CLEAVABLE PROBES FOR ISOTOPE TARGETED GLYCOPROTEOMICS AND METHODS OF USING THE SAME

摘要

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.

主权项

1. A method for producing an isotopically-labelled peptide, the method comprising:
contacting a sample including a metabolically tagged protein 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 tagL is a cleavable linker;M is an isotopic label; andZ is a chemoselective tag capable of cross-linking the metabolically tagged protein; separating the probe-protein conjugate from the sample; digesting the probe-protein conjugate to produce a probe-peptide conjugate; and cleaving the cleavable linker to release the isotopically labelled peptide.