Phosphopeptide screening using nanocrystalline titanium dioxide films as affinity matrix-assisted laser desorption ionization targets in mass spectrometry.
The use of nanocrystalline titanium dioxide films as affinity targets for the selective isolation and enrichment of phosphopeptides with subsequent analysis by matrix-assisted laser desorption ionization (MALDI) mass spectrometry is described. A strong affinity of phosphopeptides to anatase titanium dioxide surfaces is observed, and a standard protocol for the selective isolation and enrichment of phosphopeptides on titanium dioxide films using a proteolytic digest of alpha- and beta-casein was developed. All washing and elution procedures using these films can be processed directly on the MALDI target, thereby avoiding sample contamination and losses. In addition, the enrichment of the phosphopeptides was improved due to a considerable enlargement of the surface. Several film substrates compatible with routine inlet systems of mass spectrometers, as conductive glass, aluminum, and silicon, have been manufactured and tested. A biological application was examined by the human fibrinogen-thrombin system. For a quantification and comparison of different expression levels of phosphoproteins in biological systems, the peptides were labeled with S-methyl thioimidate reagents. The capability of this method for high-throughput applications make the use of mesoporous titanium dioxide films as an affinity MALDI target a promising tool in phosphoproteomics. A combination of an amidation protocol showed that a quantification of phosphorylated peptides can easily be performed using TiO(2) films.
Niklew
Marie-Luise
ML
Department of Chemistry, Humboldt-Universitaet zu Berlin, Berlin, Germany.
Hochkirch
Ulrike
U
Melikyan
Anna
A
Moritz
Thomas
T
Kurzawski
Sandra
S
Schlüter
Hartmut
H
Ebner
Ingo
I
Linscheid
Michael W
MW
D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D005340:Fibrinogen / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D053758:Nanoparticles / Q000737:chemistry*; D010748:Phosphopeptides / Q000032:analysis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013917:Thrombin / Q000378:metabolism; D014025:Titanium / Q000737:chemistry*; D014357:Trypsin / Q000378:metabolism
D002364:Caseins; D010748:Phosphopeptides; C009495:titanium dioxide; D005340:Fibrinogen; D014025:Titanium; D014357:Trypsin; D013917:Thrombin