Offline Micro-IMAC Enrichment of Phosphoproteins.

INTRODUCTIONImmobilized 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.