Cleavable probes for isotope targeted glycoproteomics and methods of using the same
申请人:The Regents of the University of California
公开号:US10114026B2
公开(公告)日:2018-10-30
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.
本文提供了生产同位素标记肽的方法。该方法的各个方面包括:将包括代谢标记蛋白质的样品与可裂解探针接触,生成探针-蛋白质共轭物;将探针-蛋白质共轭物与样品分离;消化探针-蛋白质共轭物,生成探针-肽共轭物;裂解可裂解连接体,释放同位素标记的肽。该方法可进一步包括:确定质谱中的预定同位素模式;确定同位素标记肽的氨基酸序列;以及根据确定的氨基酸序列确定蛋白质糖基化的位点。此外,还提供了用于实施上述方法的可裂解探针,其描述式如下:A-L-(M-Z),其中 A 是亲和性标记,L 是可裂解连接体,M 是同位素标记,Z 是化学选择性标记,能够交联代谢标记蛋白质。此外,还提供了用于实施上述方法的组合物和试剂盒。