Sample Preparation for Phosphoproteome and Peptidome Analysis by Capillary Liquid Chromatography Tandem Mass Spectrometry

Phosphorylation is one of the most important post-translational modifications of proteins, which modulates a wide range of biological functions and activity of proteins. A novel phosphopeptide enrichment approach based on the strong interaction of zirconium phosphonate (ZrP) modified surface with phosphopeptides has been developed. ZrP modified porous silicon (ZrP-pSi) wafer was prepared to specifically capture the phosphopeptides from complex peptide mixtures and then the captured phosphopeptides were analyzed by MALDI-TOF MS by directly placing the wafer on a MALDI target. The excellent selectivity of this approach was demonstrated by analyzing phosphopeptides in the digest mixture of β-casein and bovine serum albumin. Based on this finding, an IMAC adsorbent through Zr4+ chelating to the phosphophate modified GMA-EDMA polymer beads was prepared for the analysis of mouse liver phosphoproteome, which resulted in the identification of 194 phosphopeptides (205 phosphorylation sites) from 170 proteins in mouse liver lysate. As the serum peptidome gets increasing attention for biomarker discovery, one of the important issues is how to efficiently extract the peptides from highly complex human serum for automated peptidome analysis. Silica particles with highly ordered mesostructures (e.g. MC41s and SBA-15) have been widely applied in the fileds of separation and adsorption. The unique properties of these silica materials include high in-pore surface areas, extremely narrow pore size distribution, perfectly adjustable pore size, and the presence of silanol groups and siloxane bridges, which make them effective for selective adsorption of standard proteins with different MW based on the size-exclusion mechanism.highly ordered mesoporous silica particles were successfully applied for selective enrichment of the peptides in human plasma. The MCM-41 with pore size of 20.5 Å was proved to be effective for enriching peptides in human plasma with a wide MW range from 1 k to 12 k Da, but repelling most of other plasma proteins outside. The unique pore structures of this material make it superior for peptide enrichment to both adsorbents and ultrafiltration based methods. Fully automated on-line extraction system was developed by coupling a capillary trap column packed with restricted access material of strong cation exchange functionality (RAM/SCX) with the nano-liquid chromatography mass spectrometry (nanoLC-MS). The RAM/SCX capillary trap column can selectively extract the peptides from human serum and the trapped peptides can eluted to C18 packed capillary column for nanoLC-MS/MS. Totally more than 400 peptides can be identified with one-dimensional separation by injection of only 2 µL serum sample with the average relative standard deviation (RSD) for retention time of 0.5%. In addition, due to the high orthogonality of the RAM/SCX trap column and the C18 analytical column, multidimensional separation can also easily be conducted and 1286 peptides were identified by injection of only 20 µL serum sample with false positive rates of below 3%