Study Of Preparation Of Open Tubular Capillary Column (OTCC) Coated With Zirconium Phosphonate To Isolate 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. Global analysis of protein phosphorylation in organism is very important for exploring these critical processes. 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 approache of enrichment of phosphopeptides has been developed prior to phosphoprotein analysis by mass spectrometry.A new approach for preparation of open tubular capillary column (OTCC) coated with zirconium phosphonate for isolating phosphopeptides is described in this paper. These steps of preparation of OTCC 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 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 OTCC, such as internal diameter, capillary length and flow rate, were optimized for enrichment by the Orthogonal Test. The results indicated that the optimal parameters are 50μm of internal diameter, 150cm of capillary length and 0.3μL/min of flow rate. In addition, in order to mimic real sample preparation, urea, SDS and sodium chloride was chosen and added respectively to different aliquots of the tryptic digest ofα-casein to test the compatibility of OTCC with these reagents. The synthetical phosphopeptide (FLpTEYVATR) was also used to combine with iTRAQ reagents to evaluate the loading capacity and recovery of the OTCC(150cm×50μm). The loading capacity is averagely measured to be 32.52 pmol and the recovery is 78.1%. 20 repeated experiments of enrichment of phosphopeptide aliquots from anα-casein digest were carried out for examinateing the reproducibility of the use of OTCC. Moreover, the selectivity and limit of detection of the capture method were tested by the tryptic digest mixture ofα-casein and bovine serum albumin with molar ratio of 1:1,1:10 and 1:100, and the results show that the OTCC can still trap the phosphopeptides when the concentration is down to the level of 10-8M.At the optimization conditions, the standard phosphoproteinsα-casein andβ-casein were employed as model proteins to test the enrichment performance of the OTCC. The phosphorylation sites on phosphopeptides enriched fromα-casein were identified up to 90.9% of the theoretic phosphorylation sites and 100% forβ-casein.For further test of the enrichment method, the approach was used to successfully capture phosphopeptides from the tryptic digest of bovine milk at 10-6M as a real sample. The results indicated that the OTCC can effectively enrich the phosphopeptides from complex sample and markedly improve the identification efficiency of mass spectrometry.