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

• US 20040137427A1
• Filed: 07/23/2003
• Published: 07/15/2004
• Est. Priority Date: 06/20/1997
• Status: Abandoned Application

• Current Assignee: Ciphergen Biosystems Incorporated
• Original Assignee: Ciphergen Biosystems Incorporated
• Inventors: Hutchens, T. William, Yip, Tai-Tung
• Application Number:
  US10/626,301
• Publication Number:
  US 20040137427A1
• Time in Patent Office: Days
• US Class Current: 435/5
• 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 identifying a ligand for a receptor comprising the steps of:

a) providing a substrate comprising an adsorbent wherein the receptor is bound to the adsorbent;

b) exposing the bound receptor to a sample containing the ligand under conditions to allow binding between the receptor and the ligand; and

c) detecting bound ligand by desorption spectrometry.

2. A method of detecting a genetic package containing a polynucleotide that encodes a polypeptide agent that specifically binds to a target adsorbent, the method comprising the steps of:

a) providing a substrate comprising a target adsorbent;

b) providing a display library that comprises a plurality of different genetic packages, each different genetic package comprising a polynucleotide that comprises a nucleotide sequence that encodes a polypeptide agent, and each different genetic package having a surface on which the encoded polypeptide agent is displayed;

c) exposing the substrate to the display library under elution conditions to allow specific binding between a polypeptide agent and the target adsorbent, whereby a genetic package comprising the polypeptide agent is retained on the substrate; and

d) detecting a genetic package retained on the substrate by desorption spectrometry.

3. The method of claim 2 wherein the display library is a phage display library.

4. The method of claim 2 wherein the step of providing the substrate comprising the target adsorbent comprises the steps of:

i) providing a substrate comprising an adsorbent, wherein the adsorbent retains a target analyte under an elution condition; and

ii) exposing the adsorbent to the target analyte under the elution condition to allow retention of the target analyte by the adsorbent, whereby the target analyte becomes the target adsorbent.

5. The method of claim 2 further comprising the step of (e) sequencing the nucleotide sequence that encodes the polypeptide agent.

6. The method of claim 2 further comprising the step of (e) isolating the retained genetic package.

7. The method of claim 2 further comprising the step of (e) producing the polypeptide agent.

8. The method of claim 2 wherein the substrate comprises (1) an adsorbent that binds an anchoring polypeptide and (2) at least one target genetic package having a surface displaying the anchoring polypeptide and a target adsorbent polypeptide, the target genetic package comprising a polynucleotide that comprises a nucleotide sequence that encodes the target adsorbent, wherein the target genetic package is bound to the adsorbent through the anchoring polypeptide.

9. The method of claim 2 wherein the substrate comprises a cell or cell membrane.

10. The method of claim 2 wherein the target adsorbent comprises a polypeptide that is differentially expressed between cells of different phenotypes.

11. The method of claim 3 wherein the phage is M13.

12. The method of claim 4 wherein the target analyte is a target polypeptide and the step of ii) exposing the adsorbent comprises the step of producing the target polypeptide in situ on the adsorbent by in vitro translation of a polynucleotide encoding the target polypeptide.

13. The method of claim 5 wherein the step of sequencing comprises amplifying the polynucleotide sequence in situ on the substrate.

14. The method of claim 7 wherein the step of producing comprises reproducing the retained genetic package that displays the polypeptide agent.

15. The method of claim 7 comprising expressing the polypeptide agent from an expression vector that comprises an expression control sequence operatively linked to the nucleotide sequence encoding the polypeptide agent.

16. The method of claim 7 further comprising the step of producing a substrate comprising an adsorbent that comprises the polypeptide agent.

17. The method of claim 8 wherein the at least one target genetic package is selected from a target display library screened for genetic packages that bind at least one primary target analyte and wherein the adsorbent comprises the primary target analyte.

18. The method of claim 11 wherein the polypeptide agent is a single chain antibody.

19. The method of claim 12 wherein the target polypeptide is produced in situ by in vitro translation of a polynucleotide encoding the target polypeptide.

20. The method of claim 14 wherein the step of reproducing is carried out in situ on the substrate.

21. The method of claim 19 wherein the polynucleotide encoding the target polypeptide is produced in situ by in vitro transcription.

22. A substrate for desorption spectrometry comprising an adsorbent that binds an anchoring polypeptide displayed on a surface of a genetic package, wherein the surface of the genetic package further displays a target polypeptide and wherein the genetic package comprises a polynucleotide comprising a nucleotide sequence that encodes the target polypeptide.

23. The substrate of claim 22 wherein the genetic package is an M13 phage.

24. The substrate of claim 22 wherein the anchoring polypeptide is a fusion polypeptide with gene III protein and the target polypeptide is a fusion polypeptide with gene VIII protein.

27. A method for detecting translation of a polynucleotide comprising the steps of:

a) providing a substrate comprising an adsorbent for use in desorption spectrometry;

b) contacting the substrate with the polynucleotide encoding a polypeptide and with agents for in vitro translation of the polynucleotide, whereby the polypeptide is produced;

c) exposing the substrate to an eluant to allow retention of the polypeptide by the adsorbent; and

d) detecting retained polypeptide by desorption spectrometry;

whereby detection of the polypeptide provides detection of translation of the polynucleotide.

28. A method comprising the steps of:

a) exposing a first sample to a primary adsorbent and to an eluant to allow retention of a first analyte by the adsorbent, and detecting the adsorbed analyte by desorption spectrometry, whereby the retained first analyte becomes a secondary adsorbent;

b) exposing a second sample to the secondary adsorbent and to an eluant to allow retention of a second analyte by the secondary adsorbent, and detecting the adsorbed second analyte by desorption spectrometry, whereby the retained second analyte becomes a tertiary adsorbent.

29. The method of claim 28 further comprising repeating step (b) at least once for a subsequent sample or samples.

30. A screening method for determining whether an agent modulates binding between a target analyte and an adsorbent comprising the steps of:

a) providing a substrate comprising an adsorbent to which the target analyte binds under an elution condition;

b) exposing the substrate to the target analyte and to the agent under the elution condition to allow binding between the target analyte and the adsorbent;

c) detecting an amount of binding between the target analyte and the adsorbent by desorption spectrometry; and

d) determining whether the measured amount is different than a control amount of binding when the substrate is exposed to the target analyte under the elution condition without the agent;

whereby a difference between the measured amount and the control amount indicates that the agent modulates binding.

25. A substrate comprising an adsorbent that comprises a polypeptide agent that specifically binds to a target analyte, the polypeptide agent identified by the method of claim 33.

26. The substrate of claim 25 wherein the polypeptide agent is a single chain antibody.

31. The method of claim 30 wherein the adsorbent comprises a ligand that specifically binds the target analyte.

32. The method of claim 30 wherein the adsorbent comprises a genetic package having a surface that displays a polypeptide ligand that specifically binds the target analyte.

33. The method of claim 30 for screening a combinatorial library of agents comprising exposing each of a plurality of agents in the library to each of a plurality of the adsorbents.

34. The method of claim 31 wherein the ligand is an enzyme and the target analyte is a substrate of, or an inhibitor for, the enzyme, or vice-versa.

35. The method of claim 31 wherein the ligand is a hormone and the target analyte is a cell surface receptor or an intracellular receptor of the hormone, or vice-versa.