title: Sequential Elution from IMAC (SIMAC): An Efficient Method for Enrichment and Separation of Mono- and Multi-phosphorylated Peptides.
abstract: Phosphoproteomics relies on methods for efficient purification and sequencing of phosphopeptides from highly complex biological systems, especially when using low amounts of starting material. Current methods for phosphopeptide enrichment, e.g., Immobilized Metal ion Affinity Chromatography and titanium dioxide chromatography provide varying degrees of selectivity and specificity for phosphopeptide enrichment. The number of multi-phosphorylated peptides identified in most published studies is rather low. Here we describe a protocol for a strategy that separates mono-phosphorylated peptides from multiply phosphorylated peptides using Sequential elution from Immobilized Metal ion Affinity Chromatography. The method relies on the initial enrichment and separation of mono- and multi-phosphorylated peptides using Immobilized Metal ion Affinity Chromatography and a subsequent enrichment of the mono-phosphorylated peptides using titanium dioxide chromatography. The two separate phosphopeptide fractions are then subsequently analyzed by mass spectrometric methods optimized for mono-phosphorylated and multi-phosphorylated peptides, respectively, resulting in improved identification of especially multi-phosphorylated peptides from a minimum amount of starting material.
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 M, Denmark. mrl@bmb.sdu.dk.
authors.forename: Martin R
authors.initials: MR
authors.lastname: Larsen
mesh_terms: D000818:Animals; D002846:Chromatography, Affinity; D056148:Chromatography, Reverse-Phase; D006367:HeLa Cells; D006801:Humans; 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; D053719:Tandem Mass Spectrometry; D014025:Titanium / Q000737:chemistry; D057188:Workflow
chemical_list: D010748:Phosphopeptides; C009495:titanium dioxide; D014025:Titanium
keywords: Immobilized metal affinity chromatography; Mass spectrometry; Multi-phosphorylated peptides; Phosphopeptide enrich ment; Sequential elution; Titanium dioxide chromatography