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Journal of Biomedicine and Biotechnology
Volume 2010, Article ID 759690, 12 pages
http://dx.doi.org/10.1155/2010/759690
Methodology Report

Enhanced MALDI-TOF MS Analysis of Phosphopeptides Using an Optimized DHAP/DAHC Matrix

1Proteomic Platform and National Key Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
2Department of Biology, Graduate University of the Chinese Academy of Sciences, Beijing 100049, China

Received 30 June 2009; Revised 11 September 2009; Accepted 31 December 2009

Academic Editor: Kai Tang

Copyright © 2010 Junjie Hou et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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 -casein and -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 -casein and -casein and to characterize two phosphorylation sites from the human histone H1 treated with Cyclin-Dependent Kinase-1 (CDK1) by MALDI-TOF/TOF MS.