Multiplexed detection with isotope-coded reporters

• US 10,006,916 B2
• Filed: 03/15/2012
• Issued: 06/26/2018
• Est. Priority Date: 03/15/2011
• Status: Active Grant

• Current Assignee: Massachusetts Institute of Technology
• Original Assignee: Massachusetts Institute of Technology
• Inventors: Kwong, Gabriel A., Bhatia, Sangeeta N.
• Primary Examiner(s): Flinders, Jeremy C
• Application Number:
  US14/005,416
• Publication Number:
  US 20140303014A1
• Time in Patent Office: 2,294 Days
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 14/001 by chemical synthesis
• CPC Class Codes: C07K 7/08 having 12 to 20 amino acids gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68
• CPC Class Codes: G01N 2458/15 Non-radioactive isotope labels, e.g. for detection by mass spectrometry
• CPC Class Codes: G01N 33/58 involving labelled substances G01N33/53 takes precedence
• CPC Class Codes: G01N 33/6848 Methods of protein analysis involving mass spectrometry

Abstract

Some aspects of this invention provide reagents and methods for the sensitive, quantitative and simultaneous detection of target analytes in complex biological samples by liquid chromatography tandem mass spectrometry (LC MS/MS). Some aspects of this invention provide affinity reagents encoded with mass reporters for the sensitive and quantitative translation of an analyte of interest into a mass tag. The reagents and methods provided herein have general utility in analyte detection and encoding, for example, in biomolecular profiling, molecular diagnostics, and biochemical encoding.

Claims

1. A method for the detection of aberrant protease activity in a subject, comprising

(a) administering to the subject a pro-diagnostic reagent comprising a set of isotope-coded reporter molecules (iCOREs) each conjugated to a carrier domain, wherein each iCORE comprises a mass tag conjugated to the carrier domain via a cleavable linker, under conditions suitable for cleavage of the cleavable linker by a disease-associated protease in vivo;

(b) identifying and/or quantifying the iCOREs in a biological sample obtained from the subject; and

(c) determining that the subject has an aberrant protease activity signature based on the iCORES identified in (b).

2. The method of claim 1, wherein the set of iCORES comprises at least 5 different mass tag-conjugated cleavable linkers.

3. The method of claim 1, wherein the iCOREs are isobaric.

4. The method of claim 1, wherein the iCOREs are peptide mass tags.

5. The method of claim 3, wherein the iCOREs comprise 5-20 amino acid residues.

6. The method of claim 3, wherein the iCOREs comprise the same amino acid sequence.

7. The method of claim 6, wherein the iCOREs comprise the amino acid sequence EGVNDNEEGFFSAR (SEQ ID NO:

1).

8. The method of claim 1, wherein the iCOREs are isotope-labeled.

9. The method of claim 1, wherein (b) comprises an MS/MS assay.

10. The method of claim 1, wherein the biological sample is a urine sample.

11. The method of claim 1, wherein the disease-associated protease is associated with cancer.

12. The method of claim 1, wherein the disease-associated protease is associated with fibrosis.

13. The method of claim 1, wherein the set of iCORES comprises at least 6 different mass tag-conjugated cleavable linkers.

14. The method of claim 1, wherein the set of iCORES comprises at least 7 different mass tag-conjugated cleavable linkers.

15. The method of claim 1, wherein the set of iCORES comprises at least 8 different mass tag-conjugated cleavable linkers.

16. The method of claim 1, wherein the set of iCORES comprises at least 9 different mass tag-conjugated cleavable linkers.

17. The method of claim 1, wherein the set of iCORES comprises 10 different mass tag-conjugated cleavable linkers.

18. The method of claim 1, wherein the set of iCORES comprises 20 different mass tag-conjugated cleavable linkers.