Xiuqing Xu

Novel Fe3O4@TiO2 Core−Shell Microspheres for Selective Enrichment of Phosphopeptides in Phosphoproteome Analysis

Due to the dynamic nature and low stoichiometry of protein phosphorylation, enrichment of phosphorylated peptides from proteolytic mixtures is often necessary prior to their characterization by mass spectrometry. Immobilized metal affinity chromatography (IMAC) is a popular way to enrich phosphopeptides; however, conventional IMAC lacks enough specificity for efficient phosphoproteome analysis. In this study, novel Fe 3O 4@TiO 2 microspheres with well-defined core-shell structure were prepared and developed for highly specific purification of phosphopeptides from complex peptide mixtures. The enrichment conditions were optimized using tryptic digests of beta-casein, and the high specificity of the Fe 3O 4@TiO 2 core-shell microspheres was demonstrated by effectively enriching phosphopeptides from the digest mixture of alpha-casein and beta-casein, as well as a five-protein mixture containing nonphosphoproteins (bovine serum albumin (BSA), myoglobin, cytochrome c) and phosphoproteins (ovalbumin and beta-casein). The Fe 3O 4@TiO 2 core-shell microspheres were further successfully applied for the nano-LC-MS/MS analysis of rat liver phosphoproteome, which resulted in identification of 56 phosphopeptides (65 phosphorylation sites) in mouse liver lysate in a single run, indicating the excellent performance of the Fe 3O 4@TiO 2 core-shell microspheres.

Facile synthesis of C8-functionalized magnetic silica microspheres for enrichment of low-concentration peptides for direct MALDI-TOF MS analysis

In this study, novel C8‐functionalized magnetic polymer microspheres were prepared by coating single submicron‐sized magnetite particle with silica and subsequent modification with chloro (dimethyl) octylsilane. The resulting C8‐functionalized magnetic silica (C8‐f‐M‐S) microspheres exhibit well‐defined magnetite‐core‐silica‐shell structure and possess high content of magnetite, which endow them with high dispersibility and strong magnetic response. With their magnetic property, the synthesized C8‐f‐M‐S microspheres provide a convenient and efficient way for enrichment of low‐abundance peptides from tryptic protein digest and human serum. The enriched peptides/proteins were subjected for MALDI‐TOF MS analysis and the enrichment efficiency was documented. In a word, the facile synthesis and efficient enrichment process of the novel C8‐f‐M‐S microspheres make them promising candidates for isolation of peptides even in complex biological samples such as serum, plasma, and urine.

Comparison of 2-D LC and 3-D LC with post- and pre-tryptic-digestion SEC fractionation for proteome analysis of normal human liver tissue

The current “shotgun” proteomic analysis, strong cation exchange‐RPLC‐MS/MS system, is a widely used method for proteome research. Currently, it is not suitable for complicated protein sample analysis, like mammal tissues or cells. To increase the protein identification confidence and number, an additional separation dimension for sample fractionation is necessary to be coupled prior to current multi‐dimensional protein identification technology (MudPIT). In this work, SEC was elaborately selected and applied for sample prefractionation in consideration of its non‐bias against sample and variety of choice of mobile phases. The analysis of the global lysate of normal human liver tissue sample provided by the China Human Liver Proteome Project, were performed to compare the proteome coverage, sequence coverage (peptide per protein identification) and protein identification efficiency in MudPIT, 3‐D LC‐MS/MS identification strategy with preproteolytic and postproteolytic fractionation. It was demonstrated that 3‐D LC‐MS/MS utilizing protein level fractionation was the most effective method. A MASCOT search using the MS/MS results acquired by QSTARXL identified 1622 proteins from 3‐D LC‐MS/MS identification approaches. A primary analysis on molecular weight, pI and grand average hydrophobicity value distribution of the identified proteins in different approaches was made to further evaluate the 3‐D LC‐MS/MS analysis strategy.