title: Phosphopeptide Enrichment by Immobilized Metal Affinity Chromatography.
abstract: Immobilized metal affinity chromatography (IMAC) has been the method of choice for phosphopeptide enrichment prior to mass spectrometric analysis for many years and it is still used extensively in many laboratories. Using the affinity of negatively charged phosphate groups towards positively charged metal ions such as Fe(3+), Ga(3+), Al(3+), Zr(4+), and Ti(4+) has made it possible to enrich phosphorylated peptides from peptide samples. However, the selectivity of most of the metal ions is limited, when working with highly complex samples, e.g., whole-cell extracts, resulting in contamination from nonspecific binding of non-phosphorylated peptides. This problem is mainly caused by highly acidic peptides that also share high binding affinity towards these metal ions. By lowering the pH of the loading buffer nonspecific binding can be reduced significantly, however with the risk of reducing specific binding capacity. After binding, the enriched phosphopeptides are released from the metal ions using alkaline buffers of pH 10-11, EDTA, or phosphate-containing buffers. Here we describe a protocol for IMAC using Fe(3+) for phosphopeptide enrichment. The principles are illustrated on a semi-complex peptide mixture.
authors.affiliation: Division of Translational Cancer Research, Lund University, Lund, Sweden.|Lund Stem Cell Center, Lund University, Lund, Sweden.
authors.forename: Tine E
authors.initials: TE
authors.lastname: Thingholm
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230, Odense, Denmark. mrl@bmb.sdu.dk.
authors.forename: Martin R
authors.initials: MR
authors.lastname: Larsen
mesh_terms: D000818:Animals; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D006801:Humans; D007501:Iron / Q000737:chemistry*; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry; D057188:Workflow
chemical_list: D010748:Phosphopeptides; D007501:Iron
keywords: Immobilized metal affinity chromatography; Mass spectrometry; Phosphopeptide enrich ment; Protein phosphorylation