Methods and compositions for analyzing proteins

a protein and composition technology, applied in the field of methods and compositions for analyzing proteins, can solve the problems of limited dynamic range, poor sensitivity, and complex instrumentation, and achieve the effect of unique optical and/or charge-mass characteristics

Inactive Publication Date: 2008-12-18
MONOGRAM BIOSCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most multi-analyte assays, such as assays in the genomics area that detect multiple nucleic acid sequences, involve multiple steps, have poor sensitivity, a limited dynamic range (typically on the order of 2 to 100-fold differences, and some require sophisticated instrumentation.

Method used

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  • Methods and compositions for analyzing proteins
  • Methods and compositions for analyzing proteins
  • Methods and compositions for analyzing proteins

Examples

Experimental program
Comparison scheme
Effect test

example 1

Conjugation of Photosensitizer Molecules to Assay Reagents

[0249]Photosensitizer molecules are conjugated to a metal affinity agent, a boronic acid containing agent, a hub molecule, and the like by various conventional methods and configurations. For example, an activated (NHS ester, aldehyde, sulfonyl chloride, etc) photosensitizer (Rose Bengal, phthalocyanine, etc.) can be reacted with reactive amino-group containing moieties (aminodextran, amino-group containing agents (with appropriate protection of metal binding sites), other small and large molecules). The formed conjugates can be used directly (for example the antibody-photosensitizer conjugate, Biotin-LC-photosensitizer, etc.) in various assays. Also, the formed conjugates can be further coupled with antibody (for example, aminodextran-photosensitizer conjugate containing 20-200 photosensitizers and 200-500 amino-groups can be coupled to periodate oxidized antibody molecules to generate the antibody-dextran-sensitizer conjuga...

example 2

Conjugation of an A-Tag Moiety and Release of an E-Tag Reporter

[0250]FIG. 14 summarizes the methodology for conjugation of an e-tag moiety to an antibody or other binding moiety with a free amino group, and the reaction of the resulting conjugate with singlet oxygen to produce a sulfinic acid moiety as the released e-tag reporter.

[0251]FIG. 15 A-J shows several a-tag reagents, most of which utilize 5- or 6-carboxyfluorescein (FAM) as starting material.

example 3

Preparation of Pro2 Pro4 and Pro6 through Pro13

[0252]The scheme outlined in FIG. 16A shows a five-step procedure for the preparation of the carboxyfluorescein-derived a-tag moieties, namely, Pro2, Pro4, Pro6, Pro7, Pro8, Pro9, Pro10, Pro11, Pro12, and Pro13. The first step involves the reaction of a5- or 6-FAM withN-hydroxysuccinimide (NHS) and 1,3-dicylcohexylcarbodiimide (DCC) in DMF to give the corresponding ester, which was then treated with a variety of diamines to yield the desired amide, compound 1. Treatment of compound 1 with N-succinimidyl iodoacetate provided the expected iodoacetamide derivative, which was not isolated but was further reacted with 3-mercaptopropionic acid in the presence of triethylamine. Finally, the resulting β-thioacid (compound 2) was converted, as described above, to its NHS ester. The various e-tag moieties were synthesized starting with 5- or 6-FAM, and one of various diamines. The diamine is given H2N̂X̂NH2 in the first reaction of FIG. 16A. The ...

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Abstract

Methods, compositions and kits are disclosed for determining one or more target polypeptides in a sample where the target polypeptides have undergone a post-translational modification. A mixture comprising the sample and a first reagent comprising a cleavage-inducing moiety and a first binding agent for a binding site on a target polypeptide is subjected to conditions under which binding of respective binding moieties occurs. The binding site is the result of post-translational modification activity involving the target polypeptide. The method may be employed to determine the target polypeptide itself. In another embodiment the presence and / or amount of the target polypeptide is related to the presence and / or amount and / or activity of an agent such as an enzyme involved in the post-translational modification of the target polypeptide. The interaction between the first binding agent and the binding site brings the cleavage-inducing moiety into close proximity to a cleavable moiety, which is associated with the polypeptide and is susceptible to cleavage only when in proximity to the cleavage-inducing moiety. In this way, an electrophoretic tag for each of the polypeptides may be released. Released electrophoretic tags are separated and the presence and / or amount of the target polypeptides are determined based on the corresponding electrophoretic tags.

Description

CROSS REFERENCE TO RELATED PATENT APPLICATION[0001]This application is a divisional application of U.S. patent application Ser. No. 10 / 154,042, filed May 21, 2002, now U.S. Pat. No. ______, which claims priority to U.S. Provisional Application Ser. No. 60 / 292,548, filed May 21, 2001, and U.S. Provisional Application Ser. No. 60 / 334,901, filed Oct. 24, 2001, all of which are incorporated herein by reference.BACKGROUND OF THE INVENTION[0002]The present invention relates to separable compositions, methods, and kits for use in detection and quantitation of polypeptides. The invention finds particular application to the area of multiplexed assays for polypeptides including proteins involved in post-translational activity.[0003]The need to determine many analytes including polypeptides and nucleic acid sequences in blood or other biological fluids has become increasingly apparent in many branches of medicine. Most multi-analyte assays, such as assays in the genomics area that detect multi...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): G01N27/26G01N33/53G01N33/483G01N21/78G01N27/447G01N33/536G01N33/68
CPCG01N33/6842G01N33/6803G01N33/561G01N33/68C07K16/00C07K2317/40G01N33/582
Inventor SINGH, SHARATSALIMI-MOOSAVI, HOSSEINTAHIR, SYED HASANWALLWEBER, GERALD J.KIRAKOSSIAN, HRAIRMATRAY, TRACEY J.HERNANDEZ, VINCENT S.
Owner MONOGRAM BIOSCIENCES
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