A Ti4+-immobilized phosphate polymer-patterned silicon substrate for on-plate selective enrichment and self-desalting of phosphopeptides

Abstract

A circular hydrophobic–hydrophilic-Ti⁴⁺ immobilized phosphate polymer is patterned on a silicon wafer. Such a wafer is used as a novel sample support to allow fast selective enrichment, wash-free self-desalting and mass spectroscopy (MS) analysis of phosphopeptides, thanks to the high Ti⁴⁺ loading amount, pure phosphate polymer–Ti⁴⁺ interface, and strong hydrophobic–hydrophilic attraction pattern. The detection sensitivity was enhanced 300 folds compared with what was obtained using the common MALDI plate. Remarkable selectivity for phosphopeptides can be achieved at a molar ratio as low as 1 : 500 of phosphopeptides (casein digest)/nonphosphopeptides (BSA). High-quality mass spectra can be obtained even in the presence of NaCl (1 M), NH₄HCO₃ (100 mM), or urea (1 M). These microspots were also used to selectively capture phosphopeptides from milk and human serum, which further demonstrated that they were capable of identifying low-abundance phosphopeptides from real complex samples. They provide a low detection limit (3 fmol μL⁻¹), small sample size, and excellent enrichment and desalting efficiency. Such a method significantly simplifies the analytical procedures, reduces possible sample loss, and is relatively low cost. Therefore, this on-plate patterned technique is very promising in the high-throughput phosphoproteomic research, especially for the detection of tiny amounts of samples.