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

• US 20010014461A1
• Filed: 12/21/2000
• Published: 08/16/2001
• 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 6,844,165 B2
• Time in Patent Office: Days
• US Class Current: 435/7.92
• 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 preparing a substrate for detecting at least one analyte in a sample comprising the steps of:

a) exposing the sample 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;

b) identifying by desorption spectrometry at least one selectivity condition under which the analyte is retained; and

c) preparing a substrate comprising at least one adsorbent of an identified selectivity condition.

2. The method of

claim 1

wherein the step of identifying comprises identifying at least one selectivity condition under which a plurality of analytes are retained.

3. The method of

claim 1

wherein the step of preparing comprises preparing a substrate comprising a plurality of adsorbents that retain the analyte under an elution condition as a multiplex adsorbent.

4. A method for progressively identifying a selectivity condition with improved resolution for an analyte in a sample comprising the steps of:

(a) identify a selectivity condition that retains an analyte in a sample by;

(i) exposing a sample to a set of selectivity conditions, each selectivity condition defined by at least one binding characteristic and at least one elution characteristic;

(ii) detecting analyte retained under each selectivity condition by desorption spectrometry; and

(iii) identifying a selectivity condition that retains the analyte; and

(b) identifying a selectivity condition with improved resolution for the analyte by;

(i) selecting at least one binding characteristic or elution characteristic from the identified selectivity condition and adding it to a selectivity characteristic constant set;

(ii) exposing the sample to a modified set of selectivity conditions wherein each selectivity condition in the modified set comprises (1) the selectivity characteristics in the constant set and (2) a binding characteristic or elution characteristic that is not in the constant set; and

(iii) identifying a selectivity condition from the modified set by desorption spectrometry that retains the analyte with improved resolution compared with a prior identified selectivity condition.

5. The method of

claim 4

further comprising the step of repeating step (b) at least once.

6. The method of

claim 5

comprising repeating step (b) until a selectivity condition is identified that retains only the target analyte from the sample.

7. A substrate for desorption spectrometry comprising an adsorbent from a selectivity condition identified to resolve an analyte by the method of

claim 4

.

8. The substrate of

claim 7

in the form of a kit further comprising an eluant from the selectivity condition or instructions on using the eluant in combination with the adsorbent.

9. A method for determining whether an analyte is differentially present in a first and second biological sample comprising the steps of:

a) determining a first retention map for the analyte in the first sample for at least one selectivity condition;

b) determining a second retention map for the analyte in the second sample for the same selectivity condition; and

c) detecting a difference between the first and the second retention maps;

whereby a difference in the retention maps provides a determination that the analyte is differentially present in first and second samples.

10. The method of

claim 9

wherein the first biological sample derives from a healthy subject and the second biological sample is from a subject suffering from a pathological condition.

11. The method of

claim 9

wherein the biological samples comprise first and second cell extracts.

12. The method of

claim 9

wherein the retention map comprises a plurality of selectivity conditions.

13. The method of

claim 9

comprising, before the step of detecting, the step of converting the analyte into at least one fragment whose molecular mass smaller than the mass of the analyte.

14. The method of

claim 9

wherein the step of detecting a difference is performed in a programmable digital computer.

15. The method of

claim 9

for determining whether an agent alters the expression of a protein in a biological sample further comprising the step of administering the agent to a first biological sample but not to a second biological sample.

16. The method of

claim 10

wherein the sample is selected from the group consisting of blood, urine, serum and tissue.

17. The method of

claim 10

further comprising identifying an analyte that is present in a greater amount in second biological sample than in the first biological sample, whereby the analyte is identified as a candidate diagnostic marker for the pathological condition.

18. The method of

claim 11

wherein the first cell extract is derived from a healthy cell and the second cell extract is derived from a cancer cell.

19. A method of diagnosing in a subject a disease characterized by at least one diagnostic marker comprising the steps of:

a) providing a substrate for use in desorption spectrometry that comprises at least one addressable location, each addressable location comprising an adsorbent that resolves at least one of the diagnostic markers under an elution condition;

b) exposing the substrate to a biological sample from the subject under the elution condition to allow retention of the diagnostic marker; and

c) detecting retained diagnostic marker by desorption spectrometry;

whereby detecting retained diagnostic marker provides a diagnosis of the disease.

20. The method of

claim 19

wherein diagnosis involves detection of a plurality of diagnostic markers and the addressable locations comprise adsorbents that resolve the plurality of diagnostic markers.

21. A kit for detecting an analyte in a sample comprising (1) a substrate for use in desorption spectrometry that comprises at least one addressable location, each addressable location comprising an adsorbent that resolves an analyte under a selectivity condition comprising the adsorbent and an eluant, and (2) the eluant or instructions for exposing the sample to the selectivity condition.

22. The kit of

claim 21

for the diagnosis of a disease wherein the at least one analyte is at least one diagnostic marker for the disease.

23. The kit of

claim 22

wherein the disease characterized by a plurality of diagnostic markers and the substrate comprises a plurality of addressable locations, each addressable location comprising an adsorbent that resolves at least one of the diagnostic markers.

24. The kit of

claim 23

wherein at least one adsorbent is a multiplex adsorbent comprising adsorbent species that each retain at least one diagnostic marker.

25. The kit of

claim 23

wherein at least one adsorbent does not comprise a biopolymer.

26. The kit of

claim 23

wherein at least one addressable location comprises a ligand specific for a diagnostic marker.

27. The kit of

claim 26

wherein the ligand is an antibody.

28. A substrate for desorption spectrometry comprising at least one adsorbent in at least one addressable location wherein the at least one adsorbent resolves a plurality of diagnostic markers for a pathological condition from a patient sample.

29. The substrate of

claim 28

wherein at least one adsorbent does not comprise a biopolymer.

30. The substrate of

claim 28

wherein one adsorbent resolves the plurality of diagnostic markers.