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Photocatalytically patterned TiO2 arrays for on-plate selective enrichment of phosphopeptides and direct MALDI MS analysis.

ABSTRACT: We report the development of photocatalytically patterned TiO(2) arrays for selective on-plate enrichment and direct matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of phosphopeptides. A thin TiO(2) nanofilm with controlled porosity is prepared on gold-covered glass slides by a layer-by-layer (LbL) deposition/calcination process. The highly porous and rough nanostructure offers high surface area for selective binding of phosphorylated species. The patterned arrays are generated using an octadecyltrichlorosilane (OTS) coating in combination of UV irradiation with a photomask, followed by NaOH etching. The resulting hydrophilic TiO(2) spots are thus surrounded by a hydrophobic OTS layer, which can facilitate the enrichment of low-abundance components by confining a large volume sample into a small area. The TiO(2) arrays exhibit high specificity toward phosphopeptides in complex samples including phosphoprotein digests and human serum, and the detection can be made in the fmole range. Additional advantages of the arrays include excellent stability, reusability/reproducibility, and low cost. This method has been successfully applied to the analysis of phosphopeptides in nonfat milk. The patterned TiO(2) arrays provide an attractive interface for performing on-plate reactions, including selective capture of target species for MALDI-MS analysis, and can serve as a versatile lab-on-a-chip platform for high throughput analysis in phosphoproteome research.

SUBMITTER: Wang H

PROVIDER: S-EPMC7360113 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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Photocatalytically patterned TiO2 arrays for on-plate selective enrichment of phosphopeptides and direct MALDI MS analysis.

Wang Hui H Duan Jicheng J Cheng Quan Q

Analytical chemistry 20110209 5

We report the development of photocatalytically patterned TiO(2) arrays for selective on-plate enrichment and direct matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of phosphopeptides. A thin TiO(2) nanofilm with controlled porosity is prepared on gold-covered glass slides by a layer-by-layer (LbL) deposition/calcination process. The highly porous and rough nanostructure offers high surface area for selective binding of phosphorylated species. The patterned arra ...[more]

PMID: 21306131

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Project description:Cysteine is a rare and conserved amino acid involved in most cellular functions. The thiol group of cysteine can be subjected to diverse oxidative modifications that regulate many physio-pathological states. In the present work, a Cysteine-specific Phosphonate Adaptable Tag (CysPAT) was synthesized to selectively label cysteine-containing peptides (Cys peptides) followed by their enrichment with titanium dioxide (TiO2) and subsequent mass spectrometric analysis. The CysPAT strategy was developed using a synthetic peptide, a standard protein and subsequently the strategy was applied to protein lysates from Hela cells, achieving high specificity and enrichment efficiency. In particular, for Cys proteome analysis, the method led to the identification of 7509 unique Cys peptides from 500 ?g of HeLa cell lysate starting material. Furthermore, the method was developed to simultaneously enrich Cys peptides and phosphorylated peptides. This strategy was applied to SILAC labeled Hela cells subjected to 5 min epidermal growth factor (EGF) stimulation. In total, 10440 unique reversibly modified Cys peptides (3855 proteins) and 7339 unique phosphopeptides (2234 proteins) were simultaneously identified from 250 ?g starting material. Significant regulation was observed in both phosphorylation and reversible Cys modification of proteins involved in EGFR signaling. Our data indicates that EGF stimulation can activate the well-known phosphorylation of EGFR and downstream signaling molecules, such as mitogen-activated protein kinases (MAPK1 and MAPK3), however, it also leads to substantial modulation of reversible cysteine modifications in numerous proteins. Several protein tyrosine phosphatases (PTPs) showed a reduction of the catalytic Cys site in the conserved putative phosphatase HC(X)5R motif indicating an activation and subsequent de-phosphorylation of proteins involved in the EGF signaling pathway. Overall, the CysPAT strategy is a straight forward, easy and promising method for studying redox proteomics and the simultaneous enrichment strategy offers an excellent solution for characterization of cross-talk between phosphorylation and redox induced reversible cysteine modifications.

Dataset Information

Photocatalytically patterned TiO2 arrays for on-plate selective enrichment of phosphopeptides and direct MALDI MS analysis.

Publications

Photocatalytically patterned TiO2 arrays for on-plate selective enrichment of phosphopeptides and direct MALDI MS analysis.

Similar Datasets

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