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Anal Chem. 2009 Jun 1;81(11):4566-75. doi: 10.1021/ac9004452.

Magnetic bead processor for rapid evaluation and optimization of parameters for phosphopeptide enrichment.

Author information

1
Department of Cancer Biology and Blais Proteomics Center, Dana-Farber Cancer Institute.

Abstract

Qualitative and quantitative analysis of phosphorylation continues to be both an important and a challenging experimental paradigm in proteomics-based research. Unfortunately researchers face difficulties inherent to the optimization of complex, multivariable methods and their application to the analysis of rare and often experimentally intractable phosphorylated peptides. Here we describe a platform based on manipulation of magnetic beads in a 96-well format that facilitates rapid evaluation of experimental parameters required for enrichment of phosphopeptides. Optimized methods provided for automated enrichment and subsequent LC-MS/MS detection of over 1000 unique phosphopeptides (approximately 1% FDR) from 50 microg of cell lysates. In addition we demonstrate use of this platform for identification of phosphopeptides derived from proteins separated by SDS-PAGE and visualized near the detection limit of silver staining.

PMID:
19408940
PMCID:
PMC2692042
DOI:
10.1021/ac9004452
[Indexed for MEDLINE]
Free PMC Article

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