Specificity of Immobilized Metal Affinity-Based IMAC/C18 Tip Enrichment of Phosphopeptides for Protein Phosphorylation Analysis

• Published in: Analytical chemistry (Washington) Vol. 77; no. 16; pp. 5144 - 5154
• Main Authors: Kokubu, Makiko, Ishihama, Yasushi, Sato, Toshitaka, Nagasu, Takeshi, Oda, Yoshiya
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 15.08.2005
• Subjects: Amino Acid Sequence; Analytical chemistry; Animals; Brain - metabolism; Carbon - chemistry; Chemistry; Chromatographic methods and physical methods associated with chromatography; Chromatography, Affinity - methods; Exact sciences and technology; Hydrophobic and Hydrophilic Interactions; Metals - chemistry; Mice; Microarray Analysis - methods; Molecular Sequence Data; Other chromatographic methods; Peptides; Phosphopeptides - analysis; Phosphopeptides - chemistry; Phosphoproteins - analysis; Phosphorus; Phosphorylation; Proteins; Sensitivity and Specificity; Spectrometric and optical methods; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Phosphates; Phosphorylation; Peptides; Coupled method; Phosphopeptide; Immobilization; Hydrophobic compound; Immobilized metal affinity chromatography; Selectivity; Matrix assisted laser desorption ionization; Metal; Strong acid; Protein; Chemical enrichment; Chemical reaction; Affinity chromatography; Acetonitrile; Mass spectrometry

We have developed a simple, highly specific enrichment procedure for phosphopeptides, by increasing the specificity of an immobilized metal affinity column (IMAC) without using any chemical reaction. The method employs a biphasic IMAC-C18 tip, in which IMAC beads are packed on an Empore C18 disk in...