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

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...

Full description

Saved in:

Bibliographic Details
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
Online Access:	Get full text
Tags:	Add Tag No Tags, Be the first to tag this record!

Description
Summary:	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.
Bibliography:	istex:147BC68E1D3936D8F15DC8488089318E11D8783E ark:/67375/TPS-X3M01497-X ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 ObjectType-Article-1 ObjectType-Feature-2
ISSN:	0003-2700 1520-6882
DOI:	10.1021/ac050404f