Retentate chromatography and protein chip arrays with applications in biology and medicine

• US 20020177242A1
• Filed: 08/05/2002
• Published: 11/28/2002
• Est. Priority Date: 06/20/1997
• Status: Active Grant

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: Ciphergen Biosystems Incorporated
• Inventors: Hutchens, T. William, Yip, Tai-Tung
• Granted Patent: US 7,112,453 B2
• Time in Patent Office: Days
• US Class Current: 436/518
• CPC Class Codes: B01D 15/1878 for multi-dimensional chromatography
• CPC Class Codes: B01D 15/26 characterised by the separation mechanism
• CPC Class Codes: B01D 15/305 Hydrophilic interaction chromatography [HILIC]
• CPC Class Codes: B01D 15/322 Normal bonded phase
• CPC Class Codes: B01D 15/327 with hydrophobic interaction
• CPC Class Codes: B01D 15/362 Cation-exchange
• CPC Class Codes: B01D 15/363 Anion-exchange
• CPC Class Codes: B01D 15/38 involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
• CPC Class Codes: B01D 15/3804 Affinity chromatography
• CPC Class Codes: B01D 15/3809 of the antigen-antibody type, e.g. protein A, G, L chromatography
• CPC Class Codes: B01J 20/281 Sorbents specially adapted for preparative, analytical or investigative chromatography
• CPC Class Codes: B01J 20/286 Phases chemically bonded to a substrate, e.g. to silica or to polymers
• CPC Class Codes: B01J 20/287 Non-polar phases; Reversed phases
• CPC Class Codes: B01J 20/288 Polar phases
• CPC Class Codes: B01J 20/289 bonded via a spacer
• CPC Class Codes: B01J 20/3204 Inorganic carriers, supports or substrates
• CPC Class Codes: B01J 20/3212 consisting of a polymer obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
• CPC Class Codes: B01J 20/3219 involving a particular spacer or linking group, e.g. for attaching an active group
• CPC Class Codes: B01J 20/3236 containing metal, other than zeolites, e.g. oxides, hydroxides, sulphides or salts
• CPC Class Codes: B01J 20/3248 the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
• CPC Class Codes: B01J 20/3253 : comprising a cyclic structure not containing any of the heteroatoms nitrogen, oxygen or sulfur, e.g. aromatic structures
• CPC Class Codes: B01J 20/3265 : with an organic functional group containing a metal, e.g. a metal affinity ligand
• CPC Class Codes: B01J 20/3274 : Proteins, nucleic acids, polysaccharides, antibodies or antigens
• CPC Class Codes: B01J 20/3285 : Coating or impregnation layers comprising different type of functional groups or interactions, e.g. different ligands in various parts of the sorbent, mixed mode, dual zone, bimodal, multimodal, ionic or hydrophobic, cationic or anionic, hydrophilic or hydrophobic
• CPC Class Codes: B01J 20/3289 : Coatings involving more than one layer of same or different nature
• CPC Class Codes: B01J 2219/00605 : the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/0061 : The surface being organic
• CPC Class Codes: B01J 2219/00612 : the surface being inorganic
• CPC Class Codes: B01J 2219/00617 : by chemical means
• CPC Class Codes: B01J 2219/00619 : using hydrophilic or hydrophobic regions
• CPC Class Codes: B01J 2219/00621 : by physical means, e.g. trenches, raised areas
• CPC Class Codes: B01J 2219/00626 : Covalent
• CPC Class Codes: B01J 2219/00628 : Ionic
• CPC Class Codes: B01J 2219/0063 : Other, e.g. van der Waals forces, hydrogen bonding
• CPC Class Codes: B01J 2219/00637 : by coating it with another layer
• CPC Class Codes: B01J 2219/00641 : the porous medium being continuous, e.g. porous oxide substrates
• CPC Class Codes: B01J 2219/00644 : the porous medium being present in discrete locations, e.g. gel pads
• CPC Class Codes: B01J 2219/00659 : Two-dimensional arrays
• CPC Class Codes: B01J 2219/00702 : Processes involving means for analysing and characterising the products
• CPC Class Codes: B01J 2219/00725 : Peptides
• CPC Class Codes: B01J 2220/54 : Sorbents specially adapted for analytical or investigative chromatography
• CPC Class Codes: C12Q 1/00 : Measuring or testing processes involving enzymes, nucleic acids or microorganisms measuring or testing apparatus with condition measuring or sensing means, e.g. colony counters, C12M1/34; Compositions therefor; Processes of preparing such compositions
• CPC Class Codes: C40B 30/04 : by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
• CPC Class Codes: C40B 40/10 : Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: G01N 2030/008 : Thermal desorption
• CPC Class Codes: G01N 2030/009 : Extraction
• CPC Class Codes: G01N 30/00 : Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation G01N3/00, G01N5/00, G01N7/00, G01N9/00, G01N11/00, G01N13/00, G01N15/00, G01N17/00, G01N19/00, G01N21/00, G01N22/00, G01N23/00, G01N24/00, G01N25/00, G01N27/00, G01N29/00 take precedence
• CPC Class Codes: G01N 30/02 : Column chromatography
• CPC Class Codes: G01N 30/461 : with serial coupling of separation columns
• CPC Class Codes: G01N 30/463 : for multidimensional chromatography
• CPC Class Codes: G01N 30/466 : with separation columns in parallel
• CPC Class Codes: G01N 30/90 : Plate chromatography, e.g. thin layer or paper chromatography
• CPC Class Codes: G01N 30/95 : Detectors specially adapted therefor; Signal analysis
• CPC Class Codes: G01N 33/531 : Production of immunochemical test materials
• CPC Class Codes: G01N 33/538 : by sorbent column, particles or resin strip , i.e. sorbent materials
• CPC Class Codes: G01N 33/543 : with an insoluble carrier for immobilising immunochemicals
• CPC Class Codes: G01N 33/54306 : Solid-phase reaction mechanisms
• CPC Class Codes: G01N 33/54353 : with ligand attached to the carrier via a chemical coupling agent coatings G01N33/54393
• CPC Class Codes: G01N 33/54373 : involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
• CPC Class Codes: G01N 33/54393 : Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
• CPC Class Codes: G01N 33/566 : using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
• CPC Class Codes: G01N 33/6803 : General methods of protein analysis not limited to specific proteins or families of proteins
• CPC Class Codes: G01N 33/6845 : Methods of identifying protein-protein interactions in protein mixtures
• CPC Class Codes: G01N 33/6851 : Methods of protein analysis involving laser desorption ionisation mass spectrometry
• CPC Class Codes: Y02A 90/10 : Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation
• CPC Class Codes: Y10S 422/906 : Plasma or ion generation means
• CPC Class Codes: Y10S 435/803 : Physical recovery methods, e.g. chromatography, grinding
• CPC Class Codes: Y10S 435/81 : Packaged device or kit
• CPC Class Codes: Y10S 435/814 : Enzyme separation or purification
• CPC Class Codes: Y10S 435/815 : by sorption
• CPC Class Codes: Y10S 435/97 : Test strip or test slide
• CPC Class Codes: Y10S 435/973 : Simultaneous determination of more than one analyte
• CPC Class Codes: Y10S 435/975 : Kit
• CPC Class Codes: Y10S 436/808 : Automated or kit
• CPC Class Codes: Y10S 436/809 : Multifield plates or multicontainer arrays
• CPC Class Codes: Y10S 436/811 : Test for named disease, body condition or organ function
• CPC Class Codes: Y10S 436/813 : Cancer
• CPC Class Codes: Y10S 436/824 : Immunological separation techniques
• CPC Class Codes: Y10S 436/825 : Pretreatment for removal of interfering factors from sample
• CPC Class Codes: Y10T 436/24 : Nuclear magnetic resonance, electron spin resonance or other spin effects or mass spectrometry
• CPC Class Codes: Y10T 436/25 : including sample preparation
• CPC Class Codes: Y10T 436/25125 : Digestion or removing interfering materials
• CPC Class Codes: Y10T 436/2525 : Stabilizing or preserving
• CPC Class Codes: Y10T 436/25375 : Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
• CPC Class Codes: Y10T 436/255 : including use of a solid sorbent, semipermeable membrane, or liquid extraction

Abstract

This invention provides methods of retentate chromatography for resolving analytes in a sample. The methods involve adsorbing the analytes to a substrate under a plurality of different selectivity conditions, and detecting the analytes retained on the substrate by desorption spectrometry. The methods are useful in biology and medicine, including clinical diagnostics and drug discovery.

Claims

1. A method for high information resolution of at least one analyte in a sample comprising the steps of:

a) exposing the analyte to at least two different selectivity conditions, each selectivity condition defined by the combination of an adsorbent and an eluant, to allow retention of the analyte by the adsorbent; and

b) detecting retained analyte under the different selectivity conditions by desorption spectrometry;

whereby detection of retained analyte under the different selectivity conditions provides a high information resolution of the analyte.

2. The method of claim 1 wherein each different selectivity condition is defined at a different predetermined, addressable location for parallel processing.

3. The method of claim 1 comprising the steps of:

i) exposing the analyte to a first selectivity condition at a defined location to allow retention of the analyte by the adsorbent;

ii) detecting retained analyte under the first selectivity condition by desorption spectrometry;

iii) washing the adsorbent under a second, different selectivity condition at the defined location to allow retention of the analyte to the adsorbent; and

iv) detecting retained analyte under the second selectivity condition by desorption spectrometry.

4. The method of claim 1 wherein the analyte is an organic biomolecule.

5. The method of claim 1 wherein the analyte is a virus or a cell.

6. The method of claim 1 wherein the adsorbent comprises an anion, a cation, a hydrophobic interaction adsorbent, a polypeptide, a nucleic acid, a carbohydrate, a lectin, a dye, a reducing agent, a hydrocarbon or a combination thereof.

7. The method of claim 1 wherein the different selectivity conditions comprise different binding conditions.

8. The method of claim 1 wherein the different selectivity conditions comprise different elution conditions.

9. The method of claim 1 wherein the step of detecting comprises detecting the mass of the analyte by laser desorption mass spectrometry.

10. The method of claim 1 wherein selectivity conditions are selected to optimize retention of analyte by an adsorbent.

11. The method of claim 1 wherein the adsorbent is attached to a substrate comprising glass, ceramic, a magnetic material, an organic polymer, a conducting polymer, a native biopolymer, a metal, a metal coated with an organic polymer or a combination thereof.

12. The method of claim 1 wherein the adsorbent is in the form of a microemulsion, a latex, a layer or a bead.

13. The method of claim 2 wherein the locations are arranged in a line, an orthogonal array or a circle.

14. The method of claim 2 wherein the adsorbents are located on a substrate at different locations before the analytes are exposed to the selectivity conditions.

15. The method of claim 2 wherein the adsorbents are located on a substrate at different locations after the analytes are exposed to the selectivity conditions.

16. The method of claim 2 wherein at least one analyte is more than one analyte.

17. The method of claim 2 wherein the plurality of selectivity conditions are defined by at different adsorbents and the same eluant.

18. The method of claim 2 further comprising the step of providing a substrate comprising adsorbents at addressable locations, each adsorbent being an adsorbent from a selectivity condition identified to retain the analyte.

19. The method of claim 2 comprising the steps of:

a) exposing a sample comprising the analytes to a first selectivity condition to allow retention of analytes by a first adsorbent and to create un-retained sample;

b) collecting the un-retained sample comprising analytes, exposing the un-retained sample to a second selectivity condition to allow retention of analytes by a second adsorbent and to create a second un-retained sample; and

c) detecting retained analyte under the different selectivity conditions by desorption spectrometry.

20. The method of claim 4 wherein the organic biomolecule is an enzyme, an immunoglobulin, a cell surface receptor or an intracellular receptor.

21. The method of claim 8 wherein the elution conditions differ according to pH, buffering capacity, ionic strength, a water structure characteristic, detergent type, detergent strength, hydrophobicity or dielectric constant.

22. The method of claim 18 wherein the plurality of selectivity conditions are defined by the same eluant.

23. The method of claim 19 further comprising the steps of collecting the second un-retained sample.

27. The method of claim 26 wherein at least one physico-chemical characteristic is molecular mass.

28. The method of claim 26 wherein the database comprises, for each member of the set of reference polypeptides, an amino acid sequence of the polypeptide or a nucleotide sequence encoding the amino acid sequence of the polypeptide, and wherein the performing step further comprises providing code that derives the physico-chemical characteristics of a reference polypeptide from the amino acid sequence of the polypeptide.

29. The method of claim 27 wherein at least one of the physico-chemical characteristics is hydrophobicity, pI, number of coordinate covalent bonding residues or charge.

24. A substrate for desorption spectrometry comprising an adsorbent whose binding characteristics vary in a gradient along one or more linear axes.

25. A method for preparative purification of an analyte from an impure sample comprising the steps of:

a) exposing the sample to a substrate under a plurality of different selectivity conditions;

detecting retained analyte under the different selectivity conditions by desorption spectrometry; and

identifying selectivity conditions under which the analyte is retained;

b) purifying the analyte by repeating, for a plurality of different identified selectivity conditions, a sequence of steps comprising;

i) exposing the sample to an adsorbent under the identified selectivity condition to allow retention of the analyte by the adsorbent;

ii) separating the analyte from an impurity that is not retained by the substrate; and

iii) collecting the analyte from the adsorbent;

whereby the analyte is purified.

26. A method for selecting identity candidates for an analyte protein comprising the steps of:

a) determining a value set specifying match parameters for at least a first and second physico-chemical characteristic of a protein analyte in a sample by i) exposing the analyte to a plurality of different selectivity conditions, wherein adsorption of the protein analyte to the substrate is mediated by a basis of attraction that identifies a physico-chemical characteristic of the protein analyte; and

ii) detecting retained analyte under the different selectivity conditions by desorption spectrometry; and

b) performing, in a programmable digital computer, the steps of;

i) accessing a database comprising, for each member of a set of reference polypeptides, a value set specifying at least a first and second physico-chemical characteristic of the reference polypeptides;

ii) accessing the value set specifying the physico-chemical characteristics of the protein analyte;

iii) sorting from the database, reference polypeptides having value sets within the match parameters;

whereby the sorted reference polypeptides provide identity candidates for the protein analyte and unsorted reference polypeptides provide non-identity candidates for the protein analyte.

30. A method of detecting an enzyme in a sample comprising the steps of:

a) providing a solid phase comprising an adsorbent and an enzyme substrate bound to the adsorbent, wherein the activity of the enzyme on the enzyme substrate produces a product having a characteristic molecular mass;

b) exposing the substrate to the sample; and

c) detecting the product by desorption spectrometry;

whereby detecting the product provides a detection of the enzyme.

31. The method of claim 30 for determining the amount of the enzyme wherein the step of detecting comprises detecting an amount of the product, and comparing the detected amount to a standard that relates the detected amount to an amount of the enzyme in the sample.