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
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac050404f

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 a 200-μL pipet tip. Phosphopeptides are separated from non-phosphopeptides on the IMAC in an optimized solvent without any chemical reaction, then desorbed from the IMAC using a phosphate buffer, reconcentrated, and desalted on the C18 disk. The increase in selectivity was achieved by (a) using a strong acid to discriminate phosphates from carboxyl groups of peptides and (b) using a high concentration of acetonitrile to remove hydrophobic non-phosphopeptides. The entire procedure was optimized by using known phosphoproteins such as Akt1 kinase and protein kinase A. Although it was difficult to detect phosphopeptides in MALDI-MS spectra of tryptic peptide mixtures before enrichment, after the IMAC procedure, we could successfully detect phosphopeptides with almost no non-phosphopeptides. Next, we constructed an array of IMAC−IMAC/C18 tips, such that number of arrayed tips on a 96-well plate could easily be changed depending on the loading amount of sample. Applying this approach to mouse forebrain resulted in the identification of 162 phosphopeptides (166 phosphorylation sites) from 135 proteins using nano-LC/MS.