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 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.
Schmidt
Andreas
A
Christian Doppler Laboratory for Proteome Analysis, University of Vienna, Vienna, Austria.
Csaszar
Edina
E
Ammerer
Gustav
G
Mechtler
Karl
K
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003460:Crystallization; D007202:Indicators and Reagents; D007553:Isotope Labeling; D008969:Molecular Sequence Data; D053495:Osteopontin / Q000032:analysis; D010748:Phosphopeptides / Q000032:analysis*; D010766:Phosphorylation; D040901:Proteomics; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry; D014025:Titanium
D007202:Indicators and Reagents; D010748:Phosphopeptides; D053495:Osteopontin; C009495:titanium dioxide; D014025:Titanium