Titanium dioxide as a chemo-affinity solid phase in offline phosphopeptide chromatography prior to HPLC-MS/MS analysis.
We have developed a new offline chromatographic approach for the selective enrichment of phosphorylated peptides that is directly compatible with subsequent analysis by online nano electrospray ionization tandem mass spectrometry. In this technique, a titanium dioxide (TiO2)-packed pipette tip is used as a phosphopeptide trap that acts as an offline first-dimension separation step in a two-dimensional chromatography system. This is followed by online nano reversed-phase high-performance liquid chromatography. Here, we present suitable methods for enrichment, optimized separately for each step: sample loading, washing and elution from the TiO2-filled tips. To increase the trapping selectivity of the TiO2 column, we used the sodium salt of 1-octanesulfonic acid combined with 2,5-dihydroxybenzoic acid as ion-pairing agents and displacers for acidic peptides. These agents also improve the binding of phosphorylated peptides and block the binding of non-phosphorylated ones. This enrichment procedure takes 30 min, followed by a 100-min HPLC program, including washing and an elution gradient.
Mazanek
Michael
M
Research Institute of Molecular Pathology (IMP), Dr Bohr-Gasse 7, A-1030 Vienna, Austria.
Mituloviae
Goran
G
Herzog
Franz
F
Stingl
Christoph
C
Hutchins
James R A
JR
Peters
Jan-Michael
JM
Mechtler
Karl
K
D002845:Chromatography / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification*; D052616:Solid Phase Extraction / Q000379:methods*; D053719:Tandem Mass Spectrometry; D014025:Titanium / Q000737:chemistry*
D010748:Phosphopeptides; C009495:titanium dioxide; D014025:Titanium