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

Abstract

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.

Mesoporous TiO2 aerogel for selective enrichment of phosphopeptides in rat liver mitochondria

“…5B. The sensitivity of the on-target enrichment performed by mesoporous TiO 2 nanoparticles is at the low femtomole [21,27,28,31]. The higher sensitivity achieved by TiO 2 aerogel might be contributed to the excellent compatibility of enrichment procedure with MALDI-TOF MS and the large binding capacity of TiO 2 aerogel.…”

“…The materials with high binding capacity will bring considerable benefits, for example, smaller elution volume, lower risk for sample loss and less effort spent on desalting and solvent evaporation. Besides, higher sensitivity for phosphopeptides detection can be achieved by immobilized mesoporous metal oxides with high binding capacity on the MALDI plate [21,27,28]. However, the surface area of pure mesoporous MOAC materials is often less than 100 m 2 g −1 [24,29], and only that for those supported by silica matrices can get larger than 100 m 2 g −1 , but with complex synthesis procedure [22,23,30].…”

“…The limited specific surface area of the reported MOAC materials results in low binding capacity and low sensitivity for phosphopeptide detection. The sensitivity of the on-plate purification approach based on MOAC is at femtomole level [21,27,28,31], which may limit the application of MOAC in phosphoproteome study. Therefore, the demand is high for new materials with high surface area, in order to improve the binding capacity and detection sensitivity for phosphopeptide analysis.…”

Mesoporous TiO2-Based Experimental Layout for On-Target Enrichment and Separation of Multi- and Monophosphorylated Peptides Prior to Analysis with Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry

“…In order to enrich the phosphopeptides most of the new methods make use of the high affinity of the phosphoryl group to metals and metal oxides (e.g., on immobilized metal affinity chromatography (IMAC) [2][3][4][5] , metal oxide affinity chromatography (MOAC) 1,[6][7][8][9][10][11][12][13][14] , sequential immobilized metal affinity chromatography (SIMAC) 15 and direct on-target enrichment [16][17][18][19][20][21][22][23] ). Mass spectrometry (MS) is then commonly used to analyze the peptides.…”

Discontinuous pH gradient-mediated separation of TiO2-enriched phosphopeptides

“…Existing methodologies for phosphoproteome analysis include: fractionation of tryptically-digested complex peptide mixtures by strong cation exchange (SCX) chromatography; phosphopeptide enrichment by titanium dioxide (TiO 2 ) chromatography, immobilized metal ion affinity chromatography (IMAC), or chemistry-based modification of phosphate groups [4–7]. Indicative of the extent of interest in metal ion-mediated phosphoproteomic enrichment techniques many diverse strategies have recently been developed including the use of spectra cross-validation [8], pH modification [9], mesoporous TiO 2 [10] and even nanocrystalline TiO 2 films [11]. Employing these multiple experimental platforms, thousands of phosphopeptides and protein phosphorylation sites can be analyzed from a single experimental cell signaling study, however comprehensive and differential identification of distinct groups of phosphoproteins is still slightly beyond current technologies [12].…”