通过固定化金属亲和色谱法富集磷酸肽

Enrichment of Phosphopeptides via Immobilized Metal Affinity Chromatography.

作者信息

Swaney Danielle L, Villén Judit

机构信息

Department of Genome Sciences, University of Washington, Seattle, Washington 98195.

出版信息

Cold Spring Harb Protoc. 2016 Mar 1;2016(3):pdb.prot088005. doi: 10.1101/pdb.prot088005.

DOI:10.1101/pdb.prot088005

PMID:26933247

Abstract

Immobilized metal affinity chromatography (IMAC) is a frequently used method for the enrichment of phosphorylated peptides from complex, cellular lysate-derived peptide mixtures. Here we outline an IMAC protocol that uses iron-chelated magnetic beads to selectively isolate phosphorylated peptides for mass spectrometry-based proteomic analysis. Under acidic conditions, negatively charged phosphoryl modifications preferentially bind to positively charged metal ions (e.g., Fe(3+), Ga(3+)) on the beads. After washing away nonphosphorylated peptides, a pH shift to basic conditions causes the elution of bound phosphopeptides from the metal ion. Under optimal conditions, very high specificity for phosphopeptides can be achieved.

摘要

固定化金属亲和色谱法（IMAC）是一种常用的从复杂的细胞裂解物衍生肽混合物中富集磷酸化肽的方法。在此，我们概述了一种IMAC方案，该方案使用铁螯合磁珠选择性分离磷酸化肽，用于基于质谱的蛋白质组学分析。在酸性条件下，带负电荷的磷酸化修饰优先与磁珠上带正电荷的金属离子（如Fe(3+)、Ga(3+)）结合。洗去非磷酸化肽后，将pH值调至碱性条件会使结合的磷酸肽从金属离子上洗脱下来。在最佳条件下，可以实现对磷酸肽的非常高的特异性。