Study Of Preparation Of Multi-layer Open Tubular Capillary Column Coated With Zirconium Phosphonate(ZrP-MOTCC) For Enriching Phosphopeptides

Reversible protein phosphorylation is one of the most important post-translational modifications in mammalian. It regulates many important biological processes, including cell proliferation, differentiation, metabolism, communication, and signal transduction. In eucaryotic cells, near 25-30% protein will experience phosphorylation in their life cycle. So, research of protein phosphorylation is a hot spot nowadays. Enrichment of phosphoproteins and phosphopeptides is a very critical step for MS analysis because of low abundance of phosphoprotein in cells and the suppressive effect from the nonphosphopeptides in protein digestion.The detection of phosphopeptides has been improved by many enrichment approaches that reduce sample complexity and increase the relative concentration of phosphopeptides, but there are still needs for developing novel methods and techniques for large scale phosphoproteome analysis. In this study, a novel approach of enrichment of phosphopeptides has been developed prior to phosphoprotein analysis by mass spectrometry.A new approach for preparation of multi-layer open tubular capillary column coated with zirconium phosphonate(ZrP-MOTCC) for isolating phosphopeptides is described in this paper. These steps of preparation of ZrP-MOTCC include activation of silicon hydroxyl on inner surface of fused-silica capillary by hydrochloric acid and then sodium hydroxide; derivation by using 3-aminopropyl-triethoxysilane, methyl methacrylate,ethanediamine and then POCl3 and finally reaction of the inner surface with ZrOCl2 by incubating in its solution to form the zirconium phosphonate. The factors that affect the performance of ZrP-MOTCC were optimized for the enrichment of phosphopeptides. The results indicate that when the length of open tubular capillary columns is 50 cm, the flow rate is between 0.3 and 0.5, all of them perform well, and under the optimal flow rate, open tubular capillary columns with different internal diameter all perform well , too. In addition, in order to mimic real sample preparation, ammonium chloride was chosen and added different aliquots to the tryptic digest ofα-casein to test the compatibility of ZrP-MOTCC with this kind of reagent. The synthetical phosphopeptide (FLpTEYVATR) was also used to combine with external calibration method to evaluate the loading capacity and recovery of the MOTCC (50 cm×50μm). The loading capacity is measured to averagely be 48 pmol and the recovery is 94.1%. Moreover, the selectivity and limit of detection of the capture method were tested by the tryptic digest ofα-casein and bovine serum albumin mixture with molar ratio of 1:1,1:10,1:100 and 1:1000, respectively, and the results show that the ZrP-MOTCC can still trap the phosphopeptides when the concentration ofα-casein is down to the level of 10-9M.At the optimization conditions, the standard phosphoproteinα-casein was employed as a model protein to test the enrichment performance of the ZrP-MOTCC. The phosphorylation sites on phosphopeptides enriched fromα-casein digest were identified up to 100% compared with the theoretic phosphorylation sites.For further test of the performance of the enrichment method, the approach was used to successfully capture phosphopeptides from the tryptic digest of mouse liver proteins as a real sample. The results indicate that the ZrP-MOTCC can be effectively used to enrich the phosphopeptides from a complex sample.