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J Biomed Biotechnol. 2010;2010:759690. doi: 10.1155/2010/759690. Epub 2010 Mar 21.

Enhanced MALDI-TOF MS analysis of phosphopeptides using an optimized DHAP/DAHC matrix.

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  • 1Proteomic Platform and National Key Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.

Abstract

Selecting an appropriate matrix solution is one of the most effective means of increasing the ionization efficiency of phosphopeptides in matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). In this study, we systematically assessed matrix combinations of 2, 6-dihydroxyacetophenone (DHAP) and diammonium hydrogen citrate (DAHC), and demonstrated that the low ratio DHAP/DAHC matrix was more effective in enhancing the ionization of phosphopeptides. Low femtomole level of phosphopeptides from the tryptic digests of alpha-casein and beta-casein was readily detected by MALDI-TOF-MS in both positive and negative ion mode without desalination or phosphopeptide enrichment. Compared with the DHB/PA matrix, the optimized DHAP/DAHC matrix yielded superior sample homogeneity and higher phosphopeptide measurement sensitivity, particularly when multiple phosphorylated peptides were assessed. Finally, the DHAP/DAHC matrix was applied to identify phosphorylation sites from alpha-casein and beta-casein and to characterize two phosphorylation sites from the human histone H1 treated with Cyclin-Dependent Kinase-1 (CDK1) by MALDI-TOF/TOF MS.

PMID:
20339515
[PubMed - indexed for MEDLINE]
PMCID:
PMC2842900
Free PMC Article

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