A new ion acceleration method, namely, phase-stable acceleration, using circularly-polarized laser pulses is proposed. When the initial target density n(0) and thickness D satisfy a(L) approximately (n(0)/n(c))D/lambda(L) and D>l(s) with a(L), lambda(L), l(s), and n(c) the normalized laser amplitude, the laser wavelength in vacuum, the plasma skin depth, and the critical density of the incident laser pulse, respectively, a quasiequilibrium for the electrons is established by the light pressure and the space charge electrostatic field at the interacting front of the laser pulse. The ions within the skin depth of the laser pulse are synchronously accelerated and bunched by the electrostatic field, and thereby a high-intensity monoenergetic proton beam can be generated. The proton dynamics is investigated analytically and the results are verified by one- and two-dimensional particle-in-cell simulations.
Glycosylation analysis based on mass spectrometry (MS) of glycopeptides requires the isolation of glycopeptides from complex glycoprotein digests to facilitate structural determination of the glycopeptides. To this end, hydrophilic interaction chromatography (HILIC)-based methods have been developed to selectively enrich glycopeptides by utilizing the hydrophilicity of the glycans. However, the application of these methods is limited by the medium selectivity of HILIC matrices. To improve the effectiveness of HILIC-based methods, we introduced a customized hydrophilic matrix named "click maltose" and characterized its selectivity and glycosylation heterogeneity coverage. In the selectivity assessment, the non-glycopeptides causing ion suppression to the glycopeptides were effectively removed by click maltose, leading to the identification of 27 glycopeptides in the fractions enriched from human serum immunoglobulin G digest, compared to 13 glycopeptides enriched using Sepharose CL-6B, a commercially available matrix. For the assessment of glycosylation heterogeneity coverage, more than 140 glycopeptides covering all the five glycosites of human serum alpha(1)-acid glycoprotein were captured using click maltose. Click maltose was synthesized by linking alkynyl-derivatized maltose to azide-derivatized silica through click chemistry. The resulting flexible saccharide chain structure remarkably enhances the hydrogen-bonding interactions between the glycans of the glycopeptides and the matrix, which are responsible for the increased selectivity and glycosylation heterogeneity coverage of click maltose.
Bonded mono-, di- and oligosaccharides were developed as novel separation materials for HILIC via click chemistry and proven to have excellent chromatographic properties for separation of polar compounds.
A novel zwitterionic stationary phase with high hydrophilicity was facilely synthesized based on the "thiol-ene" click reaction between cysteine and vinyl silica, which exhibited great potential in the separation of oligosaccharides, peptides and basic compounds, as well as in the enrichment of glycopeptides.
Human milk glycans provide a broad range of carbon sources for gut microbes in infants. Levels of protein glycosylation in human milk vary during lactation and may also be affected by the stages of gestation and lactation and by the secretor status of the mother. This was the first study to evaluate systematically dynamic changes in human milk oligosaccharides and fucosylated N-glycans in the milk of Chinese mothers with different secretor statuses during 6 months of lactation. Given the unique single nucleotide polymorphism site (rs1047781, A385T) on the fucosyltransferase 2 gene among Chinese populations, our report provides a specific insight into the milk glycobiome of Chinese mothers, which may exert effects on the gut microbiota of infants that differ from findings from other study cohorts.
Sample handling procedures including protein digestion, glycopeptide enrichment, and deglycosylation have significant impact on the performance of glycoproteome analysis. Several glycoproteomic analysis systems were developed to integrate some of these sample preparation procedures. However, no microsystem integrates all of above three procedures together. In this work, we developed a glycoproteomic microreactor enabling seamless integration of all these procedures. In this reactor, trypsin digestion was accelerated by adding acetonitrile to 80%, and after acidification of protein digest by trifluoroacetic acid (TFA), the following hydrophilic interaction chromatography (HILIC) enrichment and deglycosylation were sequentially performed without any desalting, lyophilization, or buffer exchange steps. The total processing time could be as short as 1.5 h. The detection limit of human IgG as low as 30 fmol was also achieved. When applied to human serum glycoproteome analysis, a total number of 92, 178, and 221 unique N-glycosylation sites were identified from three replicate analyses of 10 nL, 100 nL, and 1 μL of human serum, respectively. It was demonstrated that the glycoproteomic microreactor based method had very high sensitivity and was well suited for glycoproteome analysis of minute protein samples.
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