Cleavable probes for isotope targeted glycoproteomics and methods of using the same

• US 10,451,632 B2
• Filed: 10/22/2018
• Issued: 10/22/2019
• Est. Priority Date: 12/05/2014
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Bertozzi, Carolyn R., Woo, Christina
• Primary Examiner(s): Haq, Shafiqul
• Application Number:
  US16/167,181
• Publication Number:
  US 20190094238A1
• Time in Patent Office: 365 Days
• CPC Class Codes: C07F 5/00 Compounds containing elements of Groups 3 or 13 of the Periodic Table
• CPC Class Codes: C07F 7/1804 Compounds having Si-O-C linkages Si-O-acyl linkages C07F7/1896
• CPC Class Codes: C07K 1/13 Labelling of peptides
• CPC Class Codes: G01N 2440/38 addition of carbohydrates, e.g. glycosylation, glycation
• CPC Class Codes: G01N 2458/15 Non-radioactive isotope labels, e.g. for detection by mass spectrometry
• CPC Class Codes: G01N 33/6848 Methods of protein analysis involving mass spectrometry

Abstract

Methods for producing isotopically-labelled peptides are provided. Aspects of the method include: contacting a sample including a metabolically tagged protein with a cleavable probe to produce a probe-protein conjugate; separating the probe-protein conjugate from the sample; digesting the probe-protein conjugate to produce a probe-peptide conjugate; and cleaving a cleavable linker to release an isotopically labelled peptide. The method may further include: identifying a predetermined isotopic pattern in a mass spectrum; determining an amino acid sequence of the isotopically labelled peptide; and identifying the site of protein glycosylation based on the determined amino acid sequence. Also provided are cleavable probes for practicing the subject methods, described by the Formula: A-L-(M-Z) where A is an affinity tag, L is a cleavable linker, M is an isotopic label and Z is a chemoselective tag capable of cross-linking a metabolically tagged protein. Compositions and kits for practicing the subject methods are also provided.

Claims

1. A cleavable probe of Formula (I):

A-L-(M-Z) 

 

(I)

wherein;

A is an affinity tag;

L is a cleavable linker;

M is an isotopic label of the formula —

(CH₂)_p—

CH(Br)═

CH(Br)—

(CH₂)_q—

wherein p and q are each independently 0-6; and

Z is a chemoselective tag comprising an alkyne.

2. The probe of claim 1, wherein A is a biotin moiety.

3. The probe of claim 1, wherein L is described by the formula:

L¹-X-L²-

wherein L¹ and L² are optional linkers and X is a cleavable group.

4. The probe of claim 1, wherein X is —

O—

Si(R)₂—

O—

, wherein each R is independently selected from hydrogen, an aryl, a substituted aryl, an alkyl and a substituted alkyl.

5. The probe of claim 1, wherein L is a cleavable silane linker.

6. The probe of claim 1, wherein the probe has the formula A-L-M-Z, wherein:

A is a biotin moiety;

L is -(PEG)_n —

NH—

(CH₂)_m—

C(CH₃)₂—

O—

Si(R)₂—

O—

;

M is —

(CH₂)_p—

CH(Br)═

CH(Br)—

(CH₂)_q—

; and

Z is an alkyne-containing group,

wherein n and m are each independently 0-20 and p and q are each independently 0-6.

7. The probe of claim 1, wherein M comprises two or more deuterium atoms.

8. The probe of claim 1, wherein M comprises a mixture of components, each component independently comprising 0, 2, 4 or 6 deuterium atoms.

10. The probe of claim 1, wherein the alkyne comprises acetylene or a cyclooctyne.

11. The probe of claim 1, wherein the probe of formula (I) is

15. The probe of claim 1, wherein M is described by the formula:

—

CH₂—

CH(Br)═

CH(Br)—

CH₂)—

.

9. A kit, comprising:

a cleavable probe of Formula (I);

A-L-(M-Z) 

 

(I)

wherein;

A is an affinity tag;

L is a cleavable linker;

M is an isotopic label of the formula —

(CH₂)_p—

CH(Br)═

CH(Br)—

(CH₂)_q—

wherein p and

q are each independently 0-6; and

Z is a chemoselective tag comprising an alkyne; and

one or more components selected from;

an enzyme, a chemical cleavage agent, a light source, a buffer, a cell, a metabolically tagged protein and a tagged sugar.

12. The kit of claim 9, wherein the metabolically tagged protein is a glycosylated protein comprising an azide-tagged sugar.

13. The kit of claim 12, wherein the azide-tagged sugar is selected from the group consisting of N-azidoacetylgalactosamine, acetylated (Ac4GaINAz);

N-azidoacetylglucosamine, acetylated (Ac4GIcNAz); and

N-azidoacetylmannosamine, acetylated (Ac4ManNAz).

14. The kit of claim 12, wherein the azide-tagged sugar comprises an isotopic label.

16. The kit of claim 9, wherein M is described by the formula:

—

CH₂—

CH(Br)═

CH(Br)—

CH₂)—

.

17. The kit of claim 9, wherein the alkyne comprises acetylene or a cyclooctyne.