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

A simple method for on-target enrichment and subsequent sepn. and anal. 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 TiO2 sintered onto a conductive glass surface. After washing with a salicylic buffer to remove the nonphosphorylated peptides, the stripe is placed in an elution chamber contg. a phosphate soln. In a way analogous to thin layer chromatog. (TLC), the phosphate soln. acts as an eluent, clearly sepg. 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 com. drinking milk, achieving successful sepn. between multi- and monophosphorylated peptides, as well as a detection limit in the femtomole range. As the enrichment, sepn., and anal. 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 a complete phosphoproteome.