`
`||||||||||||||llllllllillll|||l|||||l||l||||||||||
`US 20050] 73247Al
`
`||||||||||||||||||ll|||l|||||||||l|li
`
`(to; United States
`(12) Patent Application Publication (10) Pub. No.: US 2005/0173247 A1
`
`Jalali at al.
`(43) Pub. Date:
`Aug. 11, 2005
`
`(54) MICROTITER PLATE FORMAT DEVICE
`AND METHODS FOR SEPARATING
`DIFFERENTLY CHARGED MOLECULES
`USING AN ELECTRIC FIELD
`
`Related U.S. Application Dfittt
`
`{62] Divisiun of applictttiun No. (.i9f724336. filed ran thv.
`28. liit'iil,
`
`{75}
`
`Invcntnrs: Slliltl .Ittltlll. San Diego, CA (US);
`Karla Ewnlt. San Die-g0, CA (US);
`l’tllll Swansnrt‘ Sanlcc. CA(US)1 Brim-l
`Dwyer‘ San DlUgU‘ (“A (Us); Michael
`.I. Heller. Encinitas, CAiUS); John R.
`Havana San Diego, (.‘A (US); Etgcne
`TIL San Diego, (‘A (US)
`
`Correspondence Addretxs:
`MORGAN LEWIS & BOCKIUS LLI’
`111] PENNSYLVANMAVEN UE NW
`WASHINGTON. DC 20004 (US)
`
`(73} Antigua: Nnnogfl.‘ [nc_
`
`(2]) Appl. Nu;
`
`llll930,821
`
`{33)
`
`Filed:
`
`NIM'. 4. 3004
`
`Publication Classificaiinn
`
`7
`
`_
`‘
`GlHN 273-53
`int: (,l.
`{51]
`(52) U.5. (.1. y......"nun...“.....""....mm"... 201K450, EU‘l‘flJOll
`
`{57]
`
`ABSTRACT
`
`‘
`
`_
`_
`.
`.
`‘
`lhc present anUl‘lllLll'l relates generally tu tntcrutttur plate
`format devices and mclhuds for separating molecules having
`dil'l'crunl net charges. The devices and methods of the
`invention are particularly suited for use in high—throughput
`screening to monitor cnzymatic reactions which result in t1
`protlucl having an altered net charge For cxamplu,
`the
`systems and mclhmls {liscloscd fie-rain may he used to detect
`the activity ul‘ plmsphatascs. protcasus and kimtscs on vari~
`{ms puptitiic Slllfill’fllcs under various conditions.
`
`'
`_”-.(-):Electrode _
` Phosphorylatedi
`Microtiter Plate 0..
`
`MUN'm—lfifmm1.7!.”"“EWK?NEW
`
`
`Unphosphoryiated
`Peptide
`
`Gel —>
`
`Agilent Exhibit 1258
`
`Page 1 of 21
`
`Agilent Exhibit 1258
`Page 1 of 21
`
`

`

`Patent Application Publication Aug. 11. 2005 Sheet 1 0f10
`
`US MUS/0173247 A1
`
`FIGURE 1
`
`_'-
`
`i H Electrode
`
`Peptide
`
`Phosphorylated!
`Unphosphorylated
`
`
`
`Agilent Exhibit 1258
`
`Page 2 of 21
`
`Agilent Exhibit 1258
`Page 2 of 21
`
`

`

`Patent Application Publicatiun Aug. 11. 2005 Sheet 2 of 10
`
`US MUS/0173247 A]
`
`FIGURE 2
`
`. ‘
`
`Agilent Exhibit 1258
`
`Page 3 of 21
`
`Agilent Exhibit 1258
`Page 3 of 21
`
`

`

`Patent Application Publication Aug. 11. 2005 Sheet 3 of 10
`
`US 2005/0173247 A1
`
`FIGURE 3a
`
`Columns:
`
`1234 S 678 9101112
`
`Before
`Electrophoresis:
`
`After
`
`,Qé9399099@@9
`" ' '
`<
`7
`‘14.“
`-'
`000 96 6 ‘; Q t "J! 9 a
`_}i-f'9 999359 Magoo r:
`
`
`Electrophoresis:
`
`FIGURE 3b
`
`
`
`Co1umsf123455739101112
`
`Active
`
`Passive
`
`Unphosphorylated
` ; -—
`55$ Kemptide-L
`
`
`
`Phos horylated
`Kemgfide-L
`
`Agilent Exhibit 1258
`
`Page 4 of 21
`
`Agilent Exhibit 1258
`Page 4 of 21
`
`

`

`Patent Application Publication Aug. 11. 2005 Sheet 4 of 10
`
`US 2005/0173247 A1
`
`FIGURE 4
`
`1X Tris-Borate Buffer
`
`
`Pin Electrodes
`
`
`
`Membrane Plugs
`
`
`
`IEIIEI IEI IE]IE! IE!lEIIE!IE! l" 1'] IE!U
`
`and Gel plugs Hi
`
`Membrane
`
`Microtiter Plate
`
`Agilent Exhibit 1258
`
`Page 5 of 21
`
`Agilent Exhibit 1258
`Page 5 of 21
`
`

`

`Patent Application Publication Aug. 11. 2005 Sheet 5 0f 10
`
`US 200501173247 A1
`
`FIGURE Sa
`
`?00
`
`600
`
`500
`
`400
`
`300
`
`200 =
`
`
`
`fluorescenceintensityTR
`
`R2 =o.9997_
`
`Limit of Detection
`
`y: 3040.1x+130.47
`
`100
`
`0 0
`
`
`
`fluorescencelniensiwTR
`
`.000
`
`0.020
`
`0 .040
`
`0.060
`
`0.080
`
`0.100
`
`0.120
`
`male fraction of phosphoMaledmnphosphoMatad Kempfide
`
`FIGURE 5b
`
`2500
`
`2000
`
`E
`
`won
`
`500
`
`
`
`'
`
`.
`
`Limit of Detectiou
`
`y = 3232.31: + 273.3
`R2 = .
`a 9933
`
`0.000
`
`1,000
`0.300
`0.600
`0.400
`0.200
`mole flacfion phosphoryialadzunphosphorflated Kemp-Ede
`
`1.200
`
`Agilent Exhibit 1258
`
`Page 6 of 21
`
`Agilent Exhibit 1258
`Page 6 of 21
`
`

`

`Patent Application Publication Aug. 11. 2005 Sheet 6 0f 10
`
`US NUS/0173247 A1
`
`FIGURE 6
`
`50 mM Tris-HC1
`
`(-) Electrode
`
`Gel-filled
`
`Microcapillary
`
`ph 8.0
`
`
`Diffusion Barrier 3.:
`
`Membrane
`\‘15
`
`Microtiter Plate
`
`
`{H Electrode
`
`Agilent Exhibit 1258
`
`Page 7 of 21
`
`Agilent Exhibit 1258
`Page 7 of 21
`
`

`

`Patent Application Publication Aug. 11. 2005 Sheet 7 0f 10
`
`US NUS/0173247 A1
`
`FIGURE 7
`
` limit of detection
`
`0
`
`20
`
`40
`
`60
`
`80
`
`100
`
`Percent Concentration Phos:Unphos Kemptide
`
`Agilent Exhibit 1258
`
`Page 8 of 21
`
`Agilent Exhibit 1258
`Page 8 of 21
`
`

`

`Patent Application Publication Aug. 11, 2005 Sheet 8 of 10
`
`US 20050173247 Al
`
`FIGURE 8a
`
`FIGURE 8b
`
`133010)-
`1030100
`amen
`
`a mm
`mm
`
`2113,01
`um
`
`
`
`
`”:7 Positive Peptide % Negative Peptide
`
`FIGURE 8c
`
`FIGURE 8d
`
`moo]
`mm
`
`_ mmi
`a
`10M”:
`g 1mm
`if;
`
`
`6
`
`1G
`
`15
`
`20
`
`2
`
`acumen
`mm
`40000.00
`E acumen
`10000.00
`mom on
`O 00
`
`
`
`M9012 Concemrafim (nu)
`
`MnCIZ Concentration (mM)
`
`Agilent Exhibit 1258
`
`Page 9 of 21
`
`Agilent Exhibit 1258
`Page 9 of 21
`
`

`

`Patent Application Publication Aug. 11. 2005 Sheet 9 0f 10
`
`US 2005flll73247 Al
`
`FIGURE 9
`
`Separate Plates
`
`
`
`
`
`
`One plate
`
`Active Acme Anti“
`
`Blank
`
`Passive
`
`Active Active Active Blank Passive
`
` ~‘ Positive Peptide
`
`Negative Peptide
`
`Agilent Exhibit 1258
`
`Page 10 of 21
`
`Agilent Exhibit 1258
`Page 10 of 21
`
`

`

`Patent Application Publication Aug. 11. 2005 Sheet 10 [If 10
`
`US MUS/0173247 Al
`
`FIGURE 10
`
`Promega's peptide
`
`Texas Red Labeled
`
`Llssamine Labeled
`
`300000.
`
`250000.
`
`200000.
`
` Fluorescence
`
`150000.
`
`100000.
`
`50000.
`
`‘ 0.0
`
`Intensities
`
`Active Passive Active Passive Active Passive
`
`DUnphoephorylated lPhosphorylated
`
`Agilent Exhibit 1258
`
`Page 11 of 21
`
`Agilent Exhibit 1258
`Page 11 of 21
`
`

`

`US EGGS/0173247 A1
`
`Aug. 11, 2005
`
`MICROTITIER PLATE FORMAT DEV ICE AND
`METHODS FOR SEPARATING DIFFERENTLY
`CHARGED MOLECULES USING AN ELECTRIC
`FIELD
`
`FIELD OF INVENTION
`
`[0001] The present invention relates generally to microti-
`tcr plate format devices and methods for separating mol-
`ecules having dilTerent net charges. The devices and meth-
`ods of the invention are particularly suited for use in
`highathroughput screening to monitor enzymatic reactions
`which result in a product having an altered net charge. For
`example, the systems and methods disclosed herein may he
`used to detect the activity of phosphatascs. proteases and
`kinuses on various peptidie substrates under various condi-
`trons.
`
`BACKGROUND OF THE INVENTION
`
`[0002] Protein kinases are of particular interest in drug
`discovery research because they have been shown to be key
`regulators of many cell functions. including signal transduc-
`tion (Ullrich and Schlessingcr. 1990). transcriptional regu-
`lation {Pawson and Bernstein, 1990). cell motility (Miglietta
`and Nelson,
`[988'] and cell division (Pines and Hunter,
`1990). Protein kinnses are enzymes. which covalently
`modify proteins and peptides by the attachment of a phos-
`phate group to one or more sites on the protein. Phos-
`phaluscs perform the opposite function. Many of the known
`protein kinases use adenosine triphosphate [ATP] as the
`phosphate donor, placing the y—phosphate onto a histidine,
`tyrosine. serinc or threonine residue in the protein. The
`location of the modification site and the type of residue
`modified by the kjnase are usually specific for each particu-
`lar kinase.
`
`[0003] The added phosphate alters certain structural, ther-
`modynamic and kinetic properties of the phosphorylated
`protein. Generally. the phosphate adds two negative charges
`to the protein. This modifies the electrostatic interactions
`between the protein's constituent amino acids. in turn alter—
`ing secondary and tertiary protein structure. The phosphate
`may also form up to three hydrogen bonds or salt bridges
`with other protein residues. or may otherwise change the
`conformational equilibrium between diil‘ercnt
`fitnetiortal
`states of the protein. These structural changes provide the
`basis, in a biological system. for altering su bstrale binding
`and catalytic activity of the phosphorylatod proteins.
`
`[0004] Phosphorylation and dephosphorylation reactions,
`under the control of kinases and phosphatases. respectively,
`can occur rapidly to form stable structures. This makes the
`phosphorylation system ideal as a regulatory process. Phos-
`phorylation and dephosphoryltttion reactions may also be
`part of a cascade ol‘ reactions that can amplify a signal that
`has an extracellular origin. such as hormones and growth
`factors.
`
`phosphoamino acids with radiolabeled specific antibodies.
`or 3) Purification of phosphorylated peptides from unphos—
`phorylatcd peptides by chromatographic or eloctrophoretic
`methods, followed by quantification of the purified product.
`
`For example. in one widely used method. a sample
`[0006]
`containing the kinam of interest is incubated with activators
`and a substrate in the presence of gamma ij-A‘I'P, with an
`inexpensive substrate. such as historic or casein.11eing used.
`After a suitable incubation period, the reaction is stopped
`and an aliquot of the reaction mixture is placed directly onto
`a filter that binds the substrate. The filter is then washed
`
`several times to remove excess radioactivity. and the amount
`of radiolabellcd phosphate incorporated into the substrate is
`measured by scintillation counting {Roskoskj, 1983).
`
`[0007] The use ol‘saP in assays. however. poses significant
`disadvantages. One major problem is that,
`for sensitive
`detection, relatively high quantities of 331’ must be used
`routinely and subsequently disposed. The amount of liquid
`generated from the washings is not small, and contains 321’.
`Due to government restrictions, this waste cannot be dis-
`posed ot‘ easily. It must be allowed to decay, usually for at
`least six months, before disposal. Another disadvantage is
`the hazard posed to personnel working with the isotope.
`Shielding and special waste containers are inconvenient but
`necessary for safe handling of the isotope. Further, the lower
`detection limit of the assay is determined by the level of
`background phosphorylatiou and is
`there-fore variable.
`Although radioisotope methods have been applied in high
`throughput screening, the high cost and strict safety regu-
`lation incurred with the use ofradioisotopes in high throughv
`put screening greatly limits their use in drug discovery. For
`these and other reasons,
`it would be useful
`to develop
`alternative methods and apparatus ['or high throughput
`screening that facilitate measuring the kinase dependent
`phosphorylatiou of peptides.
`
`SUMMARY OF THE INVENTION
`
`[0008] The systems and methods of the present invention
`provide an easy—to-use, rapid system For separating differ—
`ently charged molecules and quantifying them. and can
`easily be adapted for use with standard microtiter plate
`readers and loaders. In general, the systems of the invention
`comprise a) a sample plate comprising a plurality of sub-
`stantially tubular sample wells arrayed in the sample plate.
`and at least one capture matrix. positioned in each of the
`sample wells proximate the bottom or end of the sample
`well. which comprises a diffusion—inhibiting material; and b)
`at least one first electrode in electrical contact with at least
`one sample Well at the bottom end of the sample wall, and
`at least one second electrode in electrical contact with the
`
`top end of the sample well. where both electrodes are
`coupled to a power source. The electrical contacts. with the
`bottom and top ends of the sample well may be made
`through a conductive fluid,
`
`[0005] Methods for assaying the activity of protein kinases
`often utilize a synthetic peptide substrate that can be phos-
`phorylatert by the kinasr. protein under study. The most
`common mechanisms for detecting phosphorylation of the
`peptide substrates are I) Incorporation of 32F (or 33F)
`phosphate from [5:P]1{~ATP into the peptides. purification of
`the peptides from/“1P. and scintillation or (Therenkov courtl-
`ing of the incorporated radionucleotidc, 2) Detection of
`
`[0009] The ditl'usion-inhibiting materials used in the cap-
`ture matrix in the sample wells of the system serves to
`exclude molecules which have not been selected by elee-
`trophoretic separation, and to hold or contain those mol-
`ecules of interest which have been selected, In this way, the
`nonzselccted molecules may be washed out of the wells. and
`the selected molecules retained for detection. The capture
`matrices used in the present invention may comprise more
`
`Agilent Exhibit 1258
`
`Page 12 of 21
`
`Agilent Exhibit 1258
`Page 12 of 21
`
`

`

`US EGGS/0173247 A1
`
`Aug. 11, 2005
`
`than one layer of material. with one layer being a diffusion-
`inhibiting layer of material. and another layer being a
`binding layer of material which binds the charged molecule
`of interest in a covalent or non-covalent manner. Various
`electrode assemblies are preferred for use in the systems of
`the invention. including plate electrodes. pin electrodes. and
`conductive liquid electrodes using. gels or other hydrophilic
`difl‘usion barrier materials to isolate the conductive liquid
`from the sample in the sample plate.
`
`the invention also provides
`In another aspect,
`[0010]
`methods for separating a charged molecule of interest from
`a mixture of molecules having dilfercnt chargcsin a plurality
`of samples, and quantifying the amount of the charged
`molecule of interest in the samples, the method comprising
`the steps of:
`
`(a) filling the sample wells of a system of the
`[001.1]
`invention with a liquid;
`
`(b) adding a sample containing a mixture of
`[0012]
`molecules to at least two of the sample wells of the
`device;
`
`(c) applying an electric field across the sample
`[0013]
`wells by energizing the electrodes, whereby the
`charged molecule of interest is transported by the
`electric field into the capture matrix; and
`
`(d) detecting the amount of the charged mol-
`[0014]
`ecule of interest captured within the capture matrix.
`
`the
`In preferred embodiments of the invention.
`[0015]
`system of the invention is filled with an aqueous buffer for
`use in the electrophoretic separation. The methods may be
`used effectively to separate difl'ercntly charged peptides.
`such as those formed by enzymatic reactions with peptide
`substrates which add charged moieties to or remove charged
`moieties from the peptide. Preferred embodiments of the
`methods utilize a detectable label on the charged molecule
`of interest to detect
`the amount of the charged molecule
`which is captured within the capture matrix. more preferably
`a fluorescent label. Suitable detection methods for use in the
`methods of the invention include fluorometry, colorimetry,
`luminomctry. mass spectrometry. electrochemical detection.
`and radioactivity detect ion.
`
`BRIE]? DESCRIP‘I'ION le THE DRAWINGS
`
`[0016] FIG. 1: Across sectional schematic for an embodi-
`ment of the invention utilizing a plate electrode for the first
`electrode, a pin electrode plate for the second electrode, and
`a sample plate containing a gel capture matrix at the bottoms
`of the sample wells. This type of device was utilized in the
`experiments of Example 1. Note that the sample wells are
`arrayed in a substantially parallel fashion, forming multiple
`roots of substantially parallel tubes.
`
`[0017] FIG. 2.: A photograph of the dual—plate electro-
`phoresis device for ntulti-sample electrophoresis in it 384-
`well microtiter plate format.
`
`[0018] FIG. 3a: A photograph of pre- and post-electro-
`phorcsed samples in a gel capture matrix system of the
`invention. used as described in Example 1..
`
`[0019] FIG. 3!): A graph of the fluorescence data pictured
`in Flf‘. 3d. as measured (in a fluurttmeler. Columns 7 Sr 8
`are Wells that contained buffer, but no peptide
`
`[0020] FIG. 4: A schematic of an alternative second
`electrode for use in the systems of the invention. In this
`conductive fluid electrode. a set of pin electrodes is physir
`cally isolated from the samples in the sample plate by it
`hollow support structure containing aconductive fluid {such
`as Tris-borate liuficr). and by a hydrophilic diffusion barrier
`{tiller plugs) which permits the exchange of ions between
`the conductive fluid and the sample, but which isolates the-
`electrode chamber from the wells containing the molecules
`to be separated.
`
`[0021] FIG. 5a: A graph of fluorescence data obtained by
`elcctrophorcsing samples with various mole fractions of
`phosphorylated and unphosphorylated fluorescently labeled
`Kcmptide in the device show in FIG. 4 for 5 minutes.
`
`[0022] FIG. 5b: A graph of fluorescence data obtained by
`electrophoresing samples with various mole fractions of
`phosphorylttth and unphosphorylnted Iluorescently labeled
`Ke-rnptide. in the device shown in FIG. 4 for 10 minutes.
`[0023] FIG. 6: A schematic of an alternative second
`electrode for use in the systems of the invention. In this
`conductive fluid electrode. a set plate electrode is physically
`isolated from the samples in the sample plate by a set of
`hydrogcls held in microcapillary tubes. The gels contain a
`conductive fluid (such as Tris~horatc buffer). and serve as a
`hydrophilic diffusion barrier which permits the exchange of
`ions between the conductive fluid and the sample, but which
`isolates the electrode chamber from the wells containing the
`molecules to be Separated.
`
`[0024] FIG. 7: A graph of fluorescence data obtained by
`electrophoresing samples with various mole fractions of
`phosphorylated and unphosphorylaled fluorescently labeled
`Kcrnptidc in the device shown in FIG. 6 for 5 minutes.
`[0025] FIGS. Sit-8d: Graphs showing. the efl'eet of various
`concentrations of salt ions on the ability of the systems ot‘the
`invention to electrophorctically separate phosphorylated and
`unphosphorylated Kemptide for fluorescent detection. Note
`that the systems are effective over a wide salt concentration
`range. This indicates that electrophoretic separation of prod-
`ucts using. the systems of the invention is practical in various
`salt-containing buffers used in kinasc. phosphatase. and
`protease reaction assays, demonstrating the feasibility of
`single plate reaction and separation of reaction products.
`
`[0026] FIG. 9: A graph showing one-plate enzymatic
`reaction and clectrophoretic separation in a system of the
`invention using the Kemptidetprotein kinase A system as a
`model. As compared to a two-plate (reaction in one micro-
`tilcr plate. separation in a system of the invention) assay,
`there is a higher background signal. However. phosphory-
`later] Kemptide is clearly ditl‘erentiable from unphosphory-
`lated Kemplide. as compared to the pamive ditTusion data. In
`addition.
`the background level appears to be relatively
`consistent1 which indicates that good quantitative results
`may still be obtained by subtracting out the background.
`
`[0: A graph showing the electrophoretic
`[0027] FIG.
`separation ofseveral Kemptide samples labeled with dilIer-
`ent tluorophores. These data demonstrate the compatibility
`ol‘lhe systems of the invention with several commonly Used
`fluorescent labels.
`
`DEfflNl'l‘IONS
`
`[0023] AS used herein. “tube" and “tubular" generally
`refer to any hollow elongated structure with any type of
`
`Agilent Exhibit 1258
`
`Page 13 of 21
`
`Agilent Exhibit 1258
`Page 13 of 21
`
`

`

`US EGGS/0173247 A1
`
`Aug. 11, 2005
`
`La)
`
`cross sectional shape, including circular, square, triangular.
`polygonal. ellipsoid, or irregular. Although it is preferred the
`wall thickness be less than the void in the center of a tubular
`structure. thick Walled tubes are also within the meaning of
`the term. Tubular structures may be open or closed at either
`or both ends.
`
`[0029] The term “array," as used herein, means a set of
`members. specifically tubular sample wells, deliberately
`arranged in a plane. The regular arrangement may be rect-
`angular, radial. or any other geometrically symmetric shape.
`Irregular arrays may also he use-d. although they are not
`preferred for use in the invention because they are not
`generally compatible with standard microtiter plate readers
`and loaders. Although rectangular arrays with 96, 384. or
`1536 members are-preferred because of their direct compat-
`ibility with standard microtiter plate formats, other special-
`ized rectangular arrays (tag, It] by 10) are also envisioned
`as within the scope of the term. In the sample Well arrays of
`the invention, the sample wells are arranged so that the axes
`ofthe sample wells (or length ol'the tube forming the sample
`wells) are substantially parallel (or having a greater parallel
`component than perpendicular component of any angle of
`deviation).
`
`[0030] As used herein, “diflusion—inhibitirtg material“
`means a material which under elech‘ophoretic conditions
`allows the passage through the material of small molecules
`on the scale of the charged mole-cute of interest. but which
`prevents the free diffusion of small molecules on the scale of
`the charged molecule of interest
`through the material.
`Examples of dillusion-inhihitjng materials include hydro-
`gels,
`such
`as
`agarose. polyacrylamide,
`amioopropyl—
`methacrylamide.
`3-sulfopropyldimethyl-3-melhacrylami-
`dopropylammonium inner
`salt, methaerylic
`acid,
`3-sulfopropylmethacrylate
`potassium salt,
`glyceryl-
`monomcthacrylate, and derivatives thereof; sol-gels and
`silica gels. controlled porosity glass, size-exclusion mem-
`branes, chromatography resins. and other suitable materials
`which slow the diffusion of molecules through the sample
`well by molecular sieving or other means. Cine characteristic
`ol'the (infusion-inhibiting materials is that they may hold the
`molecule of interest, and other molecules, in place in the
`absence of an electric field.
`
`[003]] As used herein. “binding layer" or “binding mate-
`rial" refers to materials which have the ability to covalently
`or non-covalently bind at
`least one molecule of interest,
`usually through covalent bonding, hydrogen bond ing. ionic
`bonding, Van de Waals interactions, and other non-covalent
`chemical interactions. 'l'hcse materials include specific affin-
`ity binding materials. such as antibodies, avidin. streptavi-
`din, haptens. biotin, and other specific interaction materials.
`Binding materials also include non-specific binding materi-
`als such as metal chelate resins, anionic resins. and cationic
`resins, polyvinylidine fluoride, nitrocellulose. and positively
`charged nylon. Binding materials preferably bind to an
`unlabeled or affinity-labeled charged molecule of interest
`with an equilibrium highly biased towards the bound state.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`[0032] The present invention provides systems and ntetha
`ads as described herein pcm‘tit simultaneous electrophoretic
`separation of peptides. and other molecules having different
`
`net charges. and the subsequent quantification of those
`charged molecules. These systems and methods are easily
`adapted to be compatible with a variety of readily available
`standard detection equipment,
`including fluoromctric or
`calorimetric microtiter plate readers. Utilizing non-radioac-
`tively labeled substrates, and standard detection systems, the
`systems of the present
`invention may easily be used to
`analyze reaction samples in a highly parallel fashion for
`high-throughput assays to determine inhibitors or stimula-
`tors of kinasc-s, phosphatases, proteases, and other biologi—
`cally active proteins.
`
`[n a general, the apparatus includes a sample plate
`[0033]
`comprising a plurality of tubular sample walls, where each
`well contains a capture matrix designed to retain the mol-
`ccule of interest upon electrophoresis of a sample. The
`system also contains at least one pair of electrodes. Each
`discrete sample well
`is in electrical contact with a first
`electrode near the bottom of the well. and a second electrode
`near the top of the wall. The capture matrix comprises a
`dill‘usion-inhibiting material that retards the free diffusion of
`molecules. This material serves two functions:
`first,
`to
`ensure that the charged molecules of interest are retained for
`detection within the capture matrix after electrophoresis: and
`second. to prevent other molecules from diffusing in to the
`capture matrix. The capture matrix preferably also contains
`other layers of material which bind the charged molecules of
`interest. Such a binding layer captures the charged molecule
`of interest in a specific or non-specific manner in order to
`hold the charge-d molecules of interest in a particular loca-
`tion for detection, which allows more facile quantification of
`the molecule of interest as compared to a diffusion-inhibit-
`ing layer only capture matrix. As the binding layer will often
`also bind other molecules in the sample. the second function
`of the diffusion-inhibiting material
`is important in these
`embodiments.
`
`individual
`invention,
`the
`In the methods of
`[0034]
`samples, containing molecules of different charges. are
`loaded into the wells of the sample plate. The samples are
`then clectrophorcscd in a liquid which supports the electros
`phoretic movement of the analytes in the sample, preferably
`an aqueous butler. Upon electrophoresis, the charged mol-
`ecules of interest are selectively transported and concen-
`trated in the capture matrix. The molecules with a negative
`charge move towards the anode and maybe sequestered by
`a capture matrix placed between the sample and the anode.
`Alternately, molecules with a positive charge move towards
`the cathode and may be sequestered by a capture matrix
`placed between the sample and the cathode. Unchanged
`molecules. and those of a charge not captured by the capture
`matrix. are washed out of the sample wells and apparatus
`with a washing buffer. Alternatively, molecules of an undes—
`ired charge are electrophorctically moved into one of the
`bufi‘cr reservoirs of the apparatus. where they may be
`removed by continuously replenishing the buffer. The mol-
`ecules of interest which are retained in the capture matrix
`may then be detected by any appropriate means, including
`lluorometry, colorimetry, luminometry, mass spectrometry,
`electrochemical detection, and radioactivity detection. Fluo-
`rornetric labels and detection are preferred for use in the-
`methods of the present invention because of their case of use
`and handling, and the fact that roost researchers are familiar
`with fluorometric detection techniques.
`
`Agilent Exhibit 1258
`
`Page 14 of 21
`
`Agilent Exhibit 1258
`Page 14 of 21
`
`

`

`US EGGS/0173247 A1
`
`Aug. 11, 2005
`
`[0035] The methods and systems described herein may be
`used to detect the activity of kinase, protease, or phosphatase
`enzymes on labeled or unlabeled substrates, and may gens
`orally be applied to monitor the Chemical modification of a
`molecule resulting in a product of altered net charge. The
`system permits simultaneous parallel analysis of many
`samples by electrophoresing multiple samples at the same
`time. This is advantageous for the screening of large num-
`bers of compounds for their elfects on various kinases,
`phosphatases. and proteases.
`In addition, as the capture
`matrix isolates the whole fraction of charged molecules of
`interest for detection, the electrophoresis and detection steps
`may be done sequentially or simultaneously. Traditionally,
`substrate conversion analysis has been done in agarosc or
`aerylamitle slab gels, which utilized the gel matrix to sepa-
`rate modified {altered charge) from unmodified substrates.
`Although the slab gel
`technique works well
`for a
`few
`samples, parallel analysis ofa large number of samples is not
`practical. In addition, the intrinsic irregularities of the slab-
`gel method make it dilficult to compare samples run in
`clifl'erent gels. Capillary gel electrophoresis devices, such as
`that described in US. Pat. No. 5.916.428, which also sepa-
`rate the charged molecules in a gel matrix, may be used to
`separate and analyze charged molecules in such samples.
`Ilowcver, these devices require somewhat specialized and
`bulky equipment to load the samples and detect the move-
`ment of the labeled substrate through the capillary. More-
`over. such devices require dynamic detection during the
`electrophoresis process. Although such devices in the art are
`useful for the separation of complex mixtures of molecules
`in which several species are to be detected, they are usually
`too costly and cumbersome for use in high-throughput
`combinatorial library screening applications.
`
`[0036] Sample Plate Design and Construction
`
`the system is com-
`[n a preferred embodiment,
`[0037]
`prised of a sample plate containing a plurality of substan—
`tially parallel sample wells. which may be arrayed in any
`configuration permitting simultaneous analysis of multiple
`samples. For example, standard 96-, 384-, lSJo-wcll mic-ro-
`titer plate formats (8.5x11 cm) may be used, and rectangu-
`larly arrayed sample plated are preferred. The sample wells
`are preferably short. being 0.5 to 3.0 cm. and more prefer-
`ably 1.0 to 2.0 cm deep, including, the capture matrix. The
`sample plate may be constructed by any usual means,
`including molding, machining, or
`laminar construction
`(which is useful for sandwiching a layer of capture matrix
`material between two layers of support material which form
`the sample plate). Suitable materiaLs for construction include
`polystyrene, polycarbonate, polypropylene and other poly-
`mers. as well as glass, quartz, and other silicate materials.
`Important considerations are that the materials should be
`insulatory, and should have a low background signal
`in
`whatever detection system is to be used with the system (i.e.,
`low fluorescence).
`
`[0038] The plurality of sample wells are open at their top
`and bottom ends. with the capture matrix positioned near
`one end of the sample well. The capture matrix forms a
`continuous layer across the sample well, as illustrated in the
`examples. Usually, a capture matrix will be positioned at or
`near the bottom of the sample well, near the first electrode.
`However, alternative embodiments are envisioned in which
`a second capture matrix is positioned. after the sample is
`loaded. at or near the top end of the sample well. With this
`
`configuration, both a positively and negatively charged
`molecule of interest could be captured from a sample for
`detection in the systems of the invention. In the assembled
`System, each sample well is in contact with a first electrode
`at its bottom end, and with a second electrode at its top end.
`The electrode contact may be dire-cl or alternately may be
`indirect, such as through a conducting, medium such as a
`conductive liquid or bullies
`
`[0039] Capture Matrix Composition
`
`[0040] The capture matrix is an integral part of the sys—
`tems of the invention, in that it has the ability to capture and
`hold the charged molecules of interest for later or simulta-
`neous detection.
`In this way,
`the capture matrix of the
`present invention dillers substantially in function from the
`gel separation matrices used in slab gel electrophoresis or in
`capillary gel electrophoresis. In those techniques, the hydro
`gel is used to separate and define groups of molecules within
`the gel matrix. As used in the present invention. the capture
`matrix is merely used to hold and segregate a single group
`ofchttt‘ged moleculesli.e., all moleculesof a Certain charge)
`from the other molecules in solution. Because of this sim-
`plilied function.
`the capture matrices used in the present
`invention have dillerent physical dimensions. In the systems
`of the present invention, preferred capture matrices have a
`thickness of less than (1.5 cm along the path of electrophore—
`sis, more preferably a thickness of less than 0.3 cm, more
`preferably a thickness of less than 0.2 cm, and most pref-
`erably a thickness of less than about 0.1 cm.
`
`[0041] The capture matrix comprises a (lilfusion inhibiting
`material which impedes the passive transport of the mol—
`ecules of interest and the other molecules in the sample. This
`serves two purposes. First,
`to separate the molecule of
`interest upon the application of an electric held across the
`sample well by selectively electrophoresing the charged
`molecule of interest into the capture matrix based upon, its
`charge, as the uncharged and oppositely charged molecules
`will be prevented from entering the capture matrix by simple
`dillusion alone. Second, alter the sample has been electro-
`phoreserl, the diH‘usion-inhibiling material holds the charged
`molecule ofinterest within the capture matrix in the absence
`of an electric field, preventing, their ditfusion back into the
`sample solution. Sol gels, cellulose, glass fiber, nylon, and
`hydrogels are preferred for use as diflttsion—inhibiting mate-
`rials in the capture matrix.
`l'lydrogels, such as agarose,
`polyacrylamide, aminopropylmethacrylamide, 3-sull'opro-
`pyldimethyl-3-methaerytarnidopropylammoniurn inner salt,
`methacrylic acid, 3—sulfopropylmethacrylate potassium salt,
`glycerylmonomethacrylate, and derivatives thereof, are par-
`ticularly preferred. The capture matrix may be comprised ol‘
`a single layer of diffusion-inhibiting material. as in the
`systems described in FIG. I and Example ]. Or the capture
`matrix may corn prise a gradient layer oldillusion-inhibiling
`material. This gradient may be a density or other physical
`property gradient, or may be a chemical property gradi