
as will be readily understood by those skilled in the art.
[0048] In general, the functionalized organic layer 112c is
characterized by having a substantially uniform hydrophilic
surface. By uniform is meant that the surface of the layer includes
substantially no irregularities, such as gaps, pinholes, etc. The
thickness of the layer 112c may vary considerably, where the
thickness may be less than about 5,000 .ANG., usually less than
about 2,000 .ANG., but can be much lower, particularly from about 5
.ANG. to about 100 .ANG., or between about 20 .ANG. to about 50
.ANG..
[0049] In at least those embodiments where the support material
includes a metallic layer beneath a functionalized organic layer,
e.g., where the support material is an amino-modified thiol layer
on a gold coated microscope slide as described above, the thickness
of the organic layer is chosen such that the layer 112c is at least
sufficiently thick to separate any fluorescently labeled moiety
that may be present to the surface a sufficient distance from the
metallic layer on the substrate surface such that significant
signal quenching (i.e., signal quenching of sufficient magnitude to
effectively preclude meaningful detection of the signal) does not
occur. In these embodiments, the functionalized organic layer may
have a thickness that is at least about 30 .ANG., usually at least
about 50 .ANG. and more usually at least about 100 .ANG..
Alternatively, in cases where quenching does occur, the slide may
be treated with a high salt solution such as 0.75 M sodium
chloride, 0.085 M sodium citrate (see. e.g., PCT Publication No. WO
01/01142).
[0050] In those embodiments where the array is employed with
fluorescently labeled target, as described in greater detail below,
the thickness of the functionalized amino-modified thiol layer may
be chosen to provide for maximum amplification of the emitted and
reflected signals. See e.g., U.S. Pat. No. 5,055,265, the
disclosure of which is herein incorporated by reference. In such
embodiments, the thickness of the organic layer will be about 1/4
of the wavelength of the emitted light from the label. While the
exact thickness of the organic layer will vary depending on the
particular label with which it is to be employed, the thickness
generally ranges from about 50 .ANG. to about 300 .ANG., usually
from about 100 .ANG. to about 200 .ANG. and more usually from about
125 .ANG. to about 150 .ANG..
[0051] In one embodiment, the functionalized amino-modified thiol
layer 112c includes at least one self-assembled monolayer (SAM). As
such, the functionalized organic layer may include one or more
different self-assembled monolayers grafted sequentially onto each
other, where when the layer includes more than one self-assembled
monolayer (see. e.g., PCT Publication No. WO 01/01142).
[0052] Among the substrates described above are some having
reflective metal (e.g., aluminum, gold, etc.) surfaces ("mirrored
substrates"). As noted above, a suitable substrate for use in
arrays in accordance with the present invention will have a
dielectric coated mirrored surface. The substrate will generally be
a composite of a plurality of different layers of material, where
the composition includes a base rigid, substantially planar solid
support material, and a plurality of layers on the solid support.
The solid support has (in the case of a reflective metal) or is
coated with a reflective metal layer. By reflective metal it is
meant a metal that reflects at least 90% incident light in the
wavelength region of interest, generally visible (400-800 nm), and
possibly including longer wavelengths in the near infrared, such as
800-1100 nm, with very little (at or near 0%) light refracted into
the medium. Suitable examples include aluminum, chromium, copper,
gold, silver, platinum, titanium, rhodium, etc.
[0053] The reflective metal is overcoated with a dielectric, e.g.,
silicon oxide or silicon dioxide (silica) or alumina or fluoride
such as MgF2 or titanium dioxide. Silicon dioxide is preferred in
many embodiments. The thickness of this layer can be adjusted to
optimize the signal from the fluorescing species, as described in
further detail in International Patent Application No. WO 98/53304,
incorporated by reference herein for all purposes. The dielectric
layer (e.g., silicon dioxide) is functionalized with a bifunctional
organic surface layer, e.g., an amino-modified silane, suitable to
facilitate the attachment of an array element 100 to the substrate
106. Suitable aluminum/oxide/amino-propyl silane (APS) coated glass
slides are commercially available from Amersham-Pharmacia,
Amersham, England.
[0054] As described below in Example 10, these mirrored substrates
have been found to have performance benefits, particularly in terms
of increased sensitivity, relative to more conventional glass slide
substrates.
[0055] B. Protein-Binding Agents
[0056] As noted above, the protein-binding agents of the present
invention are composed of three segments: a peptidomimetic, an
anchor, and a linker connecting the peptidomimetic and the
anchor.
[0057] A "peptidomimetic" as used herein refers to nonpeptide
synthetic polymers or oligomers that detectably interact with
proteins or receptors in a manner analogous to protein-protein or
protein-peptide physical chemical interactions under assay
conditions. Peptidomimetics are generally protease-resistant, and
include, for example, oligomeric species such as peptoids,
beta-peptides, and others as described in A. E. Barron & R. N.
Zuckermann, Bioinspired polymeric materials: in-between proteins
and plastics, Curr Opin Chem Biol 1999, 3(6):681-7 and K
Kirchenbaum, R. N. Zuckermann and K. A. Dill, Designing polymers
the mimic biomolecules, Curr Opin Chem Biol 1999 9(4):530-5, each
of which is incorporated by reference herein in its entirety for
all purposes, and constrained cyclic molecules such as cyclic
peptides and heterocyles. In some cases, the peptidomimetic may
also mimic the folding of natural proteins. Peptidomimetics in
accordance with the present invention comprise generally no more
than about 500 mers, more particularly no more than about 100 mers,
typically no more than about 50 mers, and usually about 10 to about
20 mers, more particularly about 12 to about 16 mers. Given the
parameters provided herein, one of skill in the art would be able
to choose an appropriate peptidomimetic length for a given
application without undue experimentation.
[0058] Examples of peptidomimetic libraries and their design and
synthesis may be found in Fahad Al-Obeidi, et al., Peptide and
Peptidomimetic Libraries, Molecular Biotechnology 1998, 9: 205-223;
Victor J. Hruby et al., Synthesis of oligopeptide and
peptidomimetic libraries, Curr Opin Chem Biol 1997, 1:114-119; and
Amy S. Ripka et al., Peptidomimetic Design, Curr Opin Chem Biol
1998, 2:441-452, incorporated by reference herein in its entirety
for all purposes.
[0059] In a preferred embodiment of the present invention, the
peptidomimetic segment of the protein-binding agent is a peptoid.
The term "peptoid" as used herein refers to polymers comprising
N.sup.58-substituted amides as described in U.S. Pat. Nos.
5,831,005, 5,877,278, 5,977,301, 5,871,387, 5,986,695, and
5,719,049; and in co-pending U.S. patent application Ser. Nos.
08/340,073, 08/836,167, 08/920,205, 08/126,539, 08/277,228,
08/454,511, 08/485,106, 09/132,828, 08/484,923, 09/704,422; and PCT
Publications Serial Nos. 96/15143 and 93/09117, each of which is
incorporated herein by reference in its entirety and for all
purposes.
[0060] The anchoring segment provides for the stable attachment of
the array element to the solid surface. This functional group is
chosen to be reactive towards a complementary group that is
displayed on the solid surface and orthogonal to (i.e.,
substantially non-reactive with) sidechains present in the ligand.
An important feature of the anchoring group is that its reaction to
the surface be sufficiently facile so that it is complete within
the average lifetime of a droplet that is deposited by the robotic
array spotter onto the surface. For example, if the surface
displays a maleimide, a suitable anchoring group is a thiol and
approximately 15-20 minutes at about 60% humidity are required for
completion of the binding reaction before the approximately 10 nL
drop evaporates. Of course, droplet lifetime varies with
temperature, humidity and other conditions, allowing more or less
time for the reaction to take place. As noted above, other surface
display/anchor combinations are possible, including a hydrazide
surface group with an aldehyde or ketone anchoring group. Or, the
anchoring group can also be a biotin molecule, that will attach
strongly (yet non-covalently) to a surface that displays an avidin
protein.
[0061] The anchoring group may be attached to the peptidomimetic at
either end (in the case of a peptoid, at either the C- or
N-terminus). It can be attached either as a submonomer (e.g., a
substituted amine), or as a modification of a peptidomimetic (e.g.,
peptoid) side chain after synthesis. Alternatively, in another
example, the anchoring group may be present as a linker connecting
the peptoid to a beaded solid support upon which it is synthesized.
This linker may be cleaved from the resin along with the peptoid,
to provide a readily available anchoring group. Some examples are
described in co-pending U.S. patent application Ser. No.
______(Attorney Docket No. 16708.001), titled Peptoids
Incorporating Chemoselective Functionalities, filed Apr. 6, 2001,
which is incorporated herein by reference in its entirety and for
all purposes.
[0062] The linking segment of the protein-binding agent molecule is
chosen to provide for separation between the solid surface and the
peptidomimetic segment sufficient to facilitate interaction between
the peptidomimetic and the components of the analyte solution
(solution with which the microarray will be contacted), for example
by providing separation between the substrate surface and the
peptidomimetic so that the surface does not interfere with protein
binding occurring (subsequently) at the peptidomimetic access for a
protein binding site to the peptidomimetic ligand displayed on the
surface, especially for proteins with deep binding pockets. The
linker may also serve to separate the peptidomimetics on the
surface from each other, thereby mitigating possible steric
hindrance between the ligand and protein binding pocket. A typical
linking segment may have a backbone of between about 2 to about 200
atoms, preferably about 6 to about 30 atoms. The linking segment
may be composed of, for example, aliphatic chains (e.g.,
aminoalkanoic acids, such as aminohexanoic acid), ethylene oxides,
sulfoxides, or "non-binding" ("orthogonal") short peptoid or
peptide elements that remain constant for each element of the
array, or some combination of these components. In one embodiment,
a 2-carbon linker may be used. In another embodiment, three
ethylene oxides may be used. An example of a non-binding short
peptoid is a 2-mer to 12-mer, for example a 4-mer of methoxyethyl
side chains that remain constant for each protein binding agent,
while the peptidomimetic segment is variable. A suitable orthogonal
peptide linker is a 2-mer to 12-mer, for example a 5-mer of
glycine.
[0063] In some embodiments, where an organic layer is present on
the substrate surface, a spacer functionality may be built into the
organic layer. In such cases, the spacer in the substrate surface
layer and the linker in the protein-binding agent may collectively
contribute to the desired spacing of the peptidomimetic from the
hard substrate surface. Further, the mitigation of steric hindrance
may also be achieved by the selection of a surface coating that
presents a particularly biocompatible format, such as an avidin
protein (coupled with a biotin anchoring group on the
protein-binding agent).
[0064] FIG. 3A illustrates an example of a small set of array
elements 300 wherein the peptidomimetic protein-binding segments
302 are 12-mer peptoids, the linker 304 is a short aliphatic chain,
and the anchoring group 306 is a thiol. The peptidomimetic segments
depicted in FIG. 3A illustrate a subset of the range of affinity
properties achievable using peptoids as the peptidomimetic segment,
in accordance with one embodiment of the present invention
[0065] As mentioned above, the number of different types of binding
agents present on the surface of the array is at least two. By
"different", it is meant that the sequence of monomeric units
between two different peptidomimetics of two different
protein-binding agents is not the same. While the number of
different species of protein-binding agents present on the surface
of the array is at least 2, at least about 10, at least about 50,
or at least about 100, it is typically much higher, generally being
at least about 1,000 usually at least about 5,000 and more usually
at least about 10,000. The number may be as high as 500,000 or
higher, but typically does not exceed about 100,000 and usually
does not exceed about 50,000.
[0066] The surface regions surrounding the protein-binding array
elements may be modified so as to minimize background non-specific
binding of proteins, allowing complex samples (e.g., lysates or
serum) to be examined in a single step. The surface may be blocked
chemically with hydrophilic termini such as alcohols,
carbohydrates, amino acids, sulfoxides, acrylamides, or ethers.
Examples of alcohol terminal groups are, for example,
mercaptoethanol, mercaptohexanol, mercapto-octanol, in the case of
a maleimide-treated surface. These block the unreacted maleimides
on the surface. The surface can also be blocked using proteins such
as solutions of 1%-10% bovine serum albumin ("BSA") or human serum
albumin ("HSA") in phosphate buffered saline, or 1%-10% non-fat dry
milk or 1%-10% casein, gelatin or other suitable blocking protein.
In some cases, the addition of detergents to the blocking protein
solution is advantageous. For example, the addition os 0.01%-0.5%
Tween-20 or Triton X-100 (particularly 0.05%), 0.1-2% SDS as are
well know in the assay development or surface chemistry arts.
[0067] C. General Features of the Array
[0068] Typically, the array is characterized by having a plurality
of protein-binding agent spots on a solid substrate, where each
spot is characterized by having one or more, usually a plurality,
of identical binding agents bound to the support surface. The
number of distinct spots on the surface of the array may or may not
be the same as the number of different protein-binding agents on
the array, e.g., the same protein-binding agent may be presented in
two or more spots on the array surface. In one embodiment, each
protein-binding agent is presented in duplicate in the array.
Depending on the nature of the binding agents, the size of the
support surface, the methods of fabrication and the intended use of
the array, the number of distinct spots on the array surface may
vary greatly. Where the support surface has the dimensions of a
standard microscope slide (about 3''.times.1''), the number of
spots on the support surface will typically be at least about
3,000, usually at least about 6,000 and more usually at least about
10,000. The number may be as high as 100,000 or higher, but
typically does not exceed about 75,000 and usually does not exceed
about 50,000.
[0069] The diameter of each spot will typically range from about
100 .mu.m to about 300 .mu.m, usually from about 200 .mu.m to about
300 .mu.m. The space between any two given spots will generally be
between about 1 .mu.m and about 50 .mu.m. The density of the spots
generally ranges from about 1 to about 5,000 spots/cm.sup.2,
usually from about 100 to 2,000 spots/cm.sup.2. Typically, the
spots are arranged across the surface of the spacer layer in the
form of a pattern. The pattern may be in the form of organized rows
and columns of spots, e.g., a grid of spots, across the substrate
surface, a series of curvilinear rows across the substrate surface,
e.g., a series of concentric circles or semi-circles of spots, and
the like. To further increase density, the spots may also be
hexagonally arranged. Still other arrangements of spots are within
the scope of the present invention.
2. Methods of Making the Protein-Binding Agent Arrays of the
Subject Invention
[0070] The arrays of the subject invention may be prepared using
any convenient protocol. One protocol of interest involves 1) the
procurement of a solid support having a surface activated for
binding of a protein-binding agent; and 2) contact of two or more
different protein-binding agents with the support surface under
conditions such that the protein-binding agents become stably
associated with the support surface.
[0071] A. Solid Support Fabrication
[0072] The solid support may be fabricated using any convenient
methodology, which methodology will vary depending the particular
nature of the solid support. In accordance with one embodiment of
the invention a glass support is coated with a layer of metal,
e.g., aluminum or gold. To prepare a solid support of glass coated
with a metal layer, the surface of the glass is coated with a thin
layer of the metal, e.g., gold, silver, platinum, copper, titanium,
or aluminum, etc. in a thickness as described above. The metal
layer may be deposited on the substrate surface using any
convenient protocol, where suitable protocols include e-beam
deposition, vapor deposition, sputtering, and the like, and are
known to those of skill in the art. See e.g., Moteshari et al., J.
Am. Chem. Soc. (1998) 120:1328-1336; Bain et al., J. Am. Chem. Soc.
(1989) 111:7155-7164; Lee et al. Langmuir (1998) 14:6419-6423;
Folkers et al., Langmuir (1992) 8:1330-1341. Where convenient, an
adhesion metal layer may be present between the metal layer and the
substrate, where adhesion metals of interest include titanium,
chromium, and the like, deposited in a thickness as described
above. In addition, oxide overlayers such as silicon dioxide or
silicon monoxide may be deposited by e-beam or sputtering
deposition on top of the metallic layer.
[0073] In one example, following preparation of a gold substrate,
if the protein-binding agent's anchoring group is a thiol and the
linking group is sufficiently long (as explained above), arrays in
accordance with the present invention can be formed by spotting
thiol-displaying protein-binding agents onto bare, clean gold. The
gold surface of the substrate may be cleaned using a chromic acid
cleaning solution (e.g., chromium oxide or sodium dichromate in
sulfuric acid, for example Nochromix, available from Fisher (50-80%
Sodium dichromate in 12 N sulfuric acid)) and rinsed with
HPLC-grade water. This has the advantage of reducing the number of
surface functionalization steps.
[0074] In another embodiment, where the protein-binding agent's
anchoring group is a functionalized modified silane (e.g., mono-,
di- or tri-functional silanes, such as chlorosilane, an
alkoxysilane, dichloro or dialkoxy silane, or trichloro or
trialkoxy silane), the anchoring group can be attached directly to
oxide-containing surfaces such as glass, titanium oxide or aluminum
oxide. Arrays in accordance with these embodiments of the present
invention can be formed by spotting active silane-displaying
protein-binding agents onto the oxidized substrate surface. A glass
substrate has surface hydroxyls available for this binding. Metal
substrate surfaces may be oxidized, for instance by thermal or
chemical treatment. For example, aluminum may be oxidized
electrochemically, thermally or chemically (e.g., with
H.sub.2O.sub.2), as is well known in the art. In addition, a
silicon dioxide or titanium oxide layer can be deposited on the
aluminum. An oxide may also be present as a native thin layer, such
as occurs with aluminum or silicon. This native oxide may then be
derivatized by the amino-silane.
[0075] In still another embodiment of the invention, a metal, e.g.,
gold or aluminum, substrate surface is first functionalized with
functionalized organic molecules that form ordered monolayers. The
termini of the organic molecules are functionalized with a reactive
group that can attach to a suitable anchoring group of a
protein-binding agent. Suitable terminal groups may be, for
example, maleimide, hydrazide, aminooxy, an activated ester such as
N-hydroxysuccinimide, anhydride, aldehyde, disulfide, thiol, azide,
phosphine or avidin, depending upon the anchor's functional
group.
[0076] In one embodiment the functionalized organic molecules are
amino-modified thiol molecules. In order to functionalize the metal
surface with the thiol molecules, the substrate having the metal
surface may be dipped in a solution of the amino-modified thiol;
the amino-modified thiol solution then may be deposited onto the
surface of the substrate. Other convenient protocols may be
employed. Typically, the gold substrate is immersed into the
amino-modified thiol solution under conditions and for a sufficient
period for the amino-modified thiol molecules to assemble into a
monolayer on the substrate surface. The temperature at which
contact is carried out typically ranges from about 10.degree. C. to
about 100.degree. C., usually from about 15.degree. C. to about
80.degree. C. Contact is maintained for a sufficient period of time
for the self-assembled monolayer to form on the gold surface, where
contact is typically maintained for at least about 20 minutes,
usually at least about 4 hours, and more usually at least about 16
hours.
[0077] Alternatively, the amino thiol can also be deposited by
vapor deposition in a vacuum oven or by spin coating. For example,
a 1-10% (e.g., 5%) solution of thiol in a volatile solvent such as
isopropanol, methanol, THF may be prepared. The slides may be spun
at about 1000-8000 rpm (e.g., 5000 rpm) to provide an even
deposition of the thiol. Then, a heterobifunctional molecule (e.g.,
succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC)
or LC (long-chain)--SMCC) that is an activated ester on one end and
a maleimide on the other is contacted with the amino group to
create the maleimide-terminated surface. Other heterobifunctional
cross-linkers could also be used with different length spacers
(e.g., a long ethylene oxide spacer (e.g., 2-4 units) between an
NHS ester on one end, and a maleimide on the other end. As noted
above, the spacer on the substrate serve the same purpose as and
the "linker" in the protein-binding agent.
[0078] In another embodiment, Aluminum slides may be used. Aluminum
metal may be deposited by e-beam deposition onto a clean glass
substrate. The aluminum is then overcoated by silicon dioxide
(SiO.sub.2) or silicon monoxide in a thickness that is the same or
thinner than 1/4 the wavelength of the emission or excitation
light. Oxide thicknesses of about 600 to 1000 Angstroms may be used
as these eliminate the need for thinning the oxide prior to
performing binding experiments. The aluminum/oxide surface may be
treated with a amino-modified silane. For example, aluminum slides
freshly coated with a 800-1,400 Angstrom layer of silicon dioxide,
may be dipped immediately into a bath of 3%-40% aminopropyl
triethoxysilane in isopropanol, that has been previously filtered
through an 0.2 uM filter membrane, and silanized for up to 1 hour,
followed by rinsing and drying. Alternatively, aluminum slides
coated with silane (APS) are available from Amersham-Pharmacia,
Amersham, England (and described in International Patent
Application No. WO 98/53304). In some cases, such commercially
available slides may require thinning of the oxide layer to between
about 200 .ANG. and about 1,000 .ANG., more particularly between
about 900 .ANG. and about 1,000 .ANG., prior to performing a
binding experiment to improve signal-to-noise ratios. The final
amino-modified Al surfaces may be functionalized with SMCC to
render a surface that presents maleimide functional groups. Here
again, the silane may also be vapor deposited or spin coated, as
described above. For example, a 1-10% (e.g., 5%) solution of silane
in a volatile solvent such as isopropanol, methanol, THF may be
prepared. The slides may be spun at 1,000 rpm (e.g., 5,000 rpm) to
provide an even deposition of the silane. Then, a
heterobifunctional molecule (e.g., succinimidyl
4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC or LC-SMCC))
that is an activated ester on one end and a maleimide on the other
is contacted with the amino group to create the
maleimide-terminated surface. Other heterobifunctional
cross-linkers could also be used with different length spacers
(e.g., a long ethylene oxide spacer (e.g., 2-4 units) between an
NHS ester on one end, and a biotin or maleimide on the other end.
As noted above, the spacer on the substrate serve the same purpose
as and the "linker" in the protein-binding agent.
[0079] Further in accordance with this embodiment, it has been
found that amplification of the fluorescent signal used in assays
conducted with microarrays in accordance with the present invention
may be enhanced by etching the commercially available
functionalized and spotted aluminum slides (Amersham) to reduce the
oxide layer thickness to about 200 .ANG. to about 900 .ANG.,
preferably about 500 .ANG.. For example, an etch solution of about
0.1-0.2% SDS/5.times.SSC (0.75 M NaCl/0.085 M sodium citrate) and
optionally 5 mM EDTA may be applied at about 50-80 degrees,
preferably about 60.degree. C. for about two to four hours. As
noted above, manual deposition of a silicon dioxide layer of a
suitable thickness may eliminate the need for the thinning of the
slide.
[0080] As noted above, where the substrate surface has an organic
self-assembled monolayer, following self assembly of the initial
monolayer, one or more additional monolayers may be grafted onto
the initial monolayer, where the additional layer(s) are made up of
molecules, e.g. alkyls, having functionalities that provide for
their covalent attachment to the surface functionalities of the
initially deposited monolayer, e.g. amino functionalities where the
initially deposited monolayer is characterized by the presence of
carboxy functionalities. Alternatively, multi-component spacers can
be constructed prior to attachment to the surface.
[0081] B. Synthesis of the Protein-Binding Agents
[0082] As noted above, protein-binding agents in accordance with
the present invention are composed of three segments: a
peptidomimetic, an anchor, and a linker connecting the
peptidomimetic and the anchor.
[0083] Peptidomimetics may have a variety of nonpeptide polymeric
structures and characteristics as long as they can mimic the
biological effects of natural peptides. References to
peptidomimetic structures have been provided above. In one
embodiment of the present invention, the peptidomimetic segment of
the protein-binding agent is a peptoid.
[0084] Libraries of peptoids may be synthesized using robotic
solid-phase synthesis techniques, such as those developed by Chiron
Corporation of Emeryville, Calif. The diversity of the library can
be controlled by the nature and arrangement of the amine
submonomers (bromoacetic acid and substituted amines) used in the
peptoid synthesis, as described in above-incorporated U.S. patent
documents. As illustrated in FIG. 2, libraries may be prepared
according to a process 200 using mix and split synthesis 202, or
parallel synthesis, such that there is a multiplicity of peptoids
synthesized, but only one species of peptoid on a given bead (i.e.,
each bead has one unique compound, repeated many times). The amount
of compound per bead may range from about 1-100 nanomoles, with
20-40 nanomoles being most typical. About 40 nanomoles per bead are
commonly obtained. This process is further described in U.S. patent
application Ser. No. 09/306,700, the disclosure of which is
incorporated by reference herein in its entirety and for all
purposes. The synthesized peptoids, still bound to resin, may then
be distributed to 96-well plates (or other multi-well plates, such
as 384-well or 1024-well plates) 204 using, for example, bead
picking technology described in the just-referenced U.S. patent
application. The use of `big bead` resin (having a diameter of
about 500.mu.) allows distribution of one bead per well.
[0085] The peptoids may then be then dried, cleaved from the resin
(e.g., with about 20-95% TFA in dichloromethane; 95% being
typical), re-dissolved in acetonitrile/water, filtered or
centrifuged, dried and re-dissolved in 50% DMSO/50% Buffer. Other
solvent/buffer systems can also be used such as 50% DMF or 50% NMP
with the remainder being PBS, TBS, Borate, Tris-HCl, etc. In some
cases, a reducing agent such as tris-carboxyethylphosphine (TCEP)
may also be added to the wells of the spotting plate to inhibit
oxidation of the thiols on the peptoids so that they retain their
reactivity toward maleimides on the substrate surface. In cases
where TCEP is added to the wells, phosphate buffers such as PBS are
to be avoided, and buffers such as TBS would be preferred. This
final solution is ready for spotting.
[0086] In one embodiment of the present invention, the
concentration of peptoids in the wells of the spotting plate should
be in the range of about 0.5-2 mM, where 2 mM is preferred. For
spotting onto the prepared planar solid support 206, the peptoids
are filtered or centrifuged, dried, and resuspended, as described
in further detail below. It is useful for the material spotted on
the slide be in an excess with respect to the amount of probe in
solution to be applied to the microarray. This is an amount that
gives a saturating fluorescence signal, so that changes in signal
intensity are due substantially to protein levels.
[0087] The anchoring and linker groups may be attached to the
peptoid at either the C- or N-terminus. They can be attached either
as a submonomer (e.g., as described in U.S. Pat. No. 5,877,278 and
above-referenced co-pending U.S. patent application Ser. No.
______(Attorney Docket No. 16708.001)) during the peptoid synthesis
as described above in the patent documents incorporated by
reference, or with in situ activated amino acid coupling steps, as
a modification of the peptoid after synthesis, according to
procedures known to those of skill in the art.
[0088] For N-terminal attachment, the peptoid may be synthesized on
a resin and then the linker and the anchor groups may be added
before the entire molecule is cleaved. For example, peptoids may be
prepared on Rink amide polystyrene resin as illustrated and
described in co-pending application Ser. No. 09/704,422 previously
incorporated by reference herein. The synthesis procedure is also
reported in Figliozzi, G. M., Goldsmith, R., Ng, S. C., Banville,
S. C., Zuckermann, R. N. Methods Enzymol. 1996, 267:437-447, the
disclosure of which is incorporated by reference herein for all
purposes. Also, before cleavage, one or more (e.g., two to four,
preferably four) submonomer hydrophilic linker groups (e.g.,
methoxyethylamine) may be added to the peptoid N-terminus. Then, a
trityl-protected cysteamine is added in order to provide a thiol
anchoring group. The peptoid with attached linker and anchoring
groups may then be cleaved from the resin, for example using 95%
TFA (v/v) in dichloromethane (CH.sub.2Cl.sub.2). The resulting
solution is then ready for application (spotting) onto microarray
slides in accordance with the present invention.
[0089] In another embodiment, an FMOC-protected beta-alanine is
attached to the N-terminus of the peptoid by in situ activation
with Hobt and DIC, as is known to those skilled in the arts of
peptide synthesis. The beta alanine functions as an adaptor
molecule between the peptoid and peptide linker. After attachment
of beta-alanine, an FMOC-protected amino acid on a peptide (e.g.,
glycolic) linker, for example, is attached to the N-terminus of the
beta-alanine. Finally, biotin is added to the N-terminus by peptide
coupling.
[0090] The thiol or biotin anchoring group can also be attached to
the end of the peptoid at the C-terminus. For example,
4-(diphenylhydroxymethyl)benzoic acid (available from Fluka) is
treated with cystamine hydrochloride in the presence of an acid
catalyst. Next the resulting amine is protected as the
N-(9-flurenylmethoxycarbonyl) (N-FMOC) derivative, and the
resulting FMOC--NH--CH.sub.2CH.sub.2--S-Tr-COOH is coupled to
aminomethyl-Big Beads (400-500 microns, Polymer Labs). The peptoid
is synthesized on the deprotected amine as described above, and
treatment with TFA results in cleavage of the thiol-modified
peptoid from the resin, while leaving the trityl protecting group
on the resin. Such procedures are described in the above-referenced
co-pending U.S. patent application Ser. No. ______ (Attorney Docket
No. PP16708.001)
[0091] C. Contacting the Solid Support with Protein-Binding
Agents
[0092] Following preparation of the substrate and protein-binding
agents, as described above, two or more different protein-binding
agents of interest that are to be bound to the surface to produce
the array are contacted with the functionalized, or otherwise
prepared (cleaned, oxidized, etc.) substrate surface. By contact is
meant that the binding agents are brought into proximity with the
surface such that they become substantially stably attached or
bound to the surface of the substrate layer.
[0093] In contacting the binding agents with the substrate surface,
any convenient means for contacting the surface with the binding
agents which results in the desired pattern of binding agent spots,
as described above, may be employed, e.g., by spotting. As
mentioned above, the term contact is used herein to refer to any
method that brings the binding agent within close proximity of the
support surface. Generally, an aqueous solution (e.g. water,
water/organic solvent (such as 50/50 water/DMSO, or the like) of
the binding agent is employed during contact where the solution may
comprise one or more components in addition to water and the
binding agent, e.g., buffering agents, salts, and the like. Other
systems include 50/50 NMP/TBS, DMF/TBS, NMP/PBS, DMF/PBS, or all
these solvents together with water. The 50/50 mix can also be
adjusted. For example, when a higher percentage of the aqueous
solution is used, the drop sizes can be smaller because of the
higher surface tension of the solution. Drop size (and therefore
density) may be controlled to some extent in this manner.
Typically, contact is achieved by depositing solutions of the
different binding agents onto discrete locations of the support
surface, such that each different type of binding agent is
deposited onto its own unique location on the substrate
surface.
[0094] The binding agents may be deposited onto the support surface
using any convenient means, e.g., by pipetting. A number of devices
and protocols have been developed for depositing aqueous solutions
onto precise locations of a support surface and may be employed in
the present methods. Such devices include "ink-jet" printing
devices, mechanical deposition or pipetting devices and the like.
See e.g., U.S. Pat. Nos. 4,877,745; 5,338,688; 5,474,796;
5,449,754; 5,658,802; 5,700,637; and 5,807,552; the disclosures of
which are herein incorporated by reference. Robotic devices for
precisely depositing aqueous volumes onto discrete locations of a
support surface, i.e., arrayers, are also commercially available
from a number of vendors, including: Genetic Microsystems;
Molecular Dynamics; Cartesian Technologies; Beecher Instruments;
Genomic Solutions; and BioRobotics. Alternatively, bubble jet
technology recently described by Okamoto, Suzuki and Yamamoto,
Nature Biotechnology, vol. 18 (April, 2000), 438, may be used.
[0095] As noted above, an important feature of a process in
accordance with the present invention is that the reaction between
the anchoring group on the protein-binding agent and the substrate
surface must be sufficiently facile so that it is complete within
the average lifetime of a droplet that is deposited by the robotic
array spotter onto the surface. For example, if the surface is
functionalized and displays a maleimide, a suitable anchoring group
is a thiol and approximately 15-20 minutes at about 60% humidity
are required for completion of the binding reaction. As noted
above, other surface display/anchor combinations are possible,
including those forming stable, yet non-covalent bonds, such as
avidin and biotin.
[0096] D. Blocking the Chip
[0097] After spotting of the peptoid library onto the array
substrate (chip), the remaining, uncoated surface of the chip may
be functionalized with a molecule that displays a hydrophilic
terminus. These hydrophilic termini are anticipated to reduce or
eliminate non-specific binding of proteins in the complex mixture.
The hydrophilic portion may consist of alcohols, sulfoxide,
carbohydrates, acrylamides, with hydrophilic termini such as
alcohols, carbohydrates, amino acids, sulfoxides, acrylamides, and
ethers or other low-protein binding group. The hydrophilic display
molecule is anchored to the chip in the same manner as the peptoid
that has already been spotted. For example, chips spotted with the
peptoid library may be chemically blocked with cysteine,
mercaptoethanol or other suitable hydrophilic thiol. The chips may
also or alternatively be blocked with protein such as 2% BSA/PBS,
10% non-fat dry milk or 1% casein for at least 1 hour, rinsed with
water and dried. Other possible blocking agents are noted above.
The blocking agents may be applied to the chips in ways well known
to those of skill in the art, such as by dipping the chips in a
solution of a blocking agent, by painting the surface of the chips
with a blocking agent solution, or by spin-coating.
[0098] Alternatively, the surface regions surrounding the array
elements may be modified with polymeric or oligomeric chemical
blocking agents so as to minimize background non-specific binding
of proteins, allowing complex samples (e.g., lysates or serum) to
be examined in a single step. The surface may be blocked chemically
following spotting of the array elements with a hydrophilic
polymeric or oligomeric molecule that reduces or eliminates
non-specific protein binding to the array. As opposed to
conventional protein blockers such as BSA and casein, the polymeric
or oligomeric chemical blocker is a synthetic molecule that may be
used alone or together with a protein blocker. In a specific
embodiment, a chemical blocker and a protein blocker may be used
together, e.g., chemical block followed by protein block in
sequence or chemical block mixed with protein block and then
applied to the array surface after spotting. The polymeric or
oligomeric chemical blocking agent may be attached to the array by
dipping the slide into the blocking agent after spotting.
[0099] For example, in a specific embodiment, the chemical blocking
agent is a polyethylene glycol (PEG) analog, modified at at least
one terminus so that it will react with and bind to the organic
functionalized substrate surface not occupied by array elements.
For example, the chemical blocking agent for a maleimide
functionalized surface may be a thiol-modified polyethylene glycol
(PEG). One specific example is a dithiol-modified PEG (SH-PEG-SH),
for example having a molecular weight of about 3400-5000 (for
example, commercially available from Shearwater Polymers). The
blocking agent may be applied with casein after the array element
spotting is completed, as described below, or in a step beforehand.
The possible functionalities for the blocker termini are the same
as those for the adapters noted above, e.g., could be biotin,
amine, activated ester, etc.
[0100] Another type of chemical blocking in accordance with the
present invention is provided by well-defined, monodisperse
oligomers of N-substituted glycines (peptoids) derivatized with
hydrophilic side chains that can be readily attached to a variety
of surface functionalities. Suitable side chains may have one or
more ethylene glycol units, or may also be composed of hydroxyls,
sulfoxides, or other hydrophilic groups (such as described above)
that resist protein adsorption. These molecules are designed to
resist protein binding and would be interspersed with the specific
protein binding molecules of the protein array (e.g., antibodies,
fusion proteins, etc). These chemical blockers may be optimal for
high density packing of the protein-resistant moieties and thus
provide improved resistance to non-specific protein binding
(NSPB).
[0101] These chemical blocker oligomers may be prepared using the
submonomer peptoid synthesis method described above. For a
peptoid-based chemical blocking agent, suitable N-substitutions are
moieties that are known to be highly resistant to non-specific
protein binding, such as ethylene oxide, sulfoxide, hydroxyl, etc.
The N-terminus can be modified with a variety of surface
immobilization groups such as biotin, thiol, hydrazide, aldehyde,
epoxide, triethoxysilane, etc., as previously described.
[0102] The length of the peptoid blocker can be readily varied from
about 2-100, where 15-30 is practical and would result in pure
materials without a subsequent purification step. The length of the
side chains can be varied from between 1-10, with 1-5 providing
facile coupling to the peptoid backbone. In addition, both the
C-terminus and N-terminus may be modified with a variety of
chemical ligation reagents. Molecular weights can be in the range
of 500-5000, generally around 2000-3000.
[0103] After synthesis has completed, the peptoid may be cleaved
using conventional cleavage reagents such as 95% TFA/5% water. This
method can yield NSPB-peptoids in multi-gram quantities which can
readily be used to coat microarray slides in fairly large batches
(e.g., 20 slides at a time, using 200 mL of coating solution). In
the course of a microarray binding experiment, the NSPB-peptoid may
be incorporated directly into a protein blocking solution such as
casein, non-fat milk, BSA, etc. Alternatively, it may be used as a
separate coating step before or after protein blocking. The mode of
attachment of the microarray element (e.g., peptoids) to the
surface would likely determine the motif of NSPB-peptoid used. For
example, if biotinylated proteins are attached via robotic spotting
to avidin-coated slides, then a biotinylated NSPB-peptoid would be
used as the coating to block NSPB.
[0104] FIG. 3B illustrates a peptoid-based chemical blocking agent
in accordance with the present invention. The peptoid-based polymer
or oligomers chemical blocker is designed to stay hydrated and
resist the nonspecific binding of proteins. In the figure,
R.sup.1=H or Me, m=2 to 100, n=1 to 10.
[0105] E. Summary
[0106] FIGS. 4A and 4B briefly illustrate processes for making
protein-binding agent arrays for some embodiments of the invention
in accordance with the procedures described above. In FIG. 4A, a
process (400) for making an array in which protein-binding agents
are bound directly to the inorganic surface of a bare planar
substrate is depicted. A planar substrate 412 with a gold or
aluminum surface is provided (410). The surface is prepared for
binding (cleaned) as described above. Protein-binding agents 422
with a thiol anchoring group 434 are spotted onto the substrate 412
(420). Once binding of the protein-binding agents is complete, a
blocking agent 432, namely a hydrophilic group, such as an alcohol,
or a protein is applied to the surface of the substrate 412 where
no protein-binding agent 422 is bound (430).
[0107] In FIG. 4B, a process (450) for making an array in which
protein-binding agents are bound to the surface of a planar
substrate via an organic surface layer is depicted. A planar
substrate 462 with a gold or aluminum surface is provided (460).
The surface is prepared for binding by applying a functionalized
amino-modified thiol or amino modified silane layer 464, as
described above. The layer 464 includes a thiol or silane
functionality 466 which binds to the gold or aluminum surface of
the substrate 462 and a binding functionality 468, such as
maleimide, for a subsequently bound protein-binding agent.
Protein-binding agents 472 with anchor group functionality 474
complementary to the substrate surface layer binding functionality
468 are spotted onto the substrate (470). Once binding of the
protein-binding agents is complete, a blocking agent 482, namely a
hydrophilic group, such as an alcohol, or a protein is applied to
the surface of the substrate where no protein-binding agent 472 is
bound (480).
3. Methods of Using the Protein-Binding Agent Arrays of the Subject
Invention
[0108] The subject arrays find use in a variety of different
applications in which binding events between the surface bound
binding agents of the array and analyte(s) of interest in a test
sample are detected. In other words, the arrays of the subject
invention find use in binding assays. In such applications, the
support bound binding agent generally acts as a "target" for the
analyte "probe" in the test sample. The analyte probe is typically
labeled, e.g., where the label may be a directly detectable label
(e.g., radioactive isotope, fluorescent label, chemiluminescent
label, etc.) or an indirectly detectable label (e.g., member of a
signal producing system, such as a ligand for a labeled antibody,
where the label may be enzymatic which converts a substrate to a
chromogenic product, etc., where the labeled antibody may be a
secondary labeled antibody) so that binding events may be readily
detected.
[0109] In particular, arrays in accordance with the present
invention are useful in performing proteomic analyses of complex
protein samples. As used herein, proteomics is the separation
and/or quantitation and/or identification of one or more proteins
in a sample. The sample may be derived from a cell (e.g., the
cell's cytosol, membrane or extra-cellular proteins), tissues
(e.g., dissected or laser-microdissected), body fluids (such as
urine, blood spinal fluid) or any other sample containing proteins.
The results of such separation/quantitation/identification may
produce novel protein targets for drug screening, proteins for
diagnostics, or novel synthetic ligands for assays or protein
purification. The arrays may very effectively be used in
differential protein binding assays. For example, two (or
more)-color fluorescent labeling of complex protein mixtures, and
the analysis of differential protein binding to the array by
fluorescence imaging may be conducted. As described below, the
arrays may be used in conjunction with other techniques to
identify, sequence and structurally characterize differentially
expressed proteins or peptides of interest. The arrays may be run
in parallel with DNA arrays and the differential binding results
compared to identify correlations between gene activity and protein
expression. Also, mixed arrays, wherein the molecules making up an
array includes antibodies, etc. may be prepared and used to conduct
binding assays.
[0110] A variety of techniques can be used to conduct differential
binding assays using arrays in accordance with the present
invention ("proteomic microarrays"). Some of these techniques, as
used in embodiments of the present invention, are described
below:
[0111] A. Protein Labeling
[0112] Complex protein samples are labeled using standard
techniques, many of which have been developed for 2-D gel analysis
of protein mixtures. For example, sample A may be labeled with an
amine reactive Cyanine 3 dye ("Cy 3") (.lamda..sub.ex=550
nm/.lamda..sub.em=570 nm), and sample B is labeled with an amine
reactive Cyanine 5 dye (Cy 5)
(.lamda..sub.ex=650/.lamda..sub.em=670 nm) (dye reagents available
from Amersham-Pharmacia). Samples A and B may be, for example, from
normal or diseased, treated or untreated, etc., tissues or cell
lines, respectively. The unreacted dye may be separated from the
labeled protein using standard methods such as gel filtration,
dialysis, etc. Of course, as noted above, a variety of different
labels, as are well known to those of skill in the art, including,
but not limited to, tetramethylrhodamine-isothiocyanate (TRITC),
fluorescein-isothiocyanate (FITC), and succidimidyl ester
derivatives, thereof, or any other dye molecule that may be reacted
to proteins via amino acid side chains such as amine side chains
(lysine), thiol side chains (cysteine) or other suitable functional
group.
[0113] B. Binding Assay and Chip Readout
[0114] Labeled protein samples are incubated with the proteomic
microarray chip for periods of time, and under a variety of
conditions of pH, salt content and temperature anticipated to
modulate the affinity of various proteins to the elements of the
array. Generally, the samples are contacted with the microarray by
introduction of an appropriate volume of the fluid sample onto the
array surface, where introduction can be flooding the surface with
the sample, deposition of the sample onto the surface, e.g., with a
pipette, immersion of the entire array in the sample, and the like.
In many embodiments, the solution is deposited onto the surface and
then sandwiched beneath a cover slip or in a sealed chamber.
[0115] For example, a 25 .mu.L-100 .mu.L (typically 50 .mu.L)
aliquot of each probe solution may be applied to the surface of a
typical microscope slide-sized chip, and a cleaned coverslip placed
on top, forming a sandwich of the probe solution on the chip
surface. The protein solutions may then be co-incubated with the
chip for at least 1 hour, or overnight. After incubation, the
coverslip is removed and the chip is washed, for example, in
1.times.PBS/0.05% Tween or other suitable buffer containing
surfactant. The chip may be washed using a variety of conditions
that decrease or increase stringency. These conditions can again be
customized to allow, for example, retention of only the most
strongly bound proteins. Or, as the case may warrant, less
stringent washing may be used to allow visualization of
comparatively weaker bound proteins. The choice is likely to be
determined by the complexity and diversity of the peptidomimetic
(e.g., peptoid) array that is displayed on the chip and the nature
of the protein mixture. The washed chips are then dried, for
example, under a stream of Argon or Nitrogen.
[0116] After suitable washing, the chip is read in an array
scanner, such as are well known in the art. The ratio of Cy 3 to Cy
5 for each spot is determined using commercially available
software. Spots that show a ratio considerably greater than or less
than one are observed, and deemed to be "differential".
[0117] FIG. 5 briefly illustrates a process for conducting a
differential proteomic binding assay using protein-binding agent
arrays for one embodiment of the invention in accordance with the
procedures described above. In FIG. 5, the process (500) begins
with the procurement of two biological samples to compare, e.g., an
"untreated" cell line 502a and a "treated" cell line 502b. Cell
lysates 504a,b are isolated from the cell line samples. The lysates
are labeled, for example, the "untreated" cell lysate 504a is
labeled with a fluorescent green dye while the "treated" cell
lysate 504b is labeled with a fluorescent red dye. The labeled
samples 506a,b are then co-incubated on a protein-binding array
chip 508 in accordance with the present invention, e.g., an array
of peptoids. The protein in the samples can either be denatured or
native. For example with the addition of 1-2% SDS the proteins in
the samples may be denatured and clusters or hydrophobic
interactions minimized or eliminated. Alternatively, the clusters,
which may be important in elucidating protein-protein binding
pathways, and proteins may be kept in their native states and the
results studied. The chip is then read in a array scanner.
[0118] C. Library Sequencing
[0119] The sequence of the peptidomimetic that binds a
differentially expressed protein may be determined by library
sequencing techniques. This can be achieved, for example, by MS/MS
methods that allow fragmentation of the peptoid along the amide
backbone. These methods are described in co-pending U.S. patent
application Ser. No. 09/580,380, the disclosure of which is
incorporated herein by reference for all purposes (see also PCT
Publication No. WO 00/72004). From the mass of the fragmentation
product, the structure and sequence of the isolated peptoid can be
determined.
[0120] D. Post-Array Processing: Protein Isolation, Purification
and Identification
[0121] Once a protein or set of proteins is determined to be
differential between samples A and B, it can be isolated by
preparing chromatographic supports composed of the same
peptidomimetic (e.g., peptoid) identified on the chip.
Peptoid-based chromatographic supports, their preparation and their
use are described in U.S. patent application Ser. No. 09/704,422,
previously incorporated by reference herein.
[0122] In accordance with one embodiment of that disclosure, a
peptoid sequence that produces a differential on the chip may be
synthesized on hydrophilic resins that are hydrophobically "masked"
during peptoid synthesis. After synthesis, the resin in "unmasked"
to reveal hydrophilic groups compatible with biological solutions.
Such a resin is immediately available for protein binding/isolation
experiments.
[0123] Once the protein is isolated, it's sequence can be
determined using standard techniques such as MALDI/TOF mass
spectrometry or trypsin digests.
[0124] FIG. 6 briefly illustrates aspects of post-array processing
in accordance with the procedures described above and below. In
FIG. 6, the process (600) begins with the preparation of
chromatographic separation columns 602 using peptidomimetic agents
601, e.g., peptoids, identified as of interest in a proteomic
differential binding assay conducted using a proteomic microarray
in accordance with the present invention. An aliquot of the complex
sample originally run on the microarray is then run through the
column. The protein of interest preferentially binds to the column
and is thereby separated form other components of the sample. The
bound protein is eluted and may then be used in further analyses
604, such as protein sequencing, tertiary structure determination,
etc. In addition, data relating to the identification of the
protein may be entered into bioinformatics databases for further
research.
[0125] Alternatively, the same peptoid that bound the
differentially expressed protein could be spotted repetitively on a
chip and incubated with the an aliquot of the same lysate. The same
protein should bind to the array, but in a much larger area than
just the one spot on the original chip. Laser desorption mass
spectrometry can then be used to sequence the protein directly from
the chip.
[0126] E. Other Applications
[0127] The anticipated uses of proteomic microarray chips in
accordance with the present invention are broad. In general, the
applications have in common the identification of a protein or set
of proteins that are over-expressed or under-expressed in one
complex mixture relative to another (or present/absent, such as in
the case of a diagnostic protein). Those skilled in the art will
recognize that embodiments of the present invention are compatible
with a wide variety of assay formats including sandwich assay, such
as ELISA.
[0128] As described above, proteomic microarrays may be used to
determine differential expression of proteins in complex solutions
by alternatively labeling (e.g., Cy 3 for one sample and Cy 5 for
another) the two or more protein solutions to be compared. The
chips may be used to find novel protein targets for later high
throughput screening assays. In another particularly powerful
application, the methodology may be used to purify a recombinant
protein that is overexpressed in a particular host such as yeast or
baculovirus. The sample that contains the expressed protein is
compared to the sample that does not by co-binding the
alternatively labeled samples on the chip, and looking for
differentials. The procedure identifies peptoids on the array that
bind with reasonable affinity and specificity to the expressed
protein. These same peptoids are then used for generating
chromatographic resins for the isolation and purification of the
recombinant protein of interest.
[0129] In an analogous manner, the proteomic microarry chips may be
used to find protein markers in plasma or serum that may be
diagnostic of particular disease states such as cancer, HIV, or
diabetes, or to find novel targets for drug screening. Also, once a
set of ligands has been identified for particular groups of
proteins, it is possible to monitor the expression levels of these
proteins to decipher mechanisms of drug action. In that regard, the
new ligands may be used as probes of protein abundance, analogous
to the ways in which antibodies are currently used to determine
protein abundance. In addition, by examining the proteins from
virulent and non-virulent strains of bacteria or viruses, one can
determine unique virulence factors that result in infectious
disease. Once these virulence factors are identified, these
proteins can be used as targets for screening new anti-bacterial or
anti-viral drugs. Alternatively, the chips provided by the present
invention may also be used to discover a variety of peptide,
antigen, or protein mimetics. For example, novel mimetic cellular
adhesion molecules, mimetic drug candidates, or protein mimetics
that may be used as chromatographic supports. In addition, the
material spotted on the chip could itself be potential drug
candidates or function as an initial scaffold for designing new
drug candidates. In such embodiments, high throughput assays to
identify potential therapeutic agents can be done directly on the
chip.
[0130] In one embodiment, the proteomic microarrays of the
invention are run in parallel with DNA arrays, and the differential
binding results derived from each are compared to identify
correlations in gene activity and protein expression. For example,
differential binding assays are conducted for complex biological
samples on both proteomic and DNA arrays. Separate aliquots from
the samples are labeled and contacted with a proteomic microarray
in accordance with the present invention and a DNA microarray, such
as are well known in the art. The differential protein expression
evidenced by the binding results on the proteomic array when
compared with those for the DNA array may elucidate relationships
between protein expression and gene families whose activation is
required for that expression.
[0131] In another embodiment of the present invention, a "mixed
array" chip of the present invention is provided. Peptoid or other
peptidomimetic binding elements attached to the chip surface may be
mixed with antibody or protein binding elements such that both
types of binding elements are present on the same chip. The
antibodies can serve as positive controls, or as a means of
monitoring the levels of specific proteins in the mixture being
analyzed. The peptoids would provide data on differentials in
unknown proteins, whereas antibody spots on the chip would provide
data on differential levels of known proteins. For example, if a
cell is treated with a certain drug, the protein levels might
change up or down. If those proteins have known antibodies, it is
possible to monitor the change of these with antibody
differentials, while at the same time, look for changes in unknown
proteins, with the peptoid differentials.
[0132] In one example of this technique, antibody solutions are
prepared in the same microtiter plates as the peptoid solutions,
but in different wells. The peptoids are already functionalized
with thiol anchoring groups. The antibodies are reacted with a
reducing agent to reduce the disulfide bonds in the antibody hinge
and Fc region (for example, as described in Levison, M. E., et al,
(1969), Experentia, vol 25, 126-127 and Blauenstein, P., et al,
(1995), Eur. J. Nuclear Med., vol 22, 690-698), thus producing a
thiol on the antibody. This allows for spotting of both peptoid and
antibody in the same spotting session, thereby creating a "mixed"
chip. Alternatively, the antibodies may be bound to immobilized
Protein A or Protein G on chip surfaces, while peptoids are
attached to avidin or streptavidin, presenting a mixed surface for
display. Or, antibodies may simply be biotinylated and spotted
together with biotinylated peptoids or peptides onto avidin-coated
chips.
[0133] Another technique that may be combined with the proteomic
microarray techniques of the present invention is the MS/MS
macromolecular structural analysis technique described in
above-referenced U.S. patent application Ser. No. 09/580,380,
previously incorporated by reference herein. In this way, the
combination of techniques can be used to identify a protein of
interest, enrich and isolate it, sequence the protein, and
elucidate aspects of its tertiary protein structure.
[0134] Data relating to the identification and post-array
processing of proteins of interest may also be entered into
bioinfomatics databases. The data may be correlated with other
biological data therein for further research.
4. Kits
[0135] Also provided by the subject invention are kits for
performing proteomic binding assays using the subject arrays. Such
kits according to the present invention will at least include an
array according to the invention. The kits may be configured for
analytical or diagnostic purposes, and may further include one or
more additional reagents employed in the method for which the array
is intended. For example, the kit may include various receptacles,
labels, buffer solutions, tools and any other material necessary to
conduct a proteomic binding assay. Kits in accordance with the
present invention may also be configured to receive samples for
analysis and thereafter perform the steps necessary for a binding
assay in accordance with the invention without further user
manipulation.
EXAMPLES
[0136] The following examples provide details concerning the
synthesis and characteristics of the proteomic arrays in accordance
with the present invention, their components, and applications. It
should be understood the following is representative only, and that
the invention is not limited by the detail set forth in these
examples.
Example 1
Preparation of Microtiter Plates Containing One Compound Per
Well
[0137] Preparation of one (1) 96-well plate containing 96 single
compounds (0.8 mM) in a 1:1 DMSO/PBS solution: A siliconized vial
(8 ml, 2 dram) was loaded with a one compound/bead library
synthesized on Rink polystyrene macro-bead solid support (66 mg,
.about.1320 beads, 40 nanomole/bead, 0.75 mmol/g, 425-500 um,
Polymer Laboratories) that contains a total of 121 possible
compounds. The beads were swollen in dichloroethane (DCE, 4 ml) for
24 hours and sieved over stainless steel mesh (600 .mu.m). Using
the resulting dichloroethane bead slurry, 96 beads were
individually picked and relocated into a polypropylene 96-well
plate (200 .mu.L, conical bottom) in order to obtain one bead per
well over 96 wells. The resulting 96-well plate, (the `grandmother`
plate), was transferred to a speed-vac evaporator Savant AES200
equipped with a 96-well plate carrier rotor and the remaining DCE
was removed from the plate. A cleavage cocktail (TFA/TES/H2O/DCE,
46:2:2:50, 75 .mu.L) was then added to each bead containing well in
the plate. After an hour the plate was transferred to the speed-vac
evaporator to remove most of the volatiles from the plate. Each
well was treated with acetonitrile (CH.sub.3CN, 10 .mu.L) and
agitated for 10 min using a microtiter plate shaker. The plate was
transferred to a speed-vac evaporator and the remaining volatiles
were removed from the plate for 10 minutes. Every single compound
in each well was dissolved in dimethylsulfoxide (DMSO, 25 .mu.L)
and relocated in a 96-well filter plate made of polystyrene,
equipped with a poly-propylene membrane (0.45 .mu.m). The 96-well
filter plate was stacked over a polypropylene 96-well plate (200
.mu.L, conical bottom) and the assembly was transferred to a
Beckman GS-6R centrifuge programmed for 5 minutes at 3000 RPM
(.about.2000 G) at 15.degree. C.
[0138] This procedure provided the `mother` plate composed of
filtered 96 DMSO solutions (1.6 mM) of one compound in 96 wells. To
form the `daughter plate` (used for spotting), an aliquot of every
DMSO solution (5 .mu.L) was transferred to a polypropylene 96-well
plate (200 .mu.L, conical bottom). Then, every well in the daughter
plate was mixed with degassed solution PBS (5 .mu.L) and stored at
-20.degree. C. Alternatively, each compound in each well may be
dissolved in 20 .mu.L of 50% DMSO/water. In the case of a plate
with a conical bottom, the plate may be centrifuged at 1,800 rpm
for 5 minutes to immobilize the bead at the bottom of the conical
well, followed by relocation of 10 uL of the clear supernatant into
a daughter plate that is ready for spotting.
[0139] The same can be done for 384 beads in 384 well plates, or
1,024 beads in 1,024-well plates, etc. For spotting, any suitable
spotting device can be used, such as a Molecular Dynamics
Generation II (for 96-well) or Generation III (384-well)
spotters.
Example 2
Functionalization of Solid Supports for Spotting
[0140] Gold or aluminum reflective microscope slides were used as
substrates for spotting a library of peptoids. In one example, the
peptoid is functionalized with a thiol endgroup and the surface is
functionalized with a maleimide. Upon spotting, the thiol on the
peptoid reacts with the maleimide on the surface to form a covalent
thioether attachment. Gold surfaces activated with maleimide were
prepared as follows: 1) Gold-coated microscope slides (1200
Angstroms Au, 30-50 Angstroms Ti or Cr) were cleaned with Chromic
acid cleaning solution for 15 minutes and rinsed with HPLC grade
water. 2) Gold slides were dipped into 1-5 mM amino-modified thiol
(1-Mercaptoundecyldiethoxyamine; a C-11 alkyl, two ethylene oxides,
and an amine; alternatively, C-2 to C-20 alkyl groups and/or ethoxy
or triethoxy groups could be used in such a compound) for 1-24
hours at room temperature or at 45 or 60.degree. C. The slides were
rinsed four times in absolute ethanol and dried under a steam of
Nitrogen. 3) The amino-modified gold slides were dipped into a
solution (50-100 uM) of succinimidyl
4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC) to render a
surface that presents the maleimide functional groups.
[0141] Aluminum slides were made or purchased from
Amersham-Pharmacia, coated with a 1000-1400 Angstrom layer of
silicon oxide, and a layer of aminopropysilane (APS). The
amino-modified Al surfaces are functionalized with SMCC as
described above.
[0142] In some cases, after spotting the peptoid library, the
aluminum slides were etched in a solution of 0.1% SDS/5.times.SSC
at 60 degrees C. for 2 hours. The etched slides exhibit a reduced
oxide layer thickness (200-900 Angstroms) to allow amplification of
both the Cy5 and Cy3 signals.
Example 3
Derivatization of Aluminum/SiO.sub.2 Surfaces with Avidin
[0143] Aluminum slides coated with aminosilane were dipped into a
solution of NHS-LC-LC-biotin ("LC" refers to 6-aminohexanoyl and
"NHS" refers to N-hydroxysuccinimidyl) (commercially available from
Pierce), depicted in FIG. 7A, that was 0.39 mM in PBS buffer. The
slides were coated for 1.5 hours with shaking at 80 rpm. After
attachment of biotin, the slides were rinsed with water, then
dipped in a solution of 1 ug/ml-1 mg/ml avidin, streptavidin or
neutravidin in PBS buffer for 2 hours, stirring at 70 rpm. The
slides were rinsed with water and ready for spotting biotinylated
peptides or peptoids. FIG. 7B depicts a representation of an
avidin-derviatized aluminum slide spotted with a biotinylated
protein-binding agent in accordance with one embodiment of the
present invention.
[0144] The various surface modification steps was followed using
ellipsometry to note the thickness changes. A thickness change
increase of 40-45 angstroms was reproducibly recorded after the
addition of avidin to the surface layers.
Example 4
Protein Labeling
[0145] Protein solutions were adjusted to a concentration of 1
mg/mL in 0.1 M sodium carbonate, pH 9.3 and a volume of 0.1-1 mL,
and mixed with bifunctional or mono-functional amine-reactive
cyanine dye (Cy3 or Cy5, Amersham Pharmacia). The protein was
purified from the unreacted dye by size exclusion chromatography
using a Sephadex G-25 packing in a 5 cm long, 1.7 cm diameter
column with a 1 mL load, 0.5 mL fractions, and a dilution factor of
3.5.
[0146] FIG. 8 provides a graph of the results illustrating that
size exclusion chromatography of the components efficiently
separates the labeled protein from the unreacted dye. The graph
shows the different elution profiles for the protein (BSA standard)
and dye molecules.
Example 5
Chip Binding Experiments
[0147] In some instances, the chips spotted with the peptoid
library are chemically blocked with cysteine, mercaptoethanol or
other suitable hydrophilic thiol. The chips are blocked with
protein such as 2% BSA/PBS, 10% non-fat dry milk or 1% casein for
at least 1 hour, rinsed with water and dried. The labeled protein
probe solution is diluted accordingly (typically to 20-1000 ng
total protein) with the blocking solution (for the etched Al
slides, detection limits of a few picograms have been observed). A
30-100 .mu.L aliquot of the probe solution is applied to the chip
surface, and a clean coverslip placed on top, forming a sandwich of
the probe solution on the chip surface. The protein solution is
incubated with the chip for at least 1 hour. The coverslip is
removed in 1.times.PBS/0.05% Tween or other suitable buffer
containing surfactant. The chip is then washed in 1.times.PBS/0.05%
Tween or other suitable buffer/surfactant system. The chips are
further rinsed with water, dried under a stream of Argon or
Nitrogen and scanned.
Example 6
Sample Chip
[0148] A microarray chip bearing a library of 1,000 peptoid-based
protein-binding agents was prepared in accordance with the present
invention. Streptavidin was spiked into cells induced to express
proteins in the wnt pathway and into control cell lysate and the
entire mixture labeled with Cy3 (control) or Cy5 (wnt). The mixture
was then incubated with the chip (6.3 ng total protein/chip; 31 pg
streptavidin/chip), which had a biotin-displaying peptoid in it's
upper left corner. FIGS. 9A and 9B show 1/6 of the incubated chip.
As shown, in addition to the biotin peptoid spots, many other
peptoid spots are binding to the protein in the lysate. By
comparing the relative signals from the Cy5 and Cy3 channels,
differentials can be identified. FIG. 9C shows 1/2 of a
streptavidin-only control chip (31 pg streptavindin/chip). Biotin
peptoid spots are visible in the upper left corner.
Example 7
Demonstration Chip
[0149] As illustrated in FIG. 10, hexameric peptides of different
and known affinity to the "anti-glu" antibody were synthesized with
a biotin anchoring group and heptameric gycyl linker. To prove the
concept of the present invention, the biotinylated peptides were
spotted onto avidin-treated slides as described in the present
disclosure. The slides were blocked with casein and probed with
Cy5-labelled antiglu antibodies. The gradations in signal intensity
correlate with the known differences (e.g., measured by direct
ELISA) in affinity between the peptides and their cognate antibody
probe.
Example 8
Comparison of Surface Attachment Systems
[0150] FIG. 11 depicts the dependence of signal strength on the
mode of surface attachment of a protein-binding agent to a
substrate. In this example, signal increases of 1000.times. are
observed for biotin/avidin immobilization compared to
thiol/maleimide (signal results depicted above surface attachment
description and molecular structure).
Example 9
Preparation of Protein-Coated Slide Surface for Antibody
Display
[0151] To prepare Protein A- or Protein G-modified side surfaces, a
slide coated with avidin (prepared as described above) was immersed
in a solution of biotinylated Protein A or Protein G (0.5-1 mg/mL
in PBS buffer, purchased from Pierce, product numbers 29989zz and
29988zz) for 2 hours at room temperature. The slides were then
rinsed with de-ionized distilled water and blown dry with Nitrogen
or Argon.
Example 10
Comparison of Glass and Mirrored Slides
[0152] Al/SiO2/APTES slides were purchased from Amersham-Pharmacia,
or home-made in a Class 100 cleanroom. Slides were prepared with a
reflective aluminum coating that is further overcoated with a thin
silicon dioxide dielectric, followed by APTES. The homemade slides
were pre-cleaned by sonication in a liquid surfactant for 10
minutes, rinsed with de-ionized water, then further cleaned in a
solution of Nochromix/H2SO4. Following a de-ionized water rinse,
the slides were immersed in isopropanol for 5 minutes and dried. A
Model CH-SEC-600-RAP e-beam equipped with rotating planetaries was
pumped to 2.times.10-6 torr prior to deposition of 1000 A of
aluminum and 800 A of SiO2. The slides were then placed in a vacuum
oven at 100 containing 25 mL of APTES, and pressure reduced to -23
mm Hg. The slides were used without further treatment. cDNA clones
were robotically spotted from 1:1 water/DMSO solution onto the
substrates using a Generation III Molecular Dynamics Spotter.
[0153] FIG. 12 shows a comparison of the image obtained from an
APTES-coated glass microscope slide to that of an APTES-coated
aluminum slide. The same amount of fluorescently labeled analyte
sample was applied to each surface. Although different input mRNA
was used, the top row depicts a standard set of spiked controls.
Spot 1, arabidopsis mRNA is spiked in at 25 ng for the Al slide and
250 ng for the glass slide, yet the signal is 2 times greater for
the aluminum slide. The second spot of the top row is spiked at the
equivalent of 1 copy per cell. On the glass substrate, the signal
is barely detectable compared to a robust signal that is about 20
times greater using the Al substrate. Background non-specific
binding is minimal in both cases, as shown by the clear background
for spots 20-22 (bacterial genes) and spots 26-29 (DMSO) and spots
31-33 (drosophila genes). The Al/SiO.sub.2 substrate amplifies the
signal from Cy3/Cy5 tagged cDNA by approximately 10-40 fold
relative to the corresponding glass substrate.
[0154] Thus, the Al/SiO.sub.2 (mirrored) slides yield array images
that are superior to their plain-glass counterparts with respect to
signal strength, as well as spot morphology and uniformity.
CONCLUSION
[0155] Although the foregoing invention has been described in some
detail for purposes of clarity of understanding, it will be
apparent that certain changes and modifications may be practiced
within the scope of the invention. For example, while the present
disclosure emphasizes the use of peptoids as the peptidomimetic
segment of the protein-binding array elements, it should be
understood that the scope of the invention is not so limited and
other molecules having the appropriate properties and function as
described herein may also be used. It should also be noted that
there are many alternative ways of implementing both the process
and apparatus of the present invention. Accordingly, the present
embodiments are to be considered as illustrative and not
restrictive, and the invention is not to be limited to the details
given herein.
* * * * *