Study of Preparation of Open Tubular Capillary Column (OTCC) Coated with Zirconium Phosphonate to Isolate Phosphopeptides

A new approach that combines the open tubular capillary column coated with zirconium phosphonate (ZrP-OTCC) and matrix-assisted laser dissociated ionization time of flight mass spectrometry (MALDI TOF MS) for selective enrichment and identification of phosphopeptides from complex samples were described . A standard phosphoprotein α-casein was chosen as the model and sample, and MALDI TOF MS was used to demonstrate the performace of the ZrP-OTCC. The optimal conditions of the ZrP-OTCC, such as interior diameter, length of the silica-fused capillary and flow rate et al, were discussed for preparation and application of the ZrP-OTCC. In addition, urea, SDS, and sodium chloride which often decrease the MS sensitivity were added into the tryptic digest of α-casein to test the compatible capacity of the ZrP-OTCC, respectively. By using the synthetical phosphopeptide (FLpTEYVATR) as a model sample, the loading capacity of the ZrP-OTCC (150 cm×50 μm) was 32.52 pmol averagely and the recovery is 78.1% by iTRAQ reagent labeling and MS. Repeated experiments of enrichment for phosphopeptides from an α-casein digest for 20 times indicate that the approach has a good reproducibility. The selectivity and sensitivity of the capturing method were also evaluated by the tryptic digest mixture of α-casein and bovine serum albumin at molar ratio of 1:1, 1:10 and 1:100, respectively, and the results show that the ZrP-OTCC can efficiently trap the phosphopeptides even when the concentration is as low as 10^－8 mol•L^－1. Most importantly, the approach was successfully used to realize automated online enrichment for phosphopeptides from the tryptic digest of 10^－6 mol•L^－1 proteins in bovine milk as a complex real world sample, making this method be promise for use in future studies of phosphoproteomes on large scale.