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

Abstract

A simple method for on-target enrichment and subsequent separation and analysis of phosphorylated peptides is presented. The tryptic digest of a phosphorylated protein, in this case β-casein, is loaded onto a spot on a thin stripe made of mesoporous TiO 2 sintered onto a conductive glass surface. After washing with a salicylic buffer in order to remove the non-phosphorylated peptides, the stripe is placed in an elution chamber containing a phosphate solution. In a way analogous to thin layer chromatography (TLC), the phosphate solution acts as an eluent, clearly separating multi-and monophosphorylated peptides. By performing matrix assisted laser desorption-ionization mass spectrometry (MALDI-MS) along the stripe, the detection of all phosphorylated peptides present in the digest is facilitated, as they are isolated from each other. The method was also tested on commercial drinking milk, achieving successful separation between mono-and multiphosphorylated peptides, as well as a detection limit in the femtomol range. As the enrichment, separation and analysis take place in the same substrate, sample handling and risk of contamination and sample loss is minimized. The results obtained suggest that the method, once optimized, may successfully provide with a complete phosphoproteome.

Enrichment Strategies for Identification and Characterization of Phosphoproteome

“…To enhance the enrichment efficiency of TiO 2 resin, the loading buffer was acidified by organic acids (e. g., TFA, acetic acid, and formic acid). 57,58 However, acidification of buffers in phosphopeptide enrichment was not enough to diminish the binding of nonphosphopeptide. 59 In many reports, acidic loading buffers on TiO 2 have been tested and optimized with the modifiers including 2, 5-dihydroxybenzoic acid (DHB), malic acid, lactic acid, and other acidic reagents to enhance the enrichment efficiency of phosphopeptide.…”

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.…”

“…A variety of materials are employed, such as titanium dioxide (TiO 2 ) [12][13][14][15][16], zirconium dioxide (ZrO 2 ) [17,18], niobium oxide (Nb 2 O 5 ) [19] and tantalum oxide (Ta 2 O 5 ) [20]. To further enhance the phosphopeptide binding capacity of MOAC materials, considerable efforts have recently been devoted to explore mesoporous metal oxides with high specific surface area [21][22][23][24][25][26]. 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.…”

“…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.…”