Phosphoric acid enhances the performance of Fe(III) affinity chromatography and matrix‐assisted laser desorption/ionization tandem mass spectrometry for recovery, detection and sequencing of phosphopeptides
- 2 July 2004
- journal article
- research article
- Published by Wiley in Rapid Communications in Mass Spectrometry
- Vol. 18 (15) , 1721-1730
Abstract
An integrated analytical strategy for enrichment, detection and sequencing of phosphorylated peptides by matrix‐assisted laser desorption/ionization (MALDI) tandem mass spectrometry (MS/MS) is reported. o ‐Phosphoric acid was found to enhance phosphopeptide ion signals in MALDI‐MS when used as the acid dopant in 2,5‐dihydroxybenzoic acid (2,5‐DHB) matrix. The effect was largest for multiply phosphorylated peptides, which exhibited an up to ten‐fold increase in ion intensity as compared with standard sample preparation methods. The enhanced phosphopeptide response was observed during MALDI‐MS analysis of several peptide mixtures derived by proteolytic digestion of phosphoproteins. Furthermore, the mixture of 2,5‐DHB and o ‐phosphoric acid was an excellent eluant for immobilized metal affinity chromatography (IMAC). Singly and multiply phosphorylated peptide species were efficiently recovered from Fe(III)‐IMAC columns, reducing sample handling for phosphopeptide mapping by MALDI‐MS and subsequent phosphopeptide sequencing by MALDI‐MS/MS. The enhanced response of phosphopeptide ions in MALDI facilitates MS/MS of large (>3 kDa) multiply phosphorylated peptide species and reduces the amount of analyte needed for complete characterization of phosphoproteins. Copyright © 2004 John Wiley & Sons, Ltd.Keywords
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