Nanostructured TiO2 thin films for phosphoproteomics studies with MALDI mass spectrometry.

Alterations in protein phosphorylation, a posttranslational modification (PTM) that regulates many -processes in living cells, is a fundamental mechanism of many diseases, including cancer. Phosphoproteomics, with the combined use of affinity chromatography and electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, is shedding light into phosphorylation signaling pathways at the proteome level and helps to solve difficulties related to sample complexity and phosphopeptide enrichment. One of the most frequent and efficient methods used to enrich samples for the phosphorylated components is titanium dioxide chromatography. Titanium dioxide has a high affinity for phosphopeptides and can also be selective in specific experimental conditions. Here, we describe a protocol for the use of a MALDI plate covered with titanium dioxide nanostructured film, a device developed for a rapid and efficient study of phosphorylated peptides.

Biosensors and molecular imaging.

The increase in the understanding of the physical and functional properties of the biological material, from the cellular level down to single molecules, owes its success to the development of suitable high-sensitivity platforms to image the biomaterial and analyze its response to specific stimuli. Imaging has indeed reached molecular capabilities, thanks to optical or magnetic markers [1], to the atomic force microscopy (AFM) in surface reconstruction [2], and is nearing success in three-dimensional (3-D) reconstruction thanks to X-ray holography [3].

Titanium dioxide coated MALDI plate for on target analysis of phosphopeptides.

Protein phosphorylation controls many cellular processes and activities. One of the major challenges in the proteomic study of phosphorylation is the enrichment of substoichiometric phosphorylated peptides from complex mixtures. Titanium dioxide (TiO2)-based chromatography is now widely applied to isolate phosphopeptides because of its efficiency and flexibility. In this study, a novel TiO2 coated matrix assisted laser desorption ionization plate is presented and tested for the purification of phosphopeptides from complex mixtures. The novel feature of this approach is the deposition of a nanostructured TiO2 film on stainless steel plates by pulsed laser deposition (PLD). By using tryptic digests of alpha-casein, beta-casein, and other nonphosphorylated proteins, the successful enrichment of phosphopeptides was possible with this novel device, called T-plate, even when working in the low fmol range, making the sample ready for mass spectrometric analysis in few minutes.