Cite as: Cold Spring Harb. Protoc.; 2007; doi:10.1101/pdb.prot4624

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Right arrow Protein Identification and Analysis
Right arrow Proteins and Proteomics, general
Right arrow Characterization of Protein Complexes
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Right arrow Phosphorylation Site Mapping
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Offline Micro-IMAC Enrichment of Phosphoproteins

Garry L. Corthals, Valerie C. Wasinger, and David R. Goodlett

This protocol was adapted from "Proteomic Methods for Phosphorylation Site Mapping," Chapter 9, in Proteins and Proteomics (ed. Simpson). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003.


INTRODUCTION

Immobilized metal affinity chromatography (IMAC) can be performed offline or online with µLC-ESI-MS/MS. The online configuration has been used successfully despite suboptimal low-level analysis due to the relative disparity between IMAC and µLC flow rates. With IMAC performed offline from µLC-ESI-MS, sample volumes from 1 to 100 µL can be loaded using a sample pressure vessel or an autosampling system equipped with a sample-trapping cartridge. This protocol describes the construction and use of a micro-IMAC column for the enrichment of phosphoproteins.


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