Shun Feng

Immobilized Zirconium Ion Affinity Chromatography for Specific Enrichment of Phosphopeptides in Phosphoproteome Analysis

Large scale characterization of phosphoproteins requires highly specific methods for purification of phosphopeptides because of the low abundance of phosphoproteins and substoichiometry of phosphorylation. Enrichment of phosphopeptides from complex peptide mixtures by IMAC is a popular way to perform phosphoproteome analysis. However, conventional IMAC adsorbents with iminodiacetic acid as the chelating group to immobilize Fe3+ lack enough specificity for efficient phosphoproteome analysis. Here we report a novel IMAC adsorbent through Zr4+ chelation to the phosphonate-modified poly(glycidyl methacrylate-co-ethylene dimethacrylate) polymer beads. The high specificity of Zr4+-IMAC adsorbent was demonstrated by effectively enriching phosphopeptides from the digest mixture of phosphoprotein (α- or β-casein) and bovine serum albumin with molar ratio at 1:100. Zr4+-IMAC adsorbent was also successfully applied for the analysis of mouse liver phosphoproteome, resulting in the identification of 153 phosphopeptides (163 phosphorylation sites) from 133 proteins in mouse liver lysate. Significantly more phosphopeptides were identified than by the conventional Fe3+-IMAC approach, indicating the excellent performance of the Zr4+-IMAC approach. The high specificity of Zr4+-IMAC adsorbent was found to mainly result from the strong interaction between chelating Zr4+ and phosphate group on phosphopeptides. Enrichment of phosphopeptides by Zr4+-IMAC provides a powerful approach for large scale phosphoproteome analysis.

One-pot synthesis of magnetic colloidal nanocrystal clusters coated with chitosan for selective enrichment of glycopeptides

Selective enrichment of glycopeptides prior to the mass spectrometry (MS) analysis is essential due to ion suppression effect during ionization caused by the co-presence of non-glycosylated peptides. Among the enrichment approaches, hydrophilic interaction liquid chromatography (HILIC) based on magnetic separation has become a popular method in recent years. As the conventional synthesis procedures of these materials are tedious and time-consuming with at least four steps. Herein, magnetic colloidal nanocrystal clusters coated with chitosan (Fe3O4@CS MCNCs) have been successfully prepared by a simple one-pot method. The resulting Fe3O4@CS MCNCs demonstrated an excellent ability for glycopeptide enrichment with high selectivity, low detection limit and high binding capacity. Furthermore, in the analysis of real complicated biological sample, 283 unique N-glycosylation sites corresponding to 175 glycosylated proteins were identified in three replicate analyses of 45μg protein sample extracted from HeLa cells, indicating the great potential in detection and identification of low abundant glycopeptides in glycoproteome analysis.

Automatic Validation of Phosphopeptide Identifications by the MS2/MS3 Target-Decoy Search Strategy

Manual checking is commonly employed to validate the phosphopeptide identifications from database searching of tandem mass spectra. It is very time-consuming and labor intensive as the number of phosphopeptide identifications increases greatly. In this study, a simple automatic validation approach was developed for phosphopeptide identification by combining consecutive stage mass spectrometry data and the target-decoy database searching strategy. Only phosphopeptides identified from both MS2 and its corresponding MS3 were accepted for further filtering, which greatly improved the reliability in phosphopeptide identification. Before database searching, the spectra were validated for charge state and neutral loss peak intensity, and then the invalid MS2/MS3 spectra were removed, which greatly reduced the database searching time. It was found that the sensitivity was significantly improved in MS2/MS3 strategy as the number of identified phosphopeptides was 2.5 times that obtained by the conventional filter-based MS2 approach. Because of the use of the target-decoy database, the false-discovery rate (FDR) of the identified phosphopeptides could be easily determined, and it was demonstrated that the determined FDR can precisely reflect the actual FDR without any manual validation stage.

Automation of nanoflow liquid chromatography-tandem mass spectrometry for proteome analysis by using a strong cation exchange trap column.

An approach was developed to automate sample introduction for nanoflow LC‐MS/MS (μLC‐MS/MS) analysis using a strong cation exchange (SCX) trap column. The system consisted of a 100 μm id×2 cm SCX trap column and a 75 μm id×12 cm C18 RP analytical column. During the sample loading step, the flow passing through the SCX trap column was directed to waste for loading a large volume of sample at high flow rate. Then the peptides bound on the SCX trap column were eluted onto the RP analytical column by a high salt buffer followed by RP chromatographic separation of the peptides at nanoliter flow rate. It was observed that higher performance of separation could be achieved with the system using SCX trap column than with the system using C18 trap column. The high proteomic coverage using this approach was demonstrated in the analysis of tryptic digest of BSA and yeast cell lysate. In addition, this system was also applied to two‐dimensional separation of tryptic digest of human hepatocellular carcinoma cell line SMMC‐7721 for large scale proteome analysis. This system was fully automated and required minimum changes on current μLC‐MS/MS system. This system represented a promising platform for routine proteome analysis.

Automation of nanoflow liquid chromatography-tandem mass spectrometry for proteome and peptide profiling analysis by using a monolithic analytical capillary column

An automated nano‐LC‐MS/MS platform without trap column was established, which only used a 20 cm lauryl methacrylate–ethylene dimethacrylate (LMA–EDMA) monolithic capillary column to allow preconcentration and separation of peptides. The monolithic column had the advantages of good permeability and low backpressure resulting in higher flow rates for capillary columns. Tryptic digests of bovine albumin and yeast protein extract were tested using the monolithic column system. High proteomic coverage using this approach were demonstrated in this study. Furthermore, peptide samples extracted from mouse liver were separated by using the monolithic column system combined with size‐exclusion chromatography prefractionation. This monolithic column system might be a promising alternative for the automated system previously using a trap column for routine proteome and peptide profiling analysis.