Sialylation of glycoproteins is vital for the function or physicochemical properties of a protein. It becomes more and more important to develop approaches that can be used to efficiently isolate and identify sialylat...Sialylation of glycoproteins is vital for the function or physicochemical properties of a protein. It becomes more and more important to develop approaches that can be used to efficiently isolate and identify sialylated glycopep- tides or glycoproteins for monitoring changes in glycoproteome. In the present study, we analyze intact structures of the enriched sialylated glycopeptides of bovine fetuin by matrix-assisted laser desorption/ionization-tandem mass spectrometry （MALDI-MS/MS）, without any chemical derivation. The experimental data show that the optimal loading buffer for TiO2 as matrix is 80% acetonitrile/2% TFA （trifluoroacetic acid）/100 mg/mL DHB （2,5-dihydroxybenzoic acid） which is also compatible with MALDI-mass spectrometric analysis. This study indi- cates that the improved enrichment approach combined with MALDI-MS/MS may be a powerful tool to analyze intact structures and components of the sialylated glycopeptides from complex peptide mixture.展开更多
Sialic acids as terminal entities of larger glycans linked to proteins and lipids are involved in multiple different pathological and physiological processes.Structural characterisation of sialoglycoconjugates is requ...Sialic acids as terminal entities of larger glycans linked to proteins and lipids are involved in multiple different pathological and physiological processes.Structural characterisation of sialoglycoconjugates is required to understand their biological function.However,a comprehensive sialylation analysis of sialoglycoconjugates has remained challenges.In this study,we employ a natural biomaterial,poplar catkin derived from white poplar tree（Populus tomentosa Carr.）,to develop a novel capturing microtip for selective and efficient enrichment of sialoglycopeptides,without losses of sialic acid residues and water molecules from sialoglycopeptides.Scanning electron microscopy and Fourier-transform infrared spectroscopy analysis,along with Maule and Wiesner staining assays,indicated that the main components on the outer layer of the poplar catkin are syringyl and guaiacyl lignins which play a key role in enriching sialoglycopeptides from complex peptide mixture.展开更多
Traditionally, herbal medicine is consumed by drinking decoctions produced by boiling herbs with water. The functional components of the decoction are heat stable. Small RNAs(sRNAs) were reported as a new class of fun...Traditionally, herbal medicine is consumed by drinking decoctions produced by boiling herbs with water. The functional components of the decoction are heat stable. Small RNAs(sRNAs) were reported as a new class of functional components in decoctions. However, the mechanisms by which sRNAs survive heat treatment of the decoction and enter cells are unclear.Previous studies showed that plant-derived exosome-like nanoparticles(ELNs), which we call botanosomes, could deliver therapeutic reagents in vivo. Here, we report that heat-stable decoctosomes(ELNs) from decoctions have more therapeutic effects than the decoctions in vitro and demonstrate therapeutic efficacy in vivo. Furthermore, sRNAs, such as HJT-sRNA-m7 and PGY-sRNA-6, in the decoctosome exhibit potent anti-fibrosis and anti-inflammatory effects, respectively. Decoctosome is comprised of lipids, chemical compounds, proteins, and s RNAs. A medical decoctosome mimic is called bencaosome. A single lipid sphinganine(d22:0) identified in the decoctosome was mixed and heated with the synthesized sRNAs to form the simplest bencaosome. This simple bencaosome structure was identified by critical micelle concentration(cmc) assay that sRNAs coassembled with sphinganine(d22:0) to form the lipid layers of vesicles. The heating process facilitates co-assembly of sRNAs and sphinganine(d22:0) until a steady state is reached. The artificially produced sphinganine-HJT-sRNA-m7 and sphinganinePGY-sRNA-6 bencaosomes could ameliorate bleomycin-induced lung fibrosis and poly(I:C)-induced lung inflammation, respectively, following oral administration in mice. Our study not only demonstrates that the herbal decoctosome may represent a combinatory remedy in precision medicine but also provides an effective oral delivery route for nucleic acid therapy.展开更多
Following the published article,we noticed an error duplication in Figure 5G“control”and“PGY-6”that was introduced during the revised process,with an attempt to replace it with higher-resolution images.Here we pro...Following the published article,we noticed an error duplication in Figure 5G“control”and“PGY-6”that was introduced during the revised process,with an attempt to replace it with higher-resolution images.Here we provide the original data in the first submitted manuscript(Figure 5G).展开更多
To develop an economical and feasible approach to probe protein complexes, differential centrifugation and three-dimensional polyacrylamide gel electrophoresis （PAGE） were performed to separate protein complexes fro...To develop an economical and feasible approach to probe protein complexes, differential centrifugation and three-dimensional polyacrylamide gel electrophoresis （PAGE） were performed to separate protein complexes from the cell lysate of human pancreatic cancer cell line, SW1990, followed by mass spectrometric identification. Four macromolecular protein complexes were separated and identified unambiguously.展开更多
Background Human papillomaviruses （HPVs） can infect squamous or mucosal epithelia and cause cervical cancer or genital warts. Coinfection with multiple HPV types is a common finding of many epidemiological studies. ...Background Human papillomaviruses （HPVs） can infect squamous or mucosal epithelia and cause cervical cancer or genital warts. Coinfection with multiple HPV types is a common finding of many epidemiological studies. Therefore, it is necessary to develop a vaccine, which can eradicate established HPV infections and prevent other HPV infections. In this study, we generated chimeric virus like particles （cVLPs） composed of HPV-6b L1, HPV-6b L2 and one artificial HPV-16 mE7 proteins. Methods The artificial HPV-16 mE7 gene was designed by codon modification, point mutation and gene shuffling then chemically synthesized and subcloned behind HPV-6b L2. HPV-6b L1 and L2-mE7 were expressed in insect cells by using Bac-to-Bac system. The generated cVLPs were purified by CsCI gradient ultracentrifuge and analyzed by immunoblot, electron microscope and haemagglutination assay. Results The HPV-6b L1 and L2-mE7 proteins were well expressed in insect cells and could selfassemble into cVLPs, whose diameter was about 55 nm and similar to that of HPV-6b L1/L2 VLPs. Intact cVLPs could be recognized by H6.M48 neutralizing monoclonal antibody and HPV-6b L2 polyclonal antibody, while the denatured cVLPs, but not the intact cVLPs, were reactive to HPV-16 E7 polyclonal antibody. HPV-6b LI/L2-mE7 cVLPs haemaggiutinated mouse erythrocytes as efficiently as HPV-6b L1/L2 VLPs did. Conclusions The insertion of the 158 amino acid HPV-16 mE7 protein behind L2 did not disrupt the correct assembling of cVLPs. The morphological characteristics and haemagglutinating activity of cVLPs were similar to those of HPV-6b LI/L2 VLPs. The cVLPs retained conformational B cell epitopes of HPV-6 VLPs and HPV-16 mE7 protein had an internal location in the cVLPs. Therefore, large modified E7 protein with higher immunogenicity could be incorporated into cVLPs by fusing to the C-terminus of L2, which would help to improve the therapeutic effects of LI/L2-E7 cVLPs.展开更多
基金Project supported by National High-Technology Research and Development Program of China （No. 2006AA02Z154） and the National Natural Science Foundation of China （No. 21075137）.
文摘Sialylation of glycoproteins is vital for the function or physicochemical properties of a protein. It becomes more and more important to develop approaches that can be used to efficiently isolate and identify sialylated glycopep- tides or glycoproteins for monitoring changes in glycoproteome. In the present study, we analyze intact structures of the enriched sialylated glycopeptides of bovine fetuin by matrix-assisted laser desorption/ionization-tandem mass spectrometry （MALDI-MS/MS）, without any chemical derivation. The experimental data show that the optimal loading buffer for TiO2 as matrix is 80% acetonitrile/2% TFA （trifluoroacetic acid）/100 mg/mL DHB （2,5-dihydroxybenzoic acid） which is also compatible with MALDI-mass spectrometric analysis. This study indi- cates that the improved enrichment approach combined with MALDI-MS/MS may be a powerful tool to analyze intact structures and components of the sialylated glycopeptides from complex peptide mixture.
基金supported by the National Natural Science Foundation of China(No.21575164)to Z.L
文摘Sialic acids as terminal entities of larger glycans linked to proteins and lipids are involved in multiple different pathological and physiological processes.Structural characterisation of sialoglycoconjugates is required to understand their biological function.However,a comprehensive sialylation analysis of sialoglycoconjugates has remained challenges.In this study,we employ a natural biomaterial,poplar catkin derived from white poplar tree（Populus tomentosa Carr.）,to develop a novel capturing microtip for selective and efficient enrichment of sialoglycopeptides,without losses of sialic acid residues and water molecules from sialoglycopeptides.Scanning electron microscopy and Fourier-transform infrared spectroscopy analysis,along with Maule and Wiesner staining assays,indicated that the main components on the outer layer of the poplar catkin are syringyl and guaiacyl lignins which play a key role in enriching sialoglycopeptides from complex peptide mixture.
基金supported by the National Natural Science Foundation of China (81788101)the Ministry of Science and Technology of China (2015CB553406)+1 种基金the National Natural Science Foundation of China (81490531)the CAMS Innovation Fund for Medical Sciences (2017-I2M-1-009)
文摘Traditionally, herbal medicine is consumed by drinking decoctions produced by boiling herbs with water. The functional components of the decoction are heat stable. Small RNAs(sRNAs) were reported as a new class of functional components in decoctions. However, the mechanisms by which sRNAs survive heat treatment of the decoction and enter cells are unclear.Previous studies showed that plant-derived exosome-like nanoparticles(ELNs), which we call botanosomes, could deliver therapeutic reagents in vivo. Here, we report that heat-stable decoctosomes(ELNs) from decoctions have more therapeutic effects than the decoctions in vitro and demonstrate therapeutic efficacy in vivo. Furthermore, sRNAs, such as HJT-sRNA-m7 and PGY-sRNA-6, in the decoctosome exhibit potent anti-fibrosis and anti-inflammatory effects, respectively. Decoctosome is comprised of lipids, chemical compounds, proteins, and s RNAs. A medical decoctosome mimic is called bencaosome. A single lipid sphinganine(d22:0) identified in the decoctosome was mixed and heated with the synthesized sRNAs to form the simplest bencaosome. This simple bencaosome structure was identified by critical micelle concentration(cmc) assay that sRNAs coassembled with sphinganine(d22:0) to form the lipid layers of vesicles. The heating process facilitates co-assembly of sRNAs and sphinganine(d22:0) until a steady state is reached. The artificially produced sphinganine-HJT-sRNA-m7 and sphinganinePGY-sRNA-6 bencaosomes could ameliorate bleomycin-induced lung fibrosis and poly(I:C)-induced lung inflammation, respectively, following oral administration in mice. Our study not only demonstrates that the herbal decoctosome may represent a combinatory remedy in precision medicine but also provides an effective oral delivery route for nucleic acid therapy.
文摘Following the published article,we noticed an error duplication in Figure 5G“control”and“PGY-6”that was introduced during the revised process,with an attempt to replace it with higher-resolution images.Here we provide the original data in the first submitted manuscript(Figure 5G).
基金Project supported by the National High-Tech R＆D Program of China （863 Program, No. 2006AA02Z154） and the National Natural Science Foundation of China （No. 21075 137）.
文摘To develop an economical and feasible approach to probe protein complexes, differential centrifugation and three-dimensional polyacrylamide gel electrophoresis （PAGE） were performed to separate protein complexes from the cell lysate of human pancreatic cancer cell line, SW1990, followed by mass spectrometric identification. Four macromolecular protein complexes were separated and identified unambiguously.
基金This work was supported by the Key Program of China International Science and Technology Cooperation(No.2005DFA30070)National Natural Sciences Foundation of China(No.30271355)
文摘Background Human papillomaviruses （HPVs） can infect squamous or mucosal epithelia and cause cervical cancer or genital warts. Coinfection with multiple HPV types is a common finding of many epidemiological studies. Therefore, it is necessary to develop a vaccine, which can eradicate established HPV infections and prevent other HPV infections. In this study, we generated chimeric virus like particles （cVLPs） composed of HPV-6b L1, HPV-6b L2 and one artificial HPV-16 mE7 proteins. Methods The artificial HPV-16 mE7 gene was designed by codon modification, point mutation and gene shuffling then chemically synthesized and subcloned behind HPV-6b L2. HPV-6b L1 and L2-mE7 were expressed in insect cells by using Bac-to-Bac system. The generated cVLPs were purified by CsCI gradient ultracentrifuge and analyzed by immunoblot, electron microscope and haemagglutination assay. Results The HPV-6b L1 and L2-mE7 proteins were well expressed in insect cells and could selfassemble into cVLPs, whose diameter was about 55 nm and similar to that of HPV-6b L1/L2 VLPs. Intact cVLPs could be recognized by H6.M48 neutralizing monoclonal antibody and HPV-6b L2 polyclonal antibody, while the denatured cVLPs, but not the intact cVLPs, were reactive to HPV-16 E7 polyclonal antibody. HPV-6b LI/L2-mE7 cVLPs haemaggiutinated mouse erythrocytes as efficiently as HPV-6b L1/L2 VLPs did. Conclusions The insertion of the 158 amino acid HPV-16 mE7 protein behind L2 did not disrupt the correct assembling of cVLPs. The morphological characteristics and haemagglutinating activity of cVLPs were similar to those of HPV-6b LI/L2 VLPs. The cVLPs retained conformational B cell epitopes of HPV-6 VLPs and HPV-16 mE7 protein had an internal location in the cVLPs. Therefore, large modified E7 protein with higher immunogenicity could be incorporated into cVLPs by fusing to the C-terminus of L2, which would help to improve the therapeutic effects of LI/L2-E7 cVLPs.
