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• W2048472959 endingPage "5154" @default.
• W2048472959 startingPage "5144" @default.
• W2048472959 abstract "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." @default.
• W2048472959 created "2016-06-24" @default.
• W2048472959 creator A5001763350 @default.
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• W2048472959 creator A5060432579 @default.
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• W2048472959 date "2005-07-08" @default.
• W2048472959 modified "2023-10-17" @default.
• W2048472959 title "Specificity of Immobilized Metal Affinity-Based IMAC/C18 Tip Enrichment of Phosphopeptides for Protein Phosphorylation Analysis" @default.
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• W2048472959 doi "https://doi.org/10.1021/ac050404f" @default.
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• W2048472959 hasPublicationYear "2005" @default.
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