Phosphopeptide Screening Using Nanocrystalline Titanium Dioxide Films as Affinity Matrix-Assisted Laser Desorption Ionization Targets in Mass Spectrometry

摘要

The use of nanocrystalline titanium dioxide films asnaffinity targets for the selective isolation and enrichmentnof phosphopeptides with subsequent analysis by matrixassistednlaser desorption ionization (MALDI) mass spectrometrynis described. A strong affinity of phosphopeptidesnto anatase titanium dioxide surfaces is observed, andna standard protocol for the selective isolation and enrichmentnof phosphopeptides on titanium dioxide films usingna proteolytic digest of r- and u0001-casein was developed. Allnwashing and elution procedures using these films can benprocessed directly on the MALDI target, thereby avoidingnsample contamination and losses. In addition, the enrichmentnof the phosphopeptides was improved due to anconsiderable enlargement of the surface. Several filmnsubstrates compatible with routine inlet systems of massnspectrometers, as conductive glass, aluminum, and silicon,nhave been manufactured and tested. A biologicalnapplication was examined by the human fibrinogenthrombinnsystem. For a quantification and comparison ofndifferent expression levels of phosphoproteins in biologicalnsystems, the peptides were labeled with S-methylnthioimidate reagents. The capability of this method fornhigh-throughput applications make the use of mesoporousntitanium dioxide films as an affinity MALDI target anpromising tool in phosphoproteomics. A combination ofnan amidation protocol showed that a quantification ofnphosphorylated peptides can easily be performed usingnTiO2 films.