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Toward a high resolution 2-DE profile of the normal human liver proteome using ultra-zoom gels

Science China(Life Sciences) 2011年第1期54卷 25-33页

作者： MI Wei LIU Xin JIA Wei LI Lei CAI Yun YING WanTao qian xiaohong Department of Biology Energy and Environment National Institute of Metrology Beijing 100013 China State Key Laboratory of Proteomics Beijing Proteome Research Center Beijing Institute of Radiation Medicine Beijing 102206 China

The human liver is the largest organ in the body and has many important physiological functions. A global analysis of human liver proteins is essential for a better understanding of the molecular basis of the normal functions of the liver and of its diseases. As part of the Human Liver Proteome Project (HLPP), the goal of the present study was to visualize and detect as many proteins as possible in normal human livers using two-dimensional gel electrophoresis (2-DE). We have constructed a reference map of the proteins of human normal liver that can be used for the comprehensive analysis of the human liver proteome and other related research. To improve the resolution and enhance the detection of low abundance proteins, we developed and optimized narrow pH range ultra-zoom 2-DE gels. High resolution patterns of human liver in pH gradients 4.5-5.5, 5-6, 5.5-6.7, 6-9 and 6-11 are presented. To improve the poor resolution in the alkaline pH range of 2-DE gels, we optimized the isoelectric focusing protocol by including sample application using cup loading at the anode and incorporating 1.2% hydroxyethyl disulfide, 15% 2-propanol and 5% glycerol in the rehydration buffer. Using the optimized protocol, we obtained reproducibly better resolution in both analytical and preparative 2-DE gels. Compared with the 2386 and 1878 protein spots resolved in the wide range 3-10 and 4-7 pH gradients respectively, we obtained 5481 protein spots from the multiple (overlapping) narrow pH range ultra-zoom gels in the range of pH 4.5-9. The visualized reference map of normal human liver proteins presented in this paper will be valuable for comparative proteomic research of the liver proteome.

关键词：蛋白质组计划高分辨率模式凝胶电泳双向电泳肝脏人类变焦剖面

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Plasma biomarker screening for liver fibrosis with the N-terminal isotope tagging strategy

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Science China(Life Sciences) 2011年第5期54卷 393-402页

作者： LI ShuLong LIU Xin WEI Lai WANG HuiFen ZHANG JiYang WEI HanDong qian xiaohong JIANG Ying HE FuChu State Key Laboratory of Proteomics Beijing Proteome Research Center Beijing Institute of Radiation Medicine Beijing 102206 China Beijing Institute of Biotechnology Beijing 100071 China People 's Hospital of Peking University Beijing 100044 China 302 Military Hospital of China Beijing 100039 China Institutes of Biomedical Sciences Fudan University Shanghai 200032 China Central Laboratory Yantai Yu-Huang-Ding Hospital Yantai 264000 China

A non-invasive diagnostic approach is crucial for the evaluation of severity of liver disease,treatment decisions,and assessing drug efficacy.This study evaluated plasma proteomic profiling via an N-terminal isotope tagging strategy coupled with liquid chromatography/Fourier transform ion cyclotron resonance mass spectrometry measurement to detect liver fibrosis staging.Pooled plasma from different liver fibrosis stages,which were assessed in advance by the current gold-standard of liver biopsy,was quantitatively analyzed.A total of 72 plasma proteins were found to be dysregulated during the fibrogenesis process,and this finding constituted a valuable candidate plasma biomarker bank for follow-up analysis.Validation results of fibronectin by Western blotting reconfirmed the mass-based data.Ingenuity Pathways Analysis showed four types of metabolic networks for the functional effect of liver fibrosis disease in chronic hepatitis B patients.Consequently,quantitative proteomics via the N-terminal acetyl isotope labeling technique provides an effective and useful tool for screening plasma candidate biomarkers for liver fibrosis.We quantitatively monitored the fibrogenesis process in CHB patients.We discovered many new valuable candidate biomarkers for the diagnosis of liver fibrosis and also partly identified the mechanism involved in liver fibrosis disease.These results provide a clearer understanding of liver fibrosis pathophysiology and will also hopefully lead to improvement of clinical diagnosis and treatment.

关键词：生物标志物同位素标记肝纤维化血浆蛋白筛选慢性乙型肝炎定量蛋白质组学离子回旋共振

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A novel method for identification and relative quantification of N-terminal peptides using metal-element-chelated tags coupled with mass spectrometry

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Science China Chemistry 2014年第5期57卷 708-717页

作者： YAN Hui HAO FeiRan CAO QiChen LI JiaBin LI NanNan TIAN Fang BAI HaiHong REN XiaoJun LI XianYu ZHANG YangJun qian xiaohong Anhui Medical University State Key Laboratory of Proteomics Beijing Proteome Research Center Beijing Institute of Radiation Medicine Affiliated Hospital of Liaoning University of Traditional Chinese Medicine

By means of the reaction between a DOTA-NHS-ester bifunctional reagent and N-terminal peptides of proteins,and then chelation of lanthanide metal ions as tags,we established a novel method for the identification of N-terminal peptides of proteins and their relative quantification using metal-element-chelated tags coupled with mass spectrometry.The experimental results indicate that metal elements are able to completely label N-terminal peptides at the protein level.The N-terminal peptides are enriched as the peptides digested with trypsin are selectively eliminated by isothiocyanate-coupled silica beads.We successfully identified the N-terminal peptides of 158 proteins of Thermoanaerobacter tengcongensis incubated at 55 and 75°C,among which N-terminal peptides of 24 proteins are partially acetylated.Moreover,metal-element tags with high molecule weights make it convenient for N-terminal peptides consisting of less than 6 amino acids to be identified;these make up 55percent of the identified proteins.Finally,we developed a general approach for the relative quantification of proteins based on N-terminal peptides.We adopted lysozyme and ribonuclease B as model proteins;the correlation coefficients(R2)of the standard curves for the quantitative method were 0.9994 and 0.9997,respectively,with each concentration ratio ranging from0.1 to 10 and both relative standard derivations(RSD)measured at less than 5%.In T.tengcongensis at two incubation temperatures,80 proteins possess quantitative information.In addition,compared with the proteins of T.tengcongensis incubated at 55°C,in T.tengcongensis incubated at 75°C,7 proteins upregulate whereas 16 proteins downregulate,and most differential proteins are related to protein synthesis.

关键词： N-末端金属元素相对定量质谱联用识别标记螯合蛋白质水平

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An ultra-fast and highly efficient multiple proteases digestion strategy using graphene-oxide-based immobilized protease reagents

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Science China Chemistry 2014年第5期57卷 695-702页

作者： BAI HaiHong PAN YiTing REN XiaoJun HAO FeiRan DENG ShanShan FAN Chao YAN Hui SHEN BingQuan MA Lin TIAN Fang PENG Bo DENG YuLin QIN WeiJie qian xiaohong School of Life Science and Technology Beijing Institute of Technology National Center for Protein Sciences Beijing State Key Laboratory of Proteomics Beijing Proteome Research CenterBeijing Institute of Radiation Medicine

Highly efficient and rapid proteolytic digestion of proteins into peptides is a crucial step in shotgun-based proteome-analysis strategy.Tandem digestion by two or more proteases is demonstrated to be helpful for increasing digestion efficiency and decreasing missed cleavages,which results in more peptides that are compatible with mass-spectrometry analysis.Compared to conventional solution digestion,immobilized protease digestion has the obvious advantages of short digestion time,no self-proteolysis,and reusability.We proposed a multiple-immobilized proteases-digestion strategy that combines the advantages of the two digestion strategies mentioned above.Graphene-oxide(GO)-based immobilized trypsin and endoproteinase Glu-C were prepared by covalently attaching them onto the GO surface.The prepared GO-trypsin and GO-Glu-C were successfully applied in standard protein digestion and multiple immobilized proteases digestion of total proteins of Thermoanaerobacter tengcongensis.Compared to 12-hour solution digestion using trypsin or Glu-C,14%and 7%improvement were obtained,respectively,in the sequence coverage of BSA by one-minute digestion using GO-trypsin and GO-Glu-C.Multiple immobilized-proteases digestion of the total proteins of Thermoanaerobacter tengcongensis showed 24.3%and 48.7%enhancement in the numbers of identified proteins than was obtained using GO-trypsin or GO-Glu-C alone.The ultra-fast and highly efficient digestion can be contributed to the high loading capacity of protease on GO,which leads to fewer missed cleavages and more complete digestion.As a result,improved protein identification and sequence coverage can be expected.

关键词：固定化蛋白酶消化时间超快速氧化物固定化胰蛋白酶石墨蛋白质组试剂

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Global characterization of modifications to the charge isomers of IgG antibody

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Journal of Pharmaceutical Analysis 2022年第1期12卷 156-163页

作者： Xinling Cui Wei Mi Zhishang Hu Xiaoyu Li Bo Meng Xinyuan Zhao xiaohong qian Tao Zhu Wantao Ying State Key Laboratory of Proteomics Beijing Proteome Research CenterNational Center for Protein Sciences(Beijing)Beijing Institute of LifeomicsBeijingChina National Institute of Metrology Center for Advanced Measurement ScienceBeijingChina National Institute of Metrology Chemical Metrology&Analytical Science DivisionBeijingChina CanSino Bio Corporation Tianjin Key Laboratory of Recombinant Bacterial Recombination and Bacterial Vaccines TianjinChina

Posttranslational modifications of antibody products affect their stability,charge distribution,and drug activity and are thus a critical quality attribute.The comprehensive mapping of antibody modifications and different charge isomers(CIs)is of utmost importance,but is challenging.We intended to quantitatively characterize the posttranslational modification status of CIs of antibody drugs and explore the impact of posttranslational modifications on charge heterogeneity.The CIs of antibodies were fractionated by strong cation exchange chromatography and verified by capillary isoelectric focusing-whole column imaging detection,followed by stepwise structural characterization at three levels.First,the differences between CIs were explored at the intact protein level using a top-down mass spectrometry approach;this showed differences in glycoforms and deamidation status.Second,at the peptide level,common modifications of oxidation,deamidation,and glycosylation were identified.Peptide mapping showed nonuniform deamidation and glycoform distribution among CIs.In total,10 N-glycoforms were detected by peptide mapping.Finally,an in-depth analysis of glycan variants of CIs was performed through the detection of enriched glycopeptides.Qualitative and quantitative analyses demonstrated the dynamics of 24 N-glycoforms.The results revealed that sialic acid modification is a critical factor accounting for charge heterogeneity,which is otherwise missed in peptide mapping and intact molecular weight analyses.This study demonstrated the importance of the comprehensive analyses of antibody CIs and provides a reference method for the quality control of biopharmaceutical analysis.

关键词： Antibody Charge isomers Mass spectrometry Posttranslational modification Glycopeptide

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Partial aging can counter-intuitively couple with sulfidation to improve the reactive durability of zerovalent iron

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Frontiers of Environmental Science & Engineering 2024年第2期18卷 1-11页

作者： Yiwei Liu Kaili Gu Jinhua Zhang Jinxiang Li Jieshu qian Jinyou Shen xiaohong Guan Jiangsu Key Laboratory of Chemical Pollution Control and Resources Reuse School of Environmental and Biological EngineeringNanjing University of Science and TechnologyNanjing 210094China School of Ecological and Environmental Sciences East China Normal UniversityShanghai 200241China

Sulfated zero-valent iron(SZVI)has shown promising applications in wastewater treatment.However,the rapid decline in the reactivity of SZVI with time limits its real practice.To mediate this problem,partial aging was proposed to improve the reactive durability of SZVI.Taking Cr(VI)as the target contaminant,we found that the aged ZVI(AZVI)gradually lost reactivity as aging time increased from 0.5 to 2 d.Counter-intuitively,the partially aged SZVI(ASZVI)showed greater reactivity than SZVI when exposed to oxygenated water for a period ranging from 0.5 to 14 d.In addition,the ASZVI with 0.5 d of aging time(ASZVI-0.5)not only maintained reactivity in successive runs but also increased the Cr(VI)removal capacity from 9.1 mg/g by SZVI to 19.1 mg/g by ASZVI-0.5.Correlation analysis further revealed that the electron transfer from the Fe0 core to the shell was mediated by the conductive FeS and FeS2 in the subshell of ASZVI.Meanwhile,the lepidocrocite and magnetite on the surface of ASZVI facilitated Cr(VI)adsorption and subsequent electron transfer for Cr(VI)reduction.Moreover,the iron(hydr)oxide shell could retain the conductive FeS and FeS2 in the subshell,allowing ASZVI to reduce Cr(VI)efficiently and sustainably.In general,partial aging can enhance the reactive durability of ZVI when coupled with sulfidation and this synergistic effect will be beneficial to the application of SZVI-based technology for wastewater treatment.

关键词： Zerovalent iron Sulfidation Partial aging Interface reconstruction Electron transfer

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Identification of hedgehog signaling as a potential oncogenic driver in an aggressive subclass of human hepatocellular carcinoma: A reanalysis of the TCGA cohort

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Science China(Life Sciences) 2019年第11期62卷 1481-1491页

作者： Yang Zhao Li Zhang Yong Zhang Bo Meng Wantao Ying xiaohong qian College of Life Science and Bioengineering Beijing University of TechnologyBeijing 100124China State Key Laboratory of Proteomics Beijing Proteome Research CenterNational Center for Protein Sciences(Beijing)Beijing Institute of LifeomicsBeijing 102206China Center for Bioinformatics and Computational Biology Institute of Biomedical SciencesSchool of Life SciencesEast China Normal UniversityShanghai 200241China School of Statistics Faculty of Economics and ManagementEast China Normal UniversityShanghai 200241China Key Lab of Transplant Engineering and Immunology West China-Washington Mitochondria and Metabolism Research CenterWest China HospitalSichuan UniversityChengdu 610041China

Hepatocellular carcinoma(HCC)is a heterogeneous disease and the second most common cause of cancer-related death worldwide.Marked developments in genomic technologies helped scientists to understand the heterogeneity of HCC and identified multiple HCC-related molecular subclasses.An integrative analysis of genomic datasets including 196 patients from The Cancer Genome Atlas(TCGA)group has recently reported a new HCC subclass,which contains three subgroups(iCluster1,iCluster2,and iCluster3).However,the transcriptional molecular characteristics underlying the iClusters have not been thoroughly investigated.Herein,we identified a more aggressive subset of HCC patients in the iCluster1,and re-clustered the TCGA samples into novel HCC subclasses referred to as aggressive(Ag),moderate-aggressive(M-Ag),and less-aggressive(L-Ag)subclasses.The Ag subclass had a greater predictive power than the TCGA iCluster1,and a higher level of alpha fetoprotein,microscopic vascular invasion,immune infiltration,isocitrate dehydrogenase 1/2 mutation status,and a worse survival than MAg and L-Ag subclasses.Global transcriptomic analysis showed that activation of hedgehog signaling in the Ag subclass may play key roles in tumor development of aggressive HCC.GLI1,a key transcriptional regulator of hedgehog signaling upregulated in the Ag subclass,was correlated with poor prognosis of HCC,and may be a potential prognostic biomarker and therapeutic target for Ag subclass HCC patients.

关键词： hepatocellular carcinoma The Cancer Genome Atlas molecular subclass Wnt hedgehog

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Electrochromism-induced adaptive fresh air pre-handling system for building energy saving

Energy and Built Environment

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Energy and Built Environment 2024年第2期5卷 300-308页

作者： Dikai Xu Mingfeng Sheng Xinpeng Zhao Hua qian Wenqing Liang xiaohong Zheng Dongliang Zhao School of Energy and Environment Southeast UniversityNanjingJiangsu 210096China Lyles School of Civil Engineering Purdue UniversityWest LafayetteIN 47907USA Department of Mechanical Engineering University of Colorado BoulderBoulderCO 80309USA

Building fresh air supply needs to meet certain regulations and fit people’s ever-growing indoor air quality de-mand.However,fresh air handling requires huge energy consumption that goes against the goal of net-zero energy buildings.Thus,in this work,an adaptive fresh air pre-handling system is designed to reduce the cool-ing and heating loads of HVAC system.The sky-facing surface of the system uses electrochromic mechanism to manipulate the optical properties and thus make full use of solar energy(solar heating)and deep space cold source(radiative cooling)by switching between heating and cooling modes.In the cooling mode,the sky-facing surface shows a transmittance of down to zero,while the reflectance is high at 0.89 on average.In the heating mode,the electrochromic glass is highly transparent,allowing the sunlight to reach the solar heat absorber.To obtain the energy-saving potential under different climates,six cities were selected from various climate regions in China.Results show that the adaptive fresh air pre-handling system can be effective in up to 55.4%time of a year.The maximum energy-saving ratios for medium office,warehouse,and single-family house can reach up to 11.52%,26.62%,and 18.29%,respectively.In addition,the system shows multi-climate adaptability and broad application scenarios,making it a potential solution to building energy saving.

关键词： Adaptive fresh air pre-handling Electrochromism Radiative sky cooling Solar air heating Building energy saving

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A New Reagent for One-Step Derivatization and Enrichment of Protein Post-Translational Modifications and Identification by Mass Spectrometry

A New Reagent for One-Step Derivatization and Enrichment of ...

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2018年中国质谱学术大会（CMSC2018）

作者： Qin Weijie Huo Bianbian Zhang Wanjun Wang Jianhua qian xiaohong National Center for Protein Sciences (Beijing) Beijing Institute of LifeomicsBeijing Proteome Research Center Research Center for Analytical Sciences College of SciencesNortheastern University

Post-translational modifications(PTMs) of proteins play fundamental roles in controlling almost every aspect of protein functions. Global mapping of PTMs is invaluable to understand the cellular and disease mechanisms regulated by various PTMs. Chemoenzymatic or metabolic attachment of an azide handle to the target PTMs provides a powerful way to enrich the PTMs that are substoichiometricly modified on low-abundance proteins before mass spectrometry(MS) analysis. However, localizing the modification site is still challenging, due to the difficulties in releasing the covalently coupled PTM peptides and their inherently poor response in MS. Therefore, we synthesized a new reagent that is capable of both chemical derivatization and reversible enrichment of azide-labeled PTM peptides for sensitive identification and site mapping by MS. The reagent is composed of a triphenylphosphine group for tagging the azide-PTM and a trimethylpiperidine group which has high affinity to an antibody for enrichment and release in mild condition. Furthermore, the hydrophobicity and alkalinity of the reagent improves ionization and fragmentation of the PTM peptides in MS, which is especially beneficial for hydrophilic glycopeptides. This reagent efficiently enriches and identifies O-GlcNAc peptides from one thousand times more abundant non-O-GlcNAc peptides with a low picogram sensitivity. In-depth O-GlcNAcylation coverage of HeLa cell is achieved by the identification of 3293 O-GlcNAc peptides and locating 1706 sites, which efficiently expands the mapping scale of O-GlcNAcylation in cell lines, providing a powerful tool for studying O-GlcNAcylation and other PTMs.

关键词： Post-translational modifications O-GlcNAc Enrichment Mass spectrometry Western-blot

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Establishment of a humanized mouse model using steady-state peripheral blood-derived hematopoietic stem and progenitor cells facilitates screening of cancer-targeted T-cell repertoires

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Cancer Innovation 2024年第3期3卷 1-21页

作者： Yulin Xu Wei Shan qian Luo Meng Zhang Dawei Huo Yijin Chen Honghu Li Yishan Ye xiaohong Yu Yi Luo He Huang Bone Marrow Transplantation Center The First Affiliated HospitalZhejiang University School of MedicineHangzhouChina Liangzhu Laboratory Zhejiang UniversityHangzhouChina Institute of Hematology Zhejiang UniversityHangzhouChina Zhejiang Province Engineering Research Center for Stem Cell and Immunity Therapy HangzhouChina School of Medicine Zhejiang UniversityHangzhouChina

Background:Cancer-targeted T-cell receptor T(TCR-T)cells hold promise in treating cancers such as hematological malignancies and breast cancers.However,approaches to obtain cancer-reactive TCR-T cells have been unsuccessful.Methods:Here,we developed a novel strategy to screen for cancer-targeted TCR-T cells using a special humanized mouse model with person-specific immune fingerprints.Rare steady-state circulating hematopoietic stem and progenitor cells were expanded via three-dimensional culture of steady-state peripheral blood mononuclear cells,and then the expanded cells were applied to establish humanized mice.The human immune system was evaluated according to the kinetics of dendritic cells,monocytes,T-cell subsets,and cytokines.To fully stimulate the immune response and to obtain B-cell precursor NAML-6-and triple-negative breast cancer MDA-MB-231-targeted TCR-T cells,we used the inactivated cells above to treat humanized mice twice a day every 7 days.Then,human T cells were processed for TCRβ-chain(TRB)sequencing analysis.After the repertoires had been constructed,features such as the fraction,diversity,and immune signature were investigated.Results:The results demonstrated an increase in diversity and clonality of T cells after treatment.The preferential usage and features of TRBV,TRBJ,and the V–J combination were also changed.The stress also induced highly clonal Science and Technology,Grant/Award Number:2021C03010;Zhejiang Provincial Natural Science Foundation of China,Grant/Award Numbers:LTGY24H080003,LY21H080004 expansion.Tumor burden and survival analysis demonstrated that stress induction could significantly inhibit the growth of subsequently transfused live tumor cells and prolong the survival of the humanized mice.Conclusions:We constructed a personalized humanized mouse model to screen cancer-targeted TCR-T pools.Our platform provides an effective source of cancer-targeted TCR-T cells and allows for the design of patient-specific engineered T cells.It therefore has the potential to greatly benefi

关键词： cancer-targeted T-cell receptor T(TCR-T)cells circulating hematopoietic stem and progenitor cells(HSPCs) humanized mouse model steady-state peripheral blood T-cell receptorβ-chain(TRB) three-dimensional culture

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