Abstract
We introduce a simplified sample preparation method using bare TiO
2
nanoparticles (NPs) to serve as multifunctional nanoprobes (desalting, accelerating, and affinity probes) for effective enrichment of phosphopeptides from microwave-assisted tryptic digestion of phosphoproteins (α-casein, β-casein and milk) in Electrospray Ionization Mass Spectrometry (ESI-MS) and Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS). The results demonstrate that TiO
2
NPs can effectively enrich and accelerate the digestion reactions of phosphoproteins in aqueous solutions and also from complex real samples. After the microwave experiments, we directly injected the resulting solutions into the ESI-MS and MALDI-MS systems for analysis, and excellent sensitivity was achieved without the need for any washing procedure or separation process. The reasons are attributed to the high binding affinity and selectivity of TiO
2
NPs toward phosphopeptides. Thus, phosphopeptides can be adsorbed onto the TiO
2
NP surface. The digested or partially digested phosphoproteins can be concentrated onto the TiO
2
NP surface. This results in the effective or complete digestion of phosphoproteins in a short period of time (45 s). In addition, high sensitivity and sequence coverage of phosphopeptide can be obtained using TiO
2
NPs as microwave absorbers and affinity probes in MALDI-MS and ESI-MS. This is due to the photocatalytic nature of the TiO
2
NPs because the absorption of microwave radiation that can accelerate the activation of trypsin for efficient digestion of phosphoproteins and enhances the ionization of phosphopeptides. The lowest concentrations detected for ESI-MS and MALDI-MS were 0.1 µM and 10 fmol, respectively, for α-casein. Comparing the two-step approach of TiO
2
NPs with microscale TiO
2
particles, the microscale TiO
2
particles shows no effect on the microwave-assisted tryptic digestion of phosphoproteins. The current approach offers multiple advantages, such as great simplicity, high sensitivity and selectivity, straightforward and separation/washing-free technique for phosphorpeptide enrichment analysis.