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

1. A method for identifying a site of glycosylation on a protein, the method comprising:

contacting a cellular sample including a metabolically tagged glycosylated protein comprising an azide-tagged sugar with a cleavable probe under conditions sufficient to produce a probe-protein conjugate, wherein the cleavable probe is described by 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 —(CH 2 ) p —CH(Br)═CH(Br)—(CH 2 ) q — wherein p and q are each independently 0-6; and

Z is an alkyne tag that cross-links the azide-tagged sugar of the metabolically tagged glycosylated protein via copper-catalyzed azide-alkyne [3+2] cycloaddition to produce the probe-protein conjugate;

separating the probe-protein conjugate from the sample;

digesting the probe-protein conjugate to produce a probe-peptide conjugate;

cleaving the cleavable linker to release an isotopically labelled glycopeptide

identifying the isotopically labelled glycopeptide via a predetermined isotopic pattern in a mass spectrum by full scan mass spectrometry prior to tandem MS analysis;

determining an amino acid sequence of the isotopically labelled glycopeptide by tandem MS analysis targeted to the identified isotopically labelled glycopeptide; and

identifying a site of glycosylation on a protein based on the determined amino acid sequence of the isotopically labelled glycopeptide.

2. The method of claim 1 , wherein the sample is obtained from a eukaryotic cell comprising the metabolically tagged glycosylated protein.

3. The method of claim 2 , wherein the method further comprises contacting the cell with the azide-tagged sugar under conditions sufficient to produce the metabolically tagged glycosylated protein.

4. The method of claim 1 , wherein A is a biotin moiety.

5. The method of claim 1 , wherein L is described by the formula:

-L 1 -X-L 2 —

wherein L 1 and L 2 are optional linkers and X is a cleavable group.

6. The method 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.

7. The method of claim 1 , wherein L is a cleavable silane linker.

8. The method of claim 3 , wherein the tagged sugar and the produced metabolically tagged protein comprise an isotopic label.

9. The method of claim 1 , further comprising quantitating a glycoprotein of the sample.

10. The method of claim 1 , further comprising:

contacting the sample with a protein probe capable of cross-linking an amino acid residue of the protein to produce a labelled protein; and

digesting the labelled protein to produce a labelled peptide.

11. The method of claim 10 , further comprising quantitating a protein of the sample.

12. The method of claim 1 , wherein:

A is a biotin moiety;

L is —(PEG) n —NH—(CH 2 ) m —C(CH 3 ) 2 —O—Si(R) 2 —O—;

M is —(CH 2 ) p —CH(Br)═CH(Br)—(CH 2 ) 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.