Enhanced detection and identification of multiply phosphorylated peptides using TiO₂ enrichment in combination with MALDI TOF/TOF MS

The analysis of PTMs such as phosphorylation has become an important field in MS because they can directly indicate protein states and interactions. Whereas the characterization of singly and doubly phosphorylated peptides has almost become routine, identifying phosphorylation events at multiple res...

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Bibliographic Details
Published in:Proteomics (Weinheim) 2008-11, Vol.8 (21), p.4577-4592
Main Authors: Schmidt, Andreas, Csaszar, Edina, Ammerer, Gustav, Mechtler, Karl
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Chromatographic enrichment
Chromatography, High Pressure Liquid
Crystallization
Indicators and Reagents
Isotope Labeling
Mass spectrometry
Molecular Sequence Data
Osteopontin - analysis
Phosphopeptides
Phosphopeptides - analysis
Phosphoproteomics
Phosphorylation
Proteomics
Spectrometry, Mass, Electrospray Ionization
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Tandem Mass Spectrometry
Titanium
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Summary:The analysis of PTMs such as phosphorylation has become an important field in MS because they can directly indicate protein states and interactions. Whereas the characterization of singly and doubly phosphorylated peptides has almost become routine, identifying phosphorylation events at multiple residues within a small region of a protein is still problematic. The identification of multiple modifications can be further hampered by low sequence information due to multiple neutral losses from phosphorylated side chains. Here we present a strategy for the analysis of complex phosphopeptides that combines peptide enrichment by titanium dioxide, separation by RP separation on monolithic columns and MS using high energy HE-CAD in a MALDI TOF/TOF analyser. Using synthetic phosphopeptides our approach is compared to multistage activation (MSA) MS/MS and the recently described electron transfer dissociation (ETD) method using an ESI-LTQ mass spectrometer.
ISSN:1615-9853
1615-9861
DOI:10.1002/pmic.200800279