A novel molybdenum disulfide nanosheet loaded Titanium/Zirconium bimetal oxide affinity probe for efficient enrichment of phosphopeptides in A549 cells.

In this paper, we developed a facile route for the preparation of a novel bimetal oxide affinity chromatography (MOAC) material. The TiO/ZrO@MoS was constructed by the electrostatic interaction between titanium oxide/zirconia (w:w, 10:1) and molybdenum disulfide nanosheet. The nanocomposite has the large specific surface area (186.30 m⋅g) and pore volume (0.37 cm⋅g). Compared with single-metal probes, the combination of bimetallic oxides probe (TiO/ZrO) and hydrophilicity MoS support offered multitudinous affinity sites for phosphopeptides capturing from tryptic digests of protein samples under 50% acetonitrile-1% trifluoroacetate conditions. Singnificant feasibility of the TiO/ZrO@MoS nanomaterial for the enrichment of phosphopeptides under optimal conditions was proved via the bovine serum albumin (BSA) and the mixtures of β-casein. The phosphopeptide expression was identified using ultra-performance liquid chromatography (uHPLC) separation and-linear ion trap mass spectrometry (MS). With these affinity characters of TiO/ZrO@MoS, it exhibited higher binding capacity (25 mg⋅g), better selectivity for phosphopeptides from β-casein/BSA (1:2000) tryptic digests, high sensitivity (1 fmol⋅µL) towards phosphopeptides from β-casein tryptic digests, and great reusability of 8 cycles test for capturing phosphopeptides. In addition, the TiO/ZrO@MoS with high sensitivity and selectivity was successfully applied to enriching phosphopeptides from nonfat milk and human serum samples. More importantly, the TiO/ZrO@MoS was further successfully applied to multi-phosphopeptides enrichment, 1779 serine, threonine and tyrosine phosphosites can be identified in A549 cell protein tryptic digest. Compared with commercial TiO from enrichening 416 phosphopeptide from A549 cell lysates, the successful locating of 44 phosphosites were overlapped.