174 results on '"Guoquan Yan"'
Search Results
2. AMPK induces degradation of the transcriptional repressor PROX1 impairing branched amino acid metabolism and tumourigenesis
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Yanan Wang, Mengjun Luo, Fan Wang, Yu Tong, Linfeng Li, Yu Shu, Ke Qiao, Lei Zhang, Guoquan Yan, Jing Liu, Hongbin Ji, Youhua Xie, Yonglong Zhang, Wei-Qiang Gao, and Yanfeng Liu
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Science - Abstract
Energy stress activates AMPK leading to metabolic plasticity and therapy resistance in cancer. Here, the authors show that AMPK activation decreases Prospero-related homeobox 1 (PROX1) levels impairing branched amino acid metabolism and tumourigenesis in liver and lung cancer models.
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- 2022
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3. GproDIA enables data-independent acquisition glycoproteomics with comprehensive statistical control
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Yi Yang, Guoquan Yan, Siyuan Kong, Mengxi Wu, Pengyuan Yang, Weiqian Cao, and Liang Qiao
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Science - Abstract
Data independent acquisition (DIA) proteomics provides deep coverage and high quantitative accuracy, but is not yet well established in glycoproteomics. Here, the authors develop a DIA-based glycoproteomics workflow with stringent statistical controls to enable accurate glycopeptide identification.
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- 2021
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4. Nascent Glycoproteome Reveals That N-Linked Glycosylation Inhibitor-1 Suppresses Expression of Glycosylated Lysosome-Associated Membrane Protein-2
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Xinyi Cao, Peiyi Meng, Yuyin Shao, Guoquan Yan, Jun Yao, Xinwen Zhou, Chao Liu, Lei Zhang, Hong Shu, and Haojie Lu
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hepatocellular carcinoma ,nascent proteome ,glycosylation ,glycoproteome ,LAMP2 ,Biology (General) ,QH301-705.5 - Abstract
Glycosylation inhibition has great potential in cancer treatment. However, the corresponding cellular response, protein expression and glycosylation changes remain unclear. As a cell-permeable small-molecule inhibitor with reduced cellular toxicity, N-linked glycosylation inhibitor-1 (NGI-1) has become a great approach to regulate glycosylation in mammalian cells. Here for the first time, we applied a nascent proteomic method to investigate the effect of NGI-1 in hepatocellular carcinoma (HCC) cell line. Besides, hydrophilic interaction liquid chromatography (HILIC) was adopted for the enrichment of glycosylated peptides. Glycoproteomic analysis revealed the abundance of glycopeptides from LAMP2, NICA, and CEIP2 was significantly changed during NGI-1 treatment. Moreover, the alterations of LAMP2 site-specific intact N-glycopeptides were comprehensively assessed. NGI-1 treatment also led to the inhibition of Cathepsin D maturation and the induction of autophagy. In summary, we provided evidence that NGI-1 repressed the expression of glycosylated LAMP2 accompanied with the occurrence of lysosomal defects and autophagy.
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- 2022
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5. Fluorescent Protein Variants Generated by Reassembly between Skeleton and Chromophore
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Tingting Sun, Tianpeng Li, Ke Yi, Guoquan Yan, and Xiaolian Gao
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Chemistry ,QD1-999 - Published
- 2021
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6. Circulating proteomic panels for risk stratification of intracranial aneurysm and its rupture
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Yueting Xiong, Yongtao Zheng, Yan Yan, Jun Yao, Hebin Liu, Fenglin Shen, Siyuan Kong, Shuang Yang, Guoquan Yan, Huanhuan Zhao, Xinwen Zhou, Jia Hu, Bin Zhou, Tao Jin, Huali Shen, Bing Leng, Pengyuan Yang, and Xiaohui Liu
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biomarker discovery ,intracranial aneurysm ,mass spectrometry ,serum proteome profiling ,Medicine (General) ,R5-920 ,Genetics ,QH426-470 - Abstract
Abstract The prevalence of intracranial aneurysm (IA) is increasing, and the consequences of its rupture are severe. This study aimed to reveal specific, sensitive, and non‐invasive biomarkers for diagnosis and classification of ruptured and unruptured IA, to benefit the development of novel treatment strategies and therapeutics altering the course of the disease. We first assembled an extensive candidate biomarker bank of IA, comprising up to 717 proteins, based on altered proteins discovered in the current tissue and serum proteomic analysis, as well as from previous studies. Mass spectrometry assays for hundreds of biomarkers were efficiently designed using our proposed deep learning‐based method, termed DeepPRM. A total of 113 potential markers were further quantitated in serum cohort I (n = 212) & II (n = 32). Combined with a machine‐learning‐based pipeline, we built two sets of biomarker combinations (P6 & P8) to accurately distinguish IA from healthy controls (accuracy: 87.50%) or classify IA rupture patients (accuracy: 91.67%) upon evaluation in the external validation set (n = 32). This extensive circulating biomarker development study provides valuable knowledge about IA biomarkers.
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- 2022
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7. Aorta smooth muscle-on-a-chip reveals impaired mitochondrial dynamics as a therapeutic target for aortic aneurysm in bicuspid aortic valve disease
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Mieradilijiang Abudupataer, Shichao Zhu, Shiqiang Yan, Kehua Xu, Jingjing Zhang, Shaman Luo, Wenrui Ma, Md Fazle Alam, Yuyi Tang, Hui Huang, Nan Chen, Li Wang, Guoquan Yan, Jun Li, Hao Lai, Chunsheng Wang, Kai Zhu, and Weijia Zhang
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organ-on-a-chip ,thoracic aortic aneurysm ,bicuspid aortic valve ,mitochondrial dynamics ,human aortic ,smooth muscle cells ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
Background: Bicuspid aortic valve (BAV) is the most common congenital cardiovascular disease in general population and is frequently associated with the development of thoracic aortic aneurysm (TAA). There is no effective strategy to intervene with TAA progression due to an incomplete understanding of the pathogenesis. Insufficiency of NOTCH1 expression is highly related to BAV-TAA, but the underlying mechanism remains to be clarified. Methods: A comparative proteomics analysis was used to explore the biological differences between non-diseased and BAV-TAA aortic tissues. A microfluidics-based aorta smooth muscle-on-a-chip model was constructed to evaluate the effect of NOTCH1 deficiency on contractile phenotype and mitochondrial dynamics of human aortic smooth muscle cells (HAoSMCs). Results: Protein analyses of human aortic tissues showed the insufficient expression of NOTCH1 and impaired mitochondrial dynamics in BAV-TAA. HAoSMCs with NOTCH1-knockdown exhibited reduced contractile phenotype and were accompanied by attenuated mitochondrial fusion. Furthermore, we identified that mitochondrial fusion activators (leflunomide and teriflunomide) or mitochondrial fission inhibitor (Mdivi-1) partially rescued the disorders of mitochondrial dynamics in HAoSMCs derived from BAV-TAA patients. Conclusions: The aorta smooth muscle-on-a-chip model simulates the human pathophysiological parameters of aorta biomechanics and provides a platform for molecular mechanism studies of aortic disease and related drug screening. This aorta smooth muscle-on-a-chip model and human tissue proteomic analysis revealed that impaired mitochondrial dynamics could be a potential therapeutic target for BAV-TAA. Funding: National Key R and D Program of China, National Natural Science Foundation of China, Shanghai Municipal Science and Technology Major Project, Shanghai Science and Technology Commission, and Shanghai Municipal Education Commission.
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- 2021
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8. Quantification of Intact O-Glycopeptides on Haptoglobin in Sera of Patients With Hepatocellular Carcinoma and Liver Cirrhosis
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Hong Shu, Lei Zhang, Yiwei Chen, Yijie Guo, Limin Li, Fanghua Chen, Zhao Cao, Guoquan Yan, Chunlai Lu, Chao Liu, and Shu Zhang
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haptoglobin ,O-glycosylation ,mass spectrometry ,lectin ,hepatocellular carcinoma ,Chemistry ,QD1-999 - Abstract
Haptoglobin (Hp) is one of the acute-phase response proteins secreted by the liver, and its aberrant N-glycosylation was previously reported in hepatocellular carcinoma (HCC). Limited studies on Hp O-glycosylation have been previously reported. In this study, we aimed to discover and confirm its O-glycosylation in HCC based on lectin binding and mass spectrometry (MS) detection. First, serum Hp was purified from patients with liver cirrhosis (LC) and HCC, respectively. Then, five lectins with Gal or GalNAc monosaccharide specificity were chosen to perform lectin blot, and the results showed that Hp in HCC bound to these lectins in a much stronger manner than that in LC. Furthermore, label-free quantification based on MS was performed. A total of 26 intact O-glycopeptides were identified on Hp, and most of them were elevated in HCC as compared to LC. Among them, the intensity of HYEGS316TVPEK (H1N1S1) on Hp was the highest in HCC patients. Increased HYEGS316TVPEK (H1N1S1) in HCC was quantified and confirmed using the MS method based on 18O/16O C-terminal labeling and multiple reaction monitoring. This study provided a comprehensive understanding of the glycosylation of Hp in liver diseases.
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- 2021
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9. Analysis of Serum Paraoxonase 1 Using Mass Spectrometry and Lectin Immunoassay in Patients With Alpha-Fetoprotein Negative Hepatocellular Carcinoma
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Xinyi Cao, Zhao Cao, Yuyin Shao, Chao Liu, Guoquan Yan, Xinmin Meng, Lei Zhang, Chen Chen, Guiyue Huang, Hong Shu, and Haojie Lu
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alpha-fetoprotein-negative hepatocellular carcinoma ,PON1 ,biomarker ,glycoproteomics ,mass spectrometry ,lectin ELISA ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,RC254-282 - Abstract
The diagnosis of AFP (alpha-fetoprotein)-negative HCC (hepatocellular carcinoma) mostly relies on imaging and pathological examinations, and it lacks valuable and practical markers. Protein N-glycosylation is a crucial post-translation modifying process related to many biological functions in an organism. Alteration of N-glycosylation correlates with inflammatory diseases and infectious diseases including hepatocellular carcinoma. Here, serum N-linked intact glycopeptides with molecular weight (MW) of 40–55 kDa were analyzed in a discovery set (n = 40) including AFP-negative HCC and liver cirrhosis (LC) patients using label-free quantification methodology. Quantitative lens culinaris agglutin (LCA) ELISA was further used to confirm the difference of glycosylation on serum PON1 in liver diseases (n = 56). Then, the alteration of site-specific intact N-glycopeptides of PON1 was comprehensively assessed by using Immunoprecipitation (IP) and mass spectrometry based 16O/18O C-terminal labeling quantification method to distinguish AFP-negative HCC from LC patients in a validation set (n = 64). Totally 195 glycopeptides were identified using a dedicated search engine pGlyco. Among them, glycopeptides from APOH, HPT/HPTR, and PON1 were significantly changed in AFP-negative HCC as compared to LC. In addition, the reactivity of PON1 with LCA in HCC patients with negative AFP was significantly elevated than that in cirrhosis patients. The two glycopeptides HAN253WTLTPLK (H5N4S2) and (H5N4S1) corresponding to PON1 were significantly increased in AFP-negative HCC patients, as compared with LC patients. Variations in PON1 glycosylation may be associated with AFP-negative HCC and might be helpful to serve as potential glycomic-based biomarkers to distinguish AFP-negative HCC from cirrhosis.
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- 2021
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10. Comparative Proteomics of Extended-Spectrum Cephalosporin-Resistant Neisseria gonorrhoeae Isolates Demonstrates Altered Protein Synthesis, Metabolism, Substance Transport, and Membrane Permeability
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Nannan Diao, Guoquan Yan, Yang Yang, Yuan Dong, Ying Wang, and Weiming Gu
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Neisseria gonorrhoeae ,extended-spectrum cephalosporin resistance ,comparative proteomics ,iTRAQ ,metabolism ,membrane permeability ,Microbiology ,QR1-502 - Abstract
Neisseria gonorrhoeae isolates exhibit resistance to extended-spectrum cephalosporins (ESCs), the last remaining option for first-line empirical monotherapy. Here, we investigated the proteomic profiles of N. gonorrhoeae clinical isolates with ESC-resistance to support exploration of the antimicrobial resistance mechanisms for N. gonorrhoeae. We used comparative iTRAQ quantitative proteomics to investigate differential protein expression of three ESC-resistant N. gonorrhoeae clinical isolates using N. gonorrhoeae ATCC49226 as a reference strain. The expression of 40 proteins was downregulated and expression of 56 proteins was upregulated in all three ESC-resistant N. gonorrhoeae isolates. Proteins with predicted function of translation, ribosomal structure and biogenesis, as well as components of the Type IV secretory systems, were significantly upregulated. Two differentially expressed proteins of ABC transporters were also reported by other teams in proteomics studies of N. gonorrhoeae isolates under antimicrobial stress conditions. Differentially expressed proteins are involved in energy production and metabolism of carbohydrates and amino acids. Our results indicated that amino acid and carbohydrate metabolism, cell membrane structure, interbacterial DNA transfer, and ribosome components might be involved in mediating ESC-resistance in N. gonorrhoeae. These findings facilitate a better understanding of the mechanisms of ESC-resistance in N. gonorrhoeae and provide useful information for identifying novel targets in the development of antimicrobials against N. gonorrhoeae.
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- 2020
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11. Acetylation accumulates PFKFB3 in cytoplasm to promote glycolysis and protects cells from cisplatin-induced apoptosis
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Fu-Long Li, Jin-Ping Liu, Ruo-Xuan Bao, GuoQuan Yan, Xu Feng, Yan-Ping Xu, Yi-Ping Sun, Weili Yan, Zhi-Qiang Ling, Yue Xiong, Kun-Liang Guan, and Hai-Xin Yuan
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Science - Abstract
Enhanced glycolysis in cancer cells has been associated with protection from DNA damage. Here the authors show that DNA damaging signals induce acetylation of PFKFB3 at lysine K472 and promote its cytosolic accumulation, which enhances glycolysis, resulting in protection from cisplatin-induced cell death.
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- 2018
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12. A Mur regulator protein in the extremophilic bacterium Deinococcus radiodurans.
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Amir Miraj Ul Hussain Shah, Ye Zhao, Yunfei Wang, Guoquan Yan, Qikun Zhang, Liangyan Wang, Bing Tian, Huan Chen, and Yuejin Hua
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Medicine ,Science - Abstract
Ferric uptake regulator (Fur) is a transcriptional regulator that controls the expression of genes involved in the uptake of iron and manganese, as well as vital nutrients, and is essential for intracellular redox cycling. We identified a unique Fur homolog (DR0865) from Deinococcus radiodurans, which is known for its extreme resistance to radiation and oxidants. A dr0865 mutant (Mt-0865) showed a higher sensitivity to manganese stress, hydrogen peroxide, gamma irradiation and ultraviolet (UV) irradiation than the wild-type R1 strain. Cellular manganese (Mn) ion (Mn2+) analysis showed that Mn2+, copper (Cu2+), and ferric (Fe3+) ions accumulated significantly in the mutant, which suggests that the dr0865 gene is not only involved in the regulation of Mn2+ homeostasis, but also affects the uptake of other ions. In addition, transcriptome profiles under MnCl2 stress showed that the expression of many genes involved in Mn metabolism was significantly different in the wild-type R1 and DR0865 mutant (Mt-0865). Furthermore, we found that the dr0865 gene serves as a positive regulator of the manganese efflux pump gene mntE (dr1236), and as a negative regulator of Mn ABC transporter genes, such as dr2283, dr2284 and dr2523. Therefore, it plays an important role in maintaining the homoeostasis of intracellular Mn (II), and also other Mn2+, zinc (Zn2+) and Cu2+ ions. Based on its role in manganese homeostasis, DR0865 likely belongs to the Mur sub-family of Fur homolog.
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- 2014
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13. Discovery and confirmation of O-GlcNAcylated proteins in rat liver mitochondria by combination of mass spectrometry and immunological methods.
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Weiqian Cao, Jing Cao, Jiangming Huang, Jun Yao, Guoquan Yan, Haoqi Xu, and Pengyuan Yang
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Medicine ,Science - Abstract
O-linked β-N-acetylglucosamine (O-GlcNAc) is an important post-translational modification (PTM) consisting of a single N-acetylglucosamine moiety attached via an O-β-glycosidic linkage to serine and threonine residues. Glycosylation with O-GlcNAc occurs on myriad nuclear and cytosolic proteins from almost all functional classes. However, with respect to O-GlcNAcylated proteins special in mitochondria, little attention has been paid. In this study, we combined mass spectrometry and immunological methods to perform global exploration of O-GlcNAcylated proteins specific in mitochondria of rat liver. First, highly purified mitochondrial proteins were obviously shown to be O-GlcNAcylated by immunoblot profiling. Then, β-elimination followed by Michael Addition with Dithiothreitol (BEMAD) treatment and LC-MS/MS were performed to enrich and identify O-GlcNAcylated mitochondrial proteins, resulting in an unambiguous assignment of 14 O-GlcNAcylation sites, mapping to 11 O-GlcNAcylated proteins. Furthermore, the identified O-GlcNAcylated mitochondrial proteins were fully validated by both electron transfer dissociation tandem mass spectrometry (ETD/MS/MS) and western blot. Thus, for the first time, our study definitely not only identified but also validated that some mitochondrial proteins in rat liver are O-GlcNAcylated. Interestingly, all of these O-GlcNAcylated mitochondrial proteins are enzymes, the majority of which are involved in a wide variety of biological processes, such as urea cycle, tricarboxylic acid cycle and lipid metabolism, indicating a role for protein O-GlcNAcylation in mitochondrial function.
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- 2013
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14. Nascent Proteome and Glycoproteome Reveal the Inhibition Role of ALG1 in Hepatocellular Carcinoma Cell Migration
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Xinyi Cao, Yuyin Shao, Peiyi Meng, Zhao Cao, Guoquan Yan, Jun Yao, Xinwen Zhou, Chao Liu, Lei Zhang, Hong Shu, and Haojie Lu
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General Engineering - Published
- 2022
15. Fast Discrimination of Sialylated N-Glycan Linkage Isomers with One-Step Derivatization by Microfluidic Capillary Electrophoresis–Mass Spectrometry
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Mengxia Cheng, Hong Shu, Maohua Yang, Guoquan Yan, Lei Zhang, Liang Wang, Wenning Wang, and Haojie Lu
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Polysaccharides ,Microfluidics ,Sialic Acids ,Electrophoresis, Capillary ,Humans ,Mass Spectrometry ,N-Acetylneuraminic Acid ,Analytical Chemistry - Abstract
Linkage isomers (α-2,3- or α-2,6-linkage) of sialylated N-glycans are involved in the emergence and progression of some diseases, so they are of great significance for diagnosing and monitoring diseases. However, the qualitative and quantitative analysis of sialylated N-glycan linkage isomers remains challenging due to their low abundance and limited isomeric separation techniques. Herein, we developed a novel strategy integrating one-step sialic acid derivatization, positive charge-sensitive separation and highly sensitive detection based on microfluidic capillary electrophoresis-mass spectrometry (MCE-MS) for fast and specific analysis of α-2,3- and α-2,6-linked sialylated N-glycan isomers. A kind of easily charged long-chain amino compound was screened first for one-step sialic acid derivatization so that only α-2,3- and α-2,6-linked isomers can be quickly and efficiently separated within 10 min by MCE due to the difference in structural conformation, whose separation mechanism was further theoretically supported by molecular dynamic simulation. In addition, different sialylated N-glycans were separated in order according to the number of sialic acids, so that a migration time-based prediction of the number of sialic acids was achieved. Finally, the sialylated N-glycome of human serum was profiled within 10 min and 6 of the 52 detected sialylated N-glycans could be potential diagnostic biomarkers of cervical cancer (CC), whose α-2,3- and α-2,6-linked isomers were distinguished by α-2,3Neuraminidase S.
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- 2022
16. Multi-comparative Thermal Proteome Profiling Uncovers New O-GlcNAc Proteins in a System-wide Method
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Guoli Wang, Yang Li, Ting Wang, Jun Wang, Jun Yao, Guoquan Yan, Ying Zhang, and Haojie Lu
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Analytical Chemistry - Abstract
Among diverse protein post-translational modifications, O-GlcNAcylation, a simple but essential monosaccharide modification, plays crucial roles in cellular processes and is closely related to various diseases. Despite its ubiquity in cells, properties of low stoichiometry and reversibility are hard nuts to crack in system-wide research of O-GlcNAc. Herein, we developed a novel method employing multi-comparative thermal proteome profiling for O-GlcNAc transferase (OGT) substrate discovery. Melting curves of proteins under different treatments were profiled and compared with high reproducibility and consistency. Consequently, proteins with significantly shifted stabilities caused by OGT and uridine-5'-diphosphate
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- 2022
17. All-in-One Strategy for Downstream Molecular Profiling of Tumor-Derived Exosomes
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Shurong Wang, Ying He, Jiayin Lu, Yuqing Wang, Xiaofeng Wu, Guoquan Yan, Xiaoni Fang, and Baohong Liu
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Proteomics ,Neoplasms ,Cell Culture Techniques ,Humans ,Proteins ,General Materials Science ,Exosomes - Abstract
In light of the significance of exosomes in cancer diagnosis and treatment, it is important to understand the components and functions of exosomes. Herein, an all-in-one strategy has been proposed for comprehensive characterization of exosomal proteins based on nanoporous TiO
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- 2022
18. Microfluidic Free‐Flow Paper Electrochromatography for Continuous Separation of Glycans
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Yingchao Liu, Yuanyu Huang, Mengxi Wu, Siyuan Kong, Weiqian Cao, Shunxiang Li, Guoquan Yan, Baohong Liu, Pengyuan Yang, Quanqing Zhang, Liang Qiao, and Huali Shen
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Electrochemistry ,Catalysis - Published
- 2022
19. Specific Analysis of α-2,3-Sialylated N-Glycan Linkage Isomers by Microchip Capillary Electrophoresis–Mass Spectrometry
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Haojie Lu, Jun Yao, Hong Shu, Xiaoxiao Feng, Ye Peng, Huimin Bao, Guoquan Yan, Mengxia Cheng, and Lei Zhang
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Glycan ,Chromatography ,biology ,Chemistry ,Methylamine ,010401 analytical chemistry ,Electrophoresis, Capillary ,010402 general chemistry ,Mass spectrometry ,01 natural sciences ,Capillary electrophoresis–mass spectrometry ,Mass Spectrometry ,0104 chemical sciences ,Analytical Chemistry ,Electrophoresis, Microchip ,carbohydrates (lipids) ,chemistry.chemical_compound ,Electrophoresis ,Polysaccharides ,Sialic Acids ,biology.protein ,Humans ,Separation method ,Linkage isomerism ,Derivatization - Abstract
Sialylated N-glycan isomers with α-2,3 and α-2,6 linkages play crucial and distinctive roles in diverse physiological and pathological processes. Changes of α-2,3-linked sialic acids in sialylated N-glycans are especially important in monitoring the initiation and progression of diseases. However, the specific analysis of α-2,3-sialylated N-glycan linkage isomers remains challenging due to their extremely low abundance and technical limitations in separation and detection. Herein, we designed an integrated strategy that combines linkage-specific derivatization and a charge-sensitive separation method based on microfluidic chip capillary electrophoresis-mass spectrometry (microchip CE-MS) for specific analysis of α-2,3-sialylated N-glycan linkage isomers for the first time. The α-2,6- and α-2,3-sialic acids were selectively labeled with methylamine (MA) and N,N-dimethylethylenediamine (DMEN), respectively, which selectively makes α-2,3-sialylated N-glycans positively charged and realizes online purification, concentration, and discrimination of α-2,3-sialylated N-glycans from other N-glycans in microchip CE-MS. This new approach was demonstrated with standard multisialylated N-glycans, and it was found that only the α-2,3-sialylated N-glycans migrated and were detected in order according to the number of α-2,3-sialic acids. Finally, this strategy was successfully applied in highly sensitive profiling and reproducible quantitation of the serum α-2,3-sialylated N-glycome from ovarian cancer (OC) patients, where 7 of 33 detected α-2,3-sialylated N-glycans significantly changed in the OC group compared with healthy controls.
- Published
- 2021
20. Characterization of Urinary Exosomes Purified with Size Exclusion Chromatography and Ultracentrifugation
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Sheng Guan, Xiangmin Zhang, Hailong Yu, Guoquan Yan, Weibing Sun, and Mingxia Gao
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Proteomics ,0301 basic medicine ,030102 biochemistry & molecular biology ,Chemistry ,media_common.quotation_subject ,Size-exclusion chromatography ,General Chemistry ,Exosomes ,Biochemistry ,Exosome ,Microvesicles ,Extracellular Vesicles ,03 medical and health sciences ,030104 developmental biology ,Cell culture ,Chromatography, Gel ,Animals ,Ultracentrifuge ,Internalization ,Ultracentrifugation ,Intracellular ,media_common - Abstract
Exosomes, a subtype of extracellular vesicles secreted by mammalian cells with a typical size range of 30-150 nm, have been implicated in many biological processes as intercellular communication carriers. The isolation of exosomes is an essential and challenging step before subsequent analysis and functional studies, due to the complexity of body fluids, as well as the small size and low density of exosomes. Ultracentrifugation (UC) and size exclusion chromatography (SEC) are two methods that have been extensively used for exosomes isolation in biological studies in recent years. In this work, we compared the characteristics of urinary exosomes extracted with SEC and UC methods in detail. Results showed that the SEC isolation method was superior to UC in the recovery of exosomal particles and proteins. The results of proteomics analysis showed that more purified exosomes were extracted with the SEC method. We also observed that parts of exosomes were ruptured and precipitated insufficiently during UC isolations. It not only led to a low recovery of exosome proteins but also resulted in a considerable loss of exosomal particles. Moreover, the exosomal rupture and particle loss in UC could not be avoided by resuspension of the exosomal particles. Our results also showed that exosomes from SEC purifications possessed a high internalization capability from 4 to 6 h when incubated with EA.hy926 and HCV29 cell lines.
- Published
- 2020
21. Magnetic metal phenolic networks: expanding the application of a promising nanoprobe to phosphoproteomics research
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Huimin Chu, Chunhui Deng, Haoyang Zheng, Jizong Yao, Guoquan Yan, and Nianrong Sun
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Phosphopeptides ,Proteomics ,Surface Properties ,Nanoprobe ,Catalysis ,Metal ,HeLa ,Organometallic Compounds ,Materials Chemistry ,Humans ,Particle Size ,Titanium ,biology ,Chemistry ,Phosphopeptide ,Magnetic Phenomena ,Extraction (chemistry) ,Metals and Alloys ,Phosphoproteomics ,General Chemistry ,Phosphoproteins ,biology.organism_classification ,Combinatorial chemistry ,Surfaces, Coatings and Films ,Electronic, Optical and Magnetic Materials ,visual_art ,Ceramics and Composites ,visual_art.visual_art_medium ,Nanoparticles ,Tannins ,HeLa Cells - Abstract
A Ti-tannic acid modified magnetic nanoprobe was synthesized via one step, and applied to phosphopeptide extraction. 3456 phosphopeptides and 1285 phosphoproteins were finally identified from 100 μg HeLa cell extracts. This opens up a new avenue for metal phenolic networks in phosphoproteomics research.
- Published
- 2020
22. A rapid and efficient method for N-termini analysis in short-lived proteins
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Xiangmin Zhang, Lanting Li, and Guoquan Yan
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Proteomics ,Proteome ,Infrared Rays ,Proteolysis ,Serum Albumin, Human ,02 engineering and technology ,Mass spectrometry ,01 natural sciences ,Analytical Chemistry ,Microsphere ,Mice ,Protein Domains ,Sequence Analysis, Protein ,Tandem Mass Spectrometry ,medicine ,Animals ,Humans ,Amino Acid Sequence ,Chromatography ,medicine.diagnostic_test ,Chemistry ,Lasers ,010401 analytical chemistry ,021001 nanoscience & nanotechnology ,Trypsin ,Human serum albumin ,Ferrosoferric Oxide ,Microspheres ,0104 chemical sciences ,Liver ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,Reagent ,Peptides ,0210 nano-technology ,Chromatography, Liquid ,medicine.drug - Abstract
A rapid and efficient method to isolate global N-termini is presented. Utilizing laser-assisted proteolysis and Fe 3 O 4 microsphere, protein N-termini could be isolated in 4 h. The amino-blocked protein was digested by trypsin assisted by laser radiation, shortening the digest time from overnight to 40 s. Non-N-terminal peptides were characterized by a tryptic free amino in their N-term, which could be derived with sulfhydryl by traut’ s reagent efficiently and then coupled with Fe 3 O 4 microspheres nearly completely in less than 4 h. The rapid method was beneficial for the identification of unstable N-termini in short-lived proteins. Human serum albumin was studied as a model. The N-terminus was successfully isolated from the digest within 4 h. Also, 2011 N-terminal peptides out of 936 proteins in mouse liver proteome sample were identified using liquid chromatography-tandem mass spectrometer (LC-MS/MS). This method was demonstrated as a facile and efficient N-termini enrichment method for targeted protein N-termini analysis, especially those with short half-life.
- Published
- 2019
23. pGlycoQuant with a deep residual network for precise and minuscule-missing-value quantitative glycoproteomics enabling the functional exploration of site-specific glycosylation
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Guoquan Yan, Siyuan Kong, H. Zhao, P. Gong, Yan Zhang, Chao Liu, X. Hou, X. Qiao, Pengyuan Yang, Mingyao Liu, Mengxi Wu, Biyun Jiang, W. Cao, and Wen-Feng Zeng
- Subjects
chemistry.chemical_compound ,Glycosylation ,chemistry ,Computer science ,Software tool ,L1 cell adhesion molecule ,Hepatic carcinoma ,Computational biology ,Residual ,Fucosylation ,Biomarker (cell) ,Glycoproteomics - Abstract
Interpreting large-scale glycoproteomic data for intact glycopeptide identification has been tremendously advanced by software tools. However, software tools for quantitative analysis of intact glycopeptides remain lagging behind, which greatly hinders exploring the differential expression and functions of site-specific glycosylation in organisms. Here, we report pGlycoQuant, a generic software tool for accurate and convenient quantitative intact glycopeptide analysis, supporting both primary and tandem mass spectrometry quantitation for multiple quantitative strategies. pGlycoQuant enables intact glycopeptide quantitation with very low missing values via a deep residual network, thus greatly expanding the quantitative function of several powerful search engines, currently including pGlyco 2.0, pGlyco3, Byonic and MSFragger-Glyco. The pGlycoQuant-based site-specific N-glycoproteomic study conducted here quantifies 6435 intact N-glycopeptides in three hepatocellular carcinoma cell lines with different metastatic potentials and, together with in vitro molecular biology experiments, illustrates core fucosylation at site 979 of the L1 cell adhesion molecule (L1CAM) as a potential regulator of HCC metastasis. pGlycoQuant is freely available at https://github.com/expellir-arma/pGlycoQuant/releases/. We have demonstrated pGlycoQuant to be a powerful tool for the quantitative analysis of site-specific glycosylation and the exploration of potential glycosylation-related biomarker candidates, and we expect further applications in glycoproteomic studies.
- Published
- 2021
24. GproDIA enables data-independent acquisition glycoproteomics with comprehensive statistical control
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Mengxi Wu, Liang Qiao, Weiqian Cao, Yi Yang, Guoquan Yan, Pengyuan Yang, and Siyuan Kong
- Subjects
Proteomics ,False discovery rate ,Proteome ,Computer science ,Science ,Glycobiology ,General Physics and Astronomy ,Inference ,Proteome informatics ,computer.software_genre ,Article ,General Biochemistry, Genetics and Molecular Biology ,Workflow ,Polysaccharides ,Humans ,Data-independent acquisition ,Glycoproteins ,Profiling (computer programming) ,Multidisciplinary ,Mass spectrometry ,Glycopeptides ,Blood Proteins ,General Chemistry ,Statistical process control ,Glycoproteomics ,Identification (information) ,Benchmark (computing) ,Schizosaccharomyces pombe Proteins ,Data mining ,computer ,Algorithms - Abstract
Large-scale profiling of intact glycopeptides is critical but challenging in glycoproteomics. Data independent acquisition (DIA) is an emerging technology with deep proteome coverage and accurate quantitative capability in proteomics studies, but is still in the early stage of development in the field of glycoproteomics. We propose GproDIA, a framework for the proteome-wide characterization of intact glycopeptides from DIA data with comprehensive statistical control by a 2-dimentional false discovery rate approach and a glycoform inference algorithm, enabling accurate identification of intact glycopeptides using wide isolation windows. We further utilize a semi-empirical spectrum prediction strategy to expand the coverage of spectral libraries of glycopeptides. We benchmark our method for N-glycopeptide profiling on DIA data of yeast and human serum samples, demonstrating that DIA with GproDIA outperforms the data-dependent acquisition-based methods for glycoproteomics in terms of capacity and data completeness of identification, as well as accuracy and precision of quantification. We expect that this work can provide a powerful tool for glycoproteomic studies., Data independent acquisition (DIA) proteomics provides deep coverage and high quantitative accuracy, but is not yet well established in glycoproteomics. Here, the authors develop a DIA-based glycoproteomics workflow with stringent statistical controls to enable accurate glycopeptide identification.
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- 2021
25. Aorta smooth muscle-on-a-chip reveals impaired mitochondrial dynamics as a therapeutic target for aortic aneurysm in bicuspid aortic valve disease
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Shichao Zhu, Hao Lai, Yuyi Tang, Li Wang, Kehua Xu, Shaman Luo, Jingjing Zhang, Chunsheng Wang, Weijia Zhang, Shiqiang Yan, Guoquan Yan, Hui Huang, Jun Li, Kai Zhu, Nan Chen, Fazle Alam, Wenrui Ma, and Mieradilijiang Abudupataer
- Subjects
Aortic valve ,Male ,thoracic aortic aneurysm ,Aortic aneurysm ,Bicuspid aortic valve ,Bicuspid Aortic Valve Disease ,Lab-On-A-Chip Devices ,Myocyte ,Biology (General) ,Aorta ,organ-on-a-chip ,General Neuroscience ,General Medicine ,Middle Aged ,Aortic Aneurysm ,smooth muscle cells ,medicine.anatomical_structure ,Cardiology ,cardiovascular system ,Medicine ,Female ,Artery ,Research Article ,Human ,Muscle tissue ,Adult ,medicine.medical_specialty ,bicuspid aortic valve ,QH301-705.5 ,Science ,Myocytes, Smooth Muscle ,Thoracic aortic aneurysm ,General Biochemistry, Genetics and Molecular Biology ,Cell Line ,medicine.artery ,Internal medicine ,medicine ,Humans ,Aged ,General Immunology and Microbiology ,business.industry ,Cardiovascular Agents ,medicine.disease ,mitochondrial dynamics ,human aortic ,Tissue Array Analysis ,business - Abstract
Background: Bicuspid aortic valve (BAV) is the most common congenital cardiovascular disease in general population and is frequently associated with the development of thoracic aortic aneurysm (TAA). There is no effective strategy to intervene with TAA progression due to an incomplete understanding of the pathogenesis. Insufficiency of NOTCH1 expression is highly related to BAV-TAA, but the underlying mechanism remains to be clarified. Methods: A comparative proteomics analysis was used to explore the biological differences between non-diseased and BAV-TAA aortic tissues. A microfluidics-based aorta smooth muscle-on-a-chip model was constructed to evaluate the effect of NOTCH1 deficiency on contractile phenotype and mitochondrial dynamics of human aortic smooth muscle cells (HAoSMCs). Results: Protein analyses of human aortic tissues showed the insufficient expression of NOTCH1 and impaired mitochondrial dynamics in BAV-TAA. HAoSMCs with NOTCH1-knockdown exhibited reduced contractile phenotype and were accompanied by attenuated mitochondrial fusion. Furthermore, we identified that mitochondrial fusion activators (leflunomide and teriflunomide) or mitochondrial fission inhibitor (Mdivi-1) partially rescued the disorders of mitochondrial dynamics in HAoSMCs derived from BAV-TAA patients. Conclusions: The aorta smooth muscle-on-a-chip model simulates the human pathophysiological parameters of aorta biomechanics and provides a platform for molecular mechanism studies of aortic disease and related drug screening. This aorta smooth muscle-on-a-chip model and human tissue proteomic analysis revealed that impaired mitochondrial dynamics could be a potential therapeutic target for BAV-TAA. Funding: National Key R and D Program of China, National Natural Science Foundation of China, Shanghai Municipal Science and Technology Major Project, Shanghai Science and Technology Commission, and Shanghai Municipal Education Commission., eLife digest To function properly, the heart must remain a one-way system, pumping out oxygenated blood into the aorta – the largest artery in the body – so it can be distributed across the organism. The aortic valve, which sits at the entrance of the aorta, is a key component of this system. Its three flaps (or ‘cusps’) are pushed open when the blood exits the heart, and they shut tightly so it does not flow back in the incorrect direction. Nearly 1.4% of people around the world are born with ‘bicuspid’ aortic valves that only have two flaps. These valves may harden or become leaky, forcing the heart to work harder. This defect is also associated with bulges on the aorta which progressively weaken the artery, sometimes causing it to rupture. Open-heart surgery is currently the only way to treat these bulges (or ‘aneurysms’), as no drug exists that could slow down disease progression. This is partly because the biological processes involved in the aneurysms worsening and bursting open is unclear. Recent studies have highlighted that many individuals with bicuspid aortic valves also have lower levels of a protein known as NOTCH1, which plays a key signalling role for cells. Problems in the mitochondria – the structures that power up a cell – are also observed. However, it is not known how these findings are connected or linked with the aneurysms developing. To answer this question, Abudupataer et al. analyzed the proteins present in diseased and healthy aortic muscle cells, confirming a lower production of NOTCH1 and impaired mitochondria in diseased tissues. They also created an ‘aorta-on-a-chip’ model where aortic muscle cells were grown in the laboratory under conditions resembling those found in the body – including the rhythmic strain that the aorta is under because of the heart beating. Abudupataer et al. then reduced NOTCH1 levels in healthy samples, which made the muscle tissue less able to contract and reduced the activity of the mitochondria. Applying drugs that tweak mitochondrial activity helped tissues from patients with bicuspid aortic valves to work better. These compounds could potentially benefit individuals with deficient aortic valves, but experiments in animals and clinical trials would be needed first to confirm the results and assess safety. The aorta-on-a-chip model developed by Abudupataer et al. also provides a platform to screen for drugs and examine the molecular mechanisms at play in aortic diseases.
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- 2021
26. Author response: Aorta smooth muscle-on-a-chip reveals impaired mitochondrial dynamics as a therapeutic target for aortic aneurysm in bicuspid aortic valve disease
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Yuyi Tang, Mieradilijiang Abudupataer, Shiqiang Yan, Li Wang, Jun Li, Hui Huang, Shaman Luo, Jingjing Zhang, Nan Chen, Guoquan Yan, Chunsheng Wang, Shichao Zhu, Hao Lai, Fazle Alam, Weijia Zhang, Wenrui Ma, Kehua Xu, and Kai Zhu
- Subjects
Aortic aneurysm ,medicine.medical_specialty ,Aorta ,Bicuspid aortic valve ,Smooth muscle ,business.industry ,Internal medicine ,medicine.artery ,medicine ,Cardiology ,Disease ,medicine.disease ,business - Published
- 2021
27. Plasma exosomal proteomic studies of corneal epithelial injury in diabetic and non-diabetic group
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Bing Li, Guoquan Yan, Minjie Sheng, Jie Zhang, and Kaichuan Chen
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Male ,Proteomics ,Proteome ,Quantitative proteomics ,Biology ,Exosomes ,Corneal Diseases ,Cellular and Molecular Neuroscience ,Western blot ,Tandem Mass Spectrometry ,medicine ,ExoCarta ,Humans ,Aged ,medicine.diagnostic_test ,Insulin receptor signaling pathway ,Epithelium, Corneal ,Middle Aged ,Molecular biology ,Sensory Systems ,Microvesicles ,Ophthalmology ,Diabetes Mellitus, Type 2 ,Female ,Target protein ,Signal transduction ,Biomarkers ,Chromatography, Liquid - Abstract
Objective Diabetic Keratopathy (DK) is one of the significant complications of type II diabetes (T2DM) with pathogenesis not yet clarified. Since hyperglycemia is able to change the protein components contained in plasma exosomes, liquid chromatography-tandem mass spectrometry (LC-MS/MS) is considered as feasible to analyze the expression of plasma exosomal proteins in patients with T2DM and non-diabetic patients respectively, find critical biological markers, and explore the mechanism of DK as well as potential therapeutic targets. Method Blood and clinical information of corneal epithelial injury in a diabetic group (the study group) and a non-diabetic group (the control group), who were patients admitted to the Department of Ophthalmology, Yangpu Hospital, Tongji University School of Medicine from July 2020 to November 2020, were collected. The qEV size exclusion method was adopted to separate exosomes from plasma. The exosomes were then identified through transmission electron microscopy (TEM), nanoparticle tracking analyzer (NTA), and Western blot. The plasma exosomes of the study group and the control group were quantitatively analyzed by proteomics. A bioinformatics method is utilized to screen differential proteins and the expression of the differential proteins was verified by Western blot. Result TEM indicated that the exosomes had a double-concave disc-like appearance, with a size of about 100 nm, and Western blot expressed as CD63 and TSG101. The plasma exosomes of the study group and the control group were analyzed by quantitative proteomics with a total number of 952 proteins detected of which 245 proteins existed in the ExoCarta exosomal protein database. Through adoption of P-value to screen credible differential proteins, the heat map displayed 28 differential proteins, 7 upregulated proteins, and 21 downregulated proteins; the volcano map displayed 7 upregulated proteins and 22 downregulated proteins; the PPI interaction map displayed 12 upregulated proteins and 18 downregulated proteins. Through GO enrichment analysis, it was identified that the differential protein participated in the main biological processes and was involved in regulating the cell's stimulation response to insulin, the insulin receptor signaling pathway, and the activity of glycosylphosphatidylinositol phospholipase D as well as anti-oxidation. The enriched cell components include main components such as exosomes, blood particles, and cytoplasm. KEGG enrichment analysis indicated that the target protein FLOT2 was mainly concentrated in insulin-related signaling pathways. Western blot indicated that the expression of FLOT2 in the study group was lower compared with the control group while the expression of Exo70 was higher. Conclusion Proteomic analysis of the study group and the control group displayed a variety of proteins in plasma exosomes. The downregulated protein FLOT2 in the study group was closely related to the occurrence, development, and complication of DK in T2DM patients. The expression status of plasma FLOT2 protein in T2DM patients is expected to be a biomarker for diagnosing and monitoring of DK.
- Published
- 2021
28. Quantification of Intact O-Glycopeptides on Haptoglobin in Sera of Patients With Hepatocellular Carcinoma and Liver Cirrhosis
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Chao Liu, Limin Li, Yijie Guo, Fanghua Chen, Zhao Cao, Hong Shu, Yiwei Chen, Chunlai Lu, Guoquan Yan, Lei Zhang, and Shu Zhang
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0301 basic medicine ,Glycosylation ,Cirrhosis ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,medicine ,QD1-999 ,Original Research ,mass spectrometry ,O-glycosylation ,biology ,Selected reaction monitoring ,Haptoglobin ,Lectin ,General Chemistry ,hepatocellular carcinoma ,medicine.disease ,Molecular biology ,Glycopeptide ,digestive system diseases ,haptoglobin ,Blot ,Chemistry ,030104 developmental biology ,chemistry ,030220 oncology & carcinogenesis ,Hepatocellular carcinoma ,biology.protein ,lectin - Abstract
Haptoglobin (Hp) is one of the acute-phase response proteins secreted by the liver, and its aberrant N-glycosylation was previously reported in hepatocellular carcinoma (HCC). Limited studies on Hp O-glycosylation have been previously reported. In this study, we aimed to discover and confirm its O-glycosylation in HCC based on lectin binding and mass spectrometry (MS) detection. First, serum Hp was purified from patients with liver cirrhosis (LC) and HCC, respectively. Then, five lectins with Gal or GalNAc monosaccharide specificity were chosen to perform lectin blot, and the results showed that Hp in HCC bound to these lectins in a much stronger manner than that in LC. Furthermore, label-free quantification based on MS was performed. A total of 26 intact O-glycopeptides were identified on Hp, and most of them were elevated in HCC as compared to LC. Among them, the intensity of HYEGS316TVPEK (H1N1S1) on Hp was the highest in HCC patients. Increased HYEGS316TVPEK (H1N1S1) in HCC was quantified and confirmed using the MS method based on 18O/16O C-terminal labeling and multiple reaction monitoring. This study provided a comprehensive understanding of the glycosylation of Hp in liver diseases.
- Published
- 2021
29. Plasma proteomic profiling reveals biomarkers associated with aortic dilation in patients with bicuspid aortic valve
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Jingjing Zhang, Shiqiang Yan, Hui Huang, Jun Li, Chunsheng Wang, Yongxin Sun, Guoquan Yan, Hao Lai, Dingqian Liu, Kehua Xu, Weijia Zhang, Wenrui Ma, Yuyi Tang, and Kai Zhu
- Subjects
medicine.medical_specialty ,business.industry ,Heart malformation ,Proteomic Profiling ,Notch signaling pathway ,General Medicine ,medicine.disease ,Actin cytoskeleton ,Thoracic aortic aneurysm ,Blood proteins ,Bicuspid aortic valve ,Internal medicine ,Cardiology ,cardiovascular system ,Medicine ,Biomarker (medicine) ,Original Article ,business - Abstract
BACKGROUND: Bicuspid aortic valve (BAV) is the most common congenital heart anomaly and is prone to cause complications, such as valvular stenosis and thoracic aortic dilation. There is currently no reliable way to predict the progression rate to thoracic aortic aneurysm. Here, we aimed to characterize the proteomic landscape in the plasma of stenotic BAV patients and provide potential biomarkers to predict progressive aortic dilation. METHODS: Plasma samples were obtained from 45 subjects (30 stenotic BAV patients and 15 healthy controls). All samples were properly prepared and analyzed using mass spectrometry (MS)-based label-free quantitative proteomics. RESULTS: A total of 748 plasma proteins had missingness
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- 2021
30. Effective Enrichment Strategy Using Boronic Acid-Functionalized Mesoporous Graphene-Silica Composites for Intact N- and O-Linked Glycopeptide Analysis in Human Serum
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Mingqi Liu, Siyuan Kong, Quanqing Zhang, Huanhuan Zhao, Guoquan Yan, Weiqian Cao, Lujie Yang, Yuanyu Huang, Mengxi Wu, Xiangmin Zhang, and Pengyuan Yang
- Subjects
chemistry.chemical_classification ,Glycosylation ,Silicon dioxide ,Graphene ,010401 analytical chemistry ,Size-exclusion chromatography ,Glycopeptides ,010402 general chemistry ,Silicon Dioxide ,01 natural sciences ,Combinatorial chemistry ,Boronic Acids ,Glycopeptide ,0104 chemical sciences ,Analytical Chemistry ,law.invention ,chemistry.chemical_compound ,chemistry ,law ,Humans ,Graphite ,Mesoporous material ,Glycoprotein ,Hydrophobic and Hydrophilic Interactions ,Boronic acid - Abstract
The heterogeneity and low abundance of protein glycosylation present challenging barriers to the analysis of intact glycopeptides, which is key to comprehensively understanding the role of glycosylation in an organism. Efficient and specific enrichment of intact glycopeptides could help greatly with this problem. Here, we propose a new enrichment strategy using a boronic acid (BA)-functionalized mesoporous graphene-silica composite (denoted as GO@mSiO2-GLYMO-APB) for isolating intact glycopeptides from complex biological samples. The merits of this composite, including high surface area and synergistic effect from size exclusion functionality of mesoporous material, hydrophilic interaction of silica, and the reversible covalent binding with BA, enable the effective and specific enrichment of both intact N- and O-glycopeptides. The results from the enrichment performance of the strategy evaluated by standard glycoproteins and the application to global N- and O-glycosylation analyses in human serum indicate the robustness and potential of the strategy for intact glycopeptide analysis.
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- 2021
31. Mettl17, a regulator of mitochondrial ribosomal RNA modifications, is required for the translation of mitochondrial coding genes
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Zeping Hu, Siyuan Xu, Shenghui Xing, Fei Lan, Ming Wang, Pengyuan Yang, Lei Zhang, Jing Liu, Ke Yao, Guoquan Yan, Peng Zhou, Luxi Xue, and Zhennan Shi
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0301 basic medicine ,S-Adenosylmethionine ,Regulator ,Mitochondrion ,Biology ,Biochemistry ,Mice ,03 medical and health sciences ,0302 clinical medicine ,RRNA modification ,Genetics ,Mitochondrial ribosome ,Animals ,Humans ,Induced pluripotent stem cell ,Molecular Biology ,Gene ,Cells, Cultured ,Embryonic Stem Cells ,Mice, Knockout ,Methyltransferases ,Ribosomal RNA ,Embryonic stem cell ,Cell biology ,Genes, Mitochondrial ,030104 developmental biology ,RNA, Ribosomal ,Protein Biosynthesis ,030217 neurology & neurosurgery ,Protein Binding ,Biotechnology - Abstract
Embryonic stem cells (ESCs) are pluripotent stem cells with the ability to self-renew and to differentiate into any cell types of the 3 germ layers. Recent studies have demonstrated that there is a strong connection between mitochondrial function and pluripotency. Here, we report that methyltransferase like (Mettl) 17, identified from the clustered regularly interspaced short palindromic repeats knockout screen, is required for proper differentiation of mouse embryonic stem cells (mESCs). Mettl17 is located in mitochondria through its N-terminal targeting sequence and specifically interacts with 12S mitochondrial ribosomal RNA (mt-rRNA) as well as small subunits of mitochondrial ribosome (MSSUs). Loss of Mettl17 affects the stability of both 12S mt-rRNA and its associated proteins of MSSUs. We further showed that Mettl17 is an S-adenosyl methionine (SAM)-binding protein and regulates mitochondrial ribosome function in a SAM-binding-dependent manner. Loss of Mettl17 leads to around 70% reduction of m4C840 and 50% reduction of m5C842 of 12S mt-rRNA, revealing the first regulator of the m4C840 and indicating a crosstalk between the 2 nearby modifications. The defects of mitochondrial ribosome caused by deletion of Mettl17 lead to the impaired translation of mitochondrial protein-coding genes, resulting in significant changes in mitochondrial oxidative phosphorylation and cellular metabolome, which are important for mESC pluripotency.-Shi, Z., Xu, S., Xing, S., Yao, K., Zhang, L., Xue, L., Zhou, P., Wang, M., Yan, G., Yang, P., Liu, J., Hu, Z., Lan, F. Mettl17, a regulator of mitochondrial ribosomal RNA modifications, is required for the translation of mitochondrial coding genes.
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- 2019
32. N-glycopeptide Signatures of IgA2 in Serum from Patients with Hepatitis B Virus-related Liver Diseases*
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Guoquan Yan, Lingyi Tang, Haojie Lu, Zefan Ge, Xinyi Cao, Hao Chi, Yinkun Liu, Xue Qin, Mingqi Liu, Shu Zhang, Chao Liu, Wei Li, Baiwen Li, Qiang Gao, and Wen-Feng Zeng
- Subjects
Hepatitis B virus ,Cirrhosis ,Carcinoma, Hepatocellular ,Glycosylation ,Proteome ,medicine.disease_cause ,Glycopeptide ,Biochemistry ,Mass Spectrometry ,Analytical Chemistry ,40-kDa Band ,03 medical and health sciences ,Liver disease ,Polysaccharides ,medicine ,Humans ,Molecular Biology ,Glycomics ,030304 developmental biology ,Biomarker: Diagnostic ,Glycoproteins ,Hepatitis ,0303 health sciences ,business.industry ,Research ,Liver Disease ,030302 biochemistry & molecular biology ,Liver Neoplasms ,Glycopeptides ,Cancer ,Hepatitis B ,Glycoproteomics ,medicine.disease ,HBV-Related Liver Diseases ,IgA2 ,Molecular biology ,digestive system diseases ,Immunoglobulin A ,Hepatocellular carcinoma ,Case-Control Studies ,business - Abstract
18O/16O labeling N-glycopeptide quantification and MRM have been performed to investigate aberrant N-glycopeptides in HBV-related liver diseases. TPLTAN205ITK (H5N5S1F1) and (H5N4S2F1) of IgA2 were significantly elevated in serum from patients with HBV infection and even higher in LC, as compared with healthy donor. In contrast, the two glycopeptides of IgA2 fell back down in HBV-related HCC. The altered N-glycopeptides might be part of a unique glycan signature indicating IgA-mediated mechanism., Graphical Abstract Highlights 18O/16O labeling N-glycopeptide quantification for 40-kDa band in HCC and LC. MRM verification of aberrant N-glycopeptides in healthy-HBV-LC-HCC cascade. TPLTAN205ITK (H5N5S1F1) and (H5N4S2F1) of IgA2 change in liver diseases. Variation in two N-glycopeptides abundance not caused by protein concentration., N-glycosylation alteration has been reported in liver diseases. Characterizing N-glycopeptides that correspond to N-glycan structure with specific site information enables better understanding of the molecular pathogenesis of liver damage and cancer. Here, unbiased quantification of N-glycopeptides of a cluster of serum glycoproteins with 40–55 kDa molecular weight (40-kDa band) was investigated in hepatitis B virus (HBV)-related liver diseases. We used an N-glycopeptide method based on 18O/16O C-terminal labeling to obtain 82 comparisons of serum from patients with HBV-related hepatocellular carcinoma (HCC) and liver cirrhosis (LC). Then, multiple reaction monitoring (MRM) was performed to quantify N-glycopeptide relative to the protein content, especially in the healthy donor-HBV-LC-HCC cascade. TPLTAN205ITK (H5N5S1F1) and (H5N4S2F1) corresponding to the glycopeptides of IgA2 were significantly elevated in serum from patients with HBV infection and even higher in HBV-related LC patients, as compared with healthy donor. In contrast, the two glycopeptides of IgA2 fell back down in HBV-related HCC patients. In addition, the variation in the abundance of two glycopeptides was not caused by its protein concentration. The altered N-glycopeptides might be part of a unique glycan signature indicating an IgA-mediated mechanism and providing potential diagnostic clues in HBV-related liver diseases.
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- 2019
33. A practical approach to enrich intact tryptic N-glycopeptides through size exclusion chromatography and hydrophilicity (SELIC) using an acrylamide-agarose composite gel system
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Cheng Zhang, Haojie Lu, Yun Xiong, Ting Zhao, Weide Ma, Guoquan Yan, Jun Yao, and Gang Chen
- Subjects
Glycosylation ,Thymoma ,Size-exclusion chromatography ,02 engineering and technology ,01 natural sciences ,Biochemistry ,Analytical Chemistry ,Abnormal glycosylation ,Mice ,chemistry.chemical_compound ,Animals ,Humans ,Environmental Chemistry ,Trypsin ,Spectroscopy ,Glycoproteins ,chemistry.chemical_classification ,Acrylamide ,Chromatography ,Chemistry ,Sepharose ,Hydrophilic interaction chromatography ,010401 analytical chemistry ,Glycopeptides ,Thymus Neoplasms ,021001 nanoscience & nanotechnology ,Glycopeptide ,0104 chemical sciences ,Proteolysis ,Chromatography, Gel ,Agarose ,0210 nano-technology ,Glycoprotein ,Hydrophobic and Hydrophilic Interactions - Abstract
Increasing researches proved that abnormal glycosylation is strongly correlated with many diseases. Specially, site-specific glycosylation and its associated heterogeneity are closely related to the function and activity of the glycoprotein. However, intact N-glycopeptide analysis still faces great challenges because the presence of highly abundant non-glycosylated peptides would suppress the ionization of lowly abundant glycopeptides. In the present study, we developed a practical intact tryptic N-glycopeptide enrichment method using acrylamide-agarose composite gel that combined the size exclusion chromatography and hydrophilic (named SELIC) effects, aimed to remove the detergent rapidly and effectively, as well as enrich intact N-glycopeptides while extracting peptides. This is a useful tool to facilitate the intact N-glycopeptides analysis of complex protein mixtures, particularly for samples that extracted from formalin-fixed and paraffin-embedded (FFPE) tissues by SDS. Using this method, we successfully identified 700 site-specific intact tryptic N-glycopeptides corresponding to 261 glycosylation sites on 191 glycoproteins from FFPE thymoma tissues.
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- 2019
34. Selective Identification and Site-Specific Quantification of 4-Hydroxy-2-nonenal-Modified Proteins
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Yi Di, Lei Zhang, Wenjuan Yuan, Siwen Zhang, Yan Cai, Caiyun Fang, Haojie Lu, Guoquan Yan, and Ying Zhang
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Models, Molecular ,Liver chemistry ,Aldehydes ,Molecular Structure ,Liver cell ,010401 analytical chemistry ,Cellular functions ,Tumor cells ,Hep G2 Cells ,010402 general chemistry ,01 natural sciences ,2-Nonenal ,Neoplasm Proteins ,0104 chemical sciences ,Analytical Chemistry ,Isobaric labeling ,chemistry.chemical_compound ,Liver ,Biochemistry ,chemistry ,Affinity chromatography ,Tumor Cells, Cultured ,Humans ,Quantitative analysis (chemistry) - Abstract
4-Hydroxy-2-nonenal (HNE)-modified proteins are closely associated with cellular functions and diseases, so qualitative and quantitative analysis of HNE-modified proteins is very necessary in order to further understand their structures and molecular functions. In this study, we described a six-plex isobaric labeling affinity purification (SiLAP) method based on the interaction of aminoxyTMT six-plex and anti-TMT antibody resin to identify and quantify the HNE modifications simultaneously. The labeling efficiency, ionization efficiency of the aminoxyTMT-tagged peptides, and reliability of the quantification method were investigated in detail. The mass tags were labeled on the modification sites, which could also significantly increase the ionization efficiency, contributing to site-specific identification and quantification of HNE peptides. The SiLAP strategy possessed high sensitivity, accuracy, and good reproducibility to qualitatively and quantitatively analyze HNE-modified proteins/peptides, which could be used to analyze both endogenously and exogenously modified proteins. Using the SiLAP strategy, 2257 HNE-modified peptides mapping 1121 proteins were collectively quantified, which was the largest data set of HNE-modified proteins with detailed modification sites, and 101 proteins were found to be differentially modified by HNE in six liver cell lines. At the same time, 33 endogenously HNE-modified peptides mapping 33 proteins were identified with modification sites.
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- 2019
35. Selective Enrichment and Quantification of N-Terminal Glycine Peptides via Sortase A Mediated Ligation
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Lei Zhang, Ting Cao, Guoquan Yan, Haojie Lu, and Jing Lv
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Proteomics ,0301 basic medicine ,Glycine ,Peptide ,01 natural sciences ,Analytical Chemistry ,03 medical and health sciences ,Bacterial Proteins ,Humans ,Amino Acid Sequence ,Threonine ,chemistry.chemical_classification ,Chemistry ,Sample complexity ,010401 analytical chemistry ,Aminoacyltransferases ,0104 chemical sciences ,Cysteine Endopeptidases ,030104 developmental biology ,Biochemistry ,Cell culture ,Covalent bond ,Sortase A ,Proteome ,MCF-7 Cells ,Peptides ,Ligation - Abstract
The identification and quantification of low-abundant proteins are always impeded by high-abundant proteins in proteomic analysis because of the extreme complexity of peptide mixtures and wide dynamic range of protein abundances. Here, we developed a novel approach to enrich and quantify N-terminal glycine peptides through sortase A mediated ligation. This strategy was based on the formation of a covalent bond between the sortase A recognition motif LPXTG and a N-terminal glycine residue. Also, the quantification was achieved by introducing isotopically labeled threonine in the motif LPXTG. In this strategy, both the enrichment of N-terminal glycine peptides and the stable isotope labeling were achieved in a single step. We applied this approach for the proteome analysis of MCF-7 cell line. It was demonstrated a significant reduction in sample complexity via highly selective and efficient enrichment of N-terminal glycine peptides, thereby detecting lots of less abundant proteins and enhancing proteome coverage. In comparison to the untreated sample, an increase of 34% of proteins was additionally identified. Furthermore, 97% of proteins were successfully quantified with high accuracy. In summary, this quantitative N-terminal glycine peptides enrichment strategy is expected for high-throughput qualitative and quantitative proteomic analysis as a complementary approach to conventional shotgun proteomics.
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- 2018
36. Orosomucoid can predict baseline peritoneal transport characteristics in peritoneal dialysis patients and reduce peritoneal proteins loss
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Zhaoxing Sun, Xiaoqiang Ding, Jun Ji, Guoquan Yan, Jianzhou Zou, Lin Zhang, Xiaoxiao Yang, Xiaofang Yu, Bo Xiang, and Manchen Bao
- Subjects
0301 basic medicine ,Proteomics ,medicine.medical_specialty ,medicine.medical_treatment ,Biophysics ,Orosomucoid ,Peritoneal equilibration test ,Biochemistry ,Gastroenterology ,Peritoneal dialysis ,03 medical and health sciences ,Mice ,Peritoneum ,Serum biomarkers ,Tandem Mass Spectrometry ,Internal medicine ,Medicine ,Animals ,Humans ,Dialysis ,Training set ,030102 biochemistry & molecular biology ,biology ,business.industry ,030104 developmental biology ,medicine.anatomical_structure ,biology.protein ,OROSOMUCOID 2 ,business ,Peritoneal Dialysis - Abstract
Background: Peritoneal dialysis (PD) is a kind of replacement therapies for end-stage renal disease (ESRD) patients. In the first 4-8 weeks of PD therapy, the patients were given an empirical dialysis prescription due to unknown peritoneal transport characteristics.Methods: Proteomic analysis was used to identify serum biomarkers which can predict the baseline peritoneal transport characteristics. Results: A discovery set of serum samples from ESRD patients were collected. After 4-8 weeks of PD therapy, these patients were divided into three groups according to the peritoneal equilibration test (PET) results: high (H), high average (HA), low average and low (LA&L) transporters. A total of 1051 differential proteins were screened by nano High Performance Liquid Chromatography - Mass Spectrometry / Mass Spectrometry (Nano HPLC-MS/MS). The top two proteins among different peritoneal transport characteristics were Orosomucoid 2 (ORM2) and C-reactive protein (CRP). To verify the above biomarkers, a larger population of PD patients were enrolled. Immunoturbidimetric assay showed that CRP was significantly elevated in H group than LA&L group, consistent with the result of proteomic analysis. Western blot validated that ORM2 in serum was increased in LA&L group compared with H and HA group. The expression of ORM2 in peritoneum also altered progressively in three groups. At last, supplying exogenous ORM could actually change peritoneal substances transport by reducing peritoneal proteins loss, without causing an excessive pro-inflammatory response in mice.Conclusions: Serum ORM2 and CRP could be used as biomarkers to predict the baseline peritoneal transport characteristics, so as to guide the early PD treatment. ORM also raised the possibility of decreasing peritoneal proteins loss in PD patients.
- Published
- 2021
37. An improved comprehensive strategy for deep and quantitative N-glycomics based on optimization of sample preparation, isotope-based data quality control and quantification, new N-glycan libraries and new algorithms
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Min Zhang, Hannah Voss, Ling Lin, Christoph Krisp, Yudong Guan, Raphael Schuster, Guoquan Yan, Pengyuan Yang, Nicolle H. Packer, Wencong Cui, Huali Shen, Weiqian Cao, Morten Thaysen-Andersen, Hartmut Schlüter, Fan Yang, Jiaxiang Hu, and Marceline Manka Fuh
- Subjects
chemistry.chemical_classification ,Glycan ,biology ,Isotope ,Computer science ,Absolute quantification ,Glycomics ,chemistry.chemical_compound ,Ovalbumin ,Biosynthesis ,chemistry ,Data quality ,biology.protein ,Sample preparation ,Biomarker discovery ,Glycoprotein ,Algorithm ,After treatment - Abstract
Global in-depth analysis of N-glycosylation, as the most complex post-translational modification of proteins, is requiring methods being as sensitive, selective and reliable as possible. Here, an enhanced strategy for N-glycomics is presented comprising optimized sample preparation yielding enhanced glycoprotein recovery and permethylation efficiency, isotopic labelling for data quality control and relative quantification, integration of new N-glycan libraries (human and mouse), newly developed R-scripts matching experimental MS1 data to theoretical N-glycan compositions and bundled sequencing algorithms for MS2-based structural identification to ultimately enhance the coverage and accuracy of N-glycans. With this strategy the numbers of identified N-glycans are more than doubled compared with previous studies, exemplified by etanercept (more than 3-fold) and chicken ovalbumin (more than 2-fold) at nanogram level. The power of this strategy and applicability to biological samples is further demonstrated by comparative N-glycomics of human acute promyelocytic leukemia cells before and after treatment with all-trans retinoic acid, showing that N-glycan biosynthesis is slowed down and 57 species are significantly altered in response to the treatment. This improved analytical platform enables deep and accurate N-glycomics for glycobiological research and biomarker discovery.
- Published
- 2020
38. Nuclear dihydroxyacetone phosphate signals nutrient sufficiency and cell cycle phase to global histone acetylation
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Jun-Li Hou, Lei Zhang, Ting-Ting Fan, Shi-Min Zhao, Min Zhang, Yiyuan Yuan, Wei Xu, Cheng Zhang, Peng-Cheng Lin, Jian-Yuan Zhao, Meng Wang, Jun Yao, Yan-Peng An, Jiao-Jiao Zhang, Yun-Zi Mao, Yan Lin, and Guoquan Yan
- Subjects
DNA Replication ,Transcription, Genetic ,Endocrinology, Diabetes and Metabolism ,Cell Cycle Proteins ,Acetates ,Triosephosphate isomerase ,Cell cycle phase ,Histones ,chemistry.chemical_compound ,Physiology (medical) ,Internal Medicine ,Humans ,Phosphorylation ,Dihydroxyacetone phosphate ,Cell Nucleus ,biology ,Chemistry ,Cyclin-dependent kinase 2 ,Cell Cycle ,Acetylation ,Cell Biology ,Nutrients ,Cell cycle ,Chromatin ,Cell biology ,Cytosol ,Histone ,Dihydroxyacetone Phosphate ,biology.protein ,Signal Transduction - Abstract
Global histone acetylation varies with changes in the nutrient and cell cycle phases; however, the mechanisms connecting these variations are not fully understood. Herein, we report that nutrient-related and cell-cycle-regulated nuclear acetate regulates global histone acetylation. Histone deacetylation-generated acetate accumulates in the nucleus and induces histone hyperacetylation. The nuclear acetate levels were controlled by glycolytic enzyme triosephosphate isomerase 1 (TPI1). Cyclin-dependent kinase 2 (CDK2), which is phosphorylated and activated by nutrient-activated mTORC1, phosphorylates TPI1 Ser 117 and promotes nuclear translocation of TPI1, decreases nuclear dihydroxyacetone phosphate (DHAP) and induces nuclear acetate accumulation because DHAP scavenges acetate via the formation of 1-acetyl-DHAP. CDK2 accumulates in the cytosol during the late G1/S phases. Inactivation or blockade of nuclear translocation of TPI1 abrogates nutrient-dependent and cell-cycle-dependent global histone acetylation, chromatin condensation, gene transcription and DNA replication. These results identify the mechanism of maintaining global histone acetylation by nutrient and cell cycle signals. Zhang et al. identify a nuclear role for the glycolytic enzyme TPI1 in connecting nutrient status and cell cycle status to global histone acetylation.
- Published
- 2020
39. Disturbed mitochondrial acetylation in accordance with the availability of acetyl groups in hepatocellular carcinoma
- Author
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Xiaowen Hu, Guoquan Yan, Zhiyun Wei, Jie Jiang, Jing Li, Lin He, Dandan Wang, Ying Qing, Chunling Wan, Xuemei Tong, Hui Lin, Yan Gao, Xuhan Yang, Daizhan Zhou, Shujiao Du, Qingyu Wang, Juan Zhang, and Liya Sun
- Subjects
Adult ,Male ,Carcinoma, Hepatocellular ,Mitochondria, Liver ,medicine.disease_cause ,Acetyl Coenzyme A ,medicine ,Humans ,Epigenetics ,Nuclear protein ,Molecular Biology ,Principal Component Analysis ,biology ,ATP synthase ,Chemistry ,Liver Neoplasms ,Acetylation ,Cell Biology ,Metabolism ,Middle Aged ,Cell biology ,Histone ,Proteome ,biology.protein ,Molecular Medicine ,Female ,Carcinogenesis - Abstract
As an essential post-translational modification, acetylation participates in various cellular processes and shows aberrances during tumorigenesis. Owing to its modification substrate, acetyl-CoA, acetylation is postulated as a depot for acetyl groups and evolve to build a connection between epigenetics and metabolism. Here we depict a distinct acetylome atlas of hepatocellular carcinoma from the perspectives of both protein acetylation and acetyl-CoA metabolism. We found that tumor acetylome demonstrated a compartment-dependent alteration that the acetylation level of mitochondrial proteins tended to be decreased while nuclear proteins were highly acetylated. In addition, elevated expression of ATP-citrate synthase (ACLY) was observed in tumors, which would facilitate histone acetylation by transporting mitochondrial acetyl coenzyme A to the nucleus. A hypothetical model of the oncogenic acetylome was proposed that growing demands for histone acetylation in tumor cells would drive the relocalization of acetyl-CoA to the nucleus, which may contribute to the global deacetylation of mitochondrial proteins to support the nuclear acetyl-CoA pool in an ACLY-dependent manner. Our findings are thought-provoking on the potential linkage between epigenetics and metabolism in the progression of tumorigenesis.
- Published
- 2020
40. Rapid and Easy Enrichment Strategy for Naturally Acetylated N Termini Based on LysN Digestion and Amine-Reactive Resin Capture
- Author
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Xiaoxian Du, Lei Zhang, Guoquan Yan, Huimin Bao, Jingtian Lu, Lijun Yang, Haojie Lu, and Caiyun Fang
- Subjects
Protease ,medicine.medical_treatment ,Lysine ,010401 analytical chemistry ,Acetylation ,Alkylation ,010402 general chemistry ,Mass spectrometry ,Proteomics ,Trypsin ,01 natural sciences ,0104 chemical sciences ,Analytical Chemistry ,chemistry.chemical_compound ,Resins, Synthetic ,Biochemistry ,chemistry ,medicine ,Humans ,Amine gas treating ,Amines ,Derivatization ,medicine.drug ,HeLa Cells - Abstract
Protein N-terminal acetylation (Nα-acetylation) is one of the most common modifications in both eukaryotes and prokaryotes. Although studies have shown that Nα-acetylation plays important roles in protein assembly, stability, and location, the physiological role has not been fully elucidated. Therefore, a robust and large-scale analytical method is important for a better understanding of Nα-acetylation. Here, an enrichment strategy was presented based on LysN digestion and amine-reactive resin capture to study naturally acetylated protein N termini. Since LysN protease cleaves at the amino-terminus of the lysine residue, all resulting peptides except naturally acetylated N-terminal peptides contain free amino groups and can be removed by coupling with AminoLink Resin. Therefore, the naturally acetylated N-terminal peptides were left in solution and enriched for further liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The method was very simple and fast, which contained no additional chemical derivatization except protein reduction and alkylation necessarily needed in bottom-up proteomics. It could be used to study acetylated N termini from complex biological samples without bias toward different peptides with various physicochemical properties. The enrichment specificity was above 99% when it was applied in HeLa cell lysates. Neo-N termini generated by endogenous degradation could be directly distinguished without the use of stable-isotope labeling because no chemical derivatization was introduced in this method. Furthermore, this method was highly complementary to the traditional analytical methods for protein N termini based on trypsin only with ArgC-like activity. Therefore, the described method was beneficial to naturally acetylated protein N termini profiling.
- Published
- 2020
41. Ribosome 18S m
- Author
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Bowen, Rong, Qian, Zhang, Jinkai, Wan, Shenghui, Xing, Ruofei, Dai, Yuan, Li, Jiabin, Cai, Jiaying, Xie, Yang, Song, Jiawei, Chen, Lei, Zhang, Guoquan, Yan, Wen, Zhang, Hai, Gao, Jing-Dong J, Han, Qianhui, Qu, Honghui, Ma, Ye, Tian, and Fei, Lan
- Subjects
Adenosine ,Breast Neoplasms ,Cell Growth Processes ,Methyltransferases ,Methylation ,HEK293 Cells ,Cell Line, Tumor ,MCF-7 Cells ,RNA, Ribosomal, 18S ,Animals ,Humans ,Female ,Caenorhabditis elegans ,HeLa Cells - Abstract
N6 methylation at adenosine 1832 (m
- Published
- 2020
42. Comparative Proteomics of Extended-Spectrum Cephalosporin-Resistant Neisseria gonorrhoeae Isolates Demonstrates Altered Protein Synthesis, Metabolism, Substance Transport, and Membrane Permeability
- Author
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Yang Yang, Nannan Diao, Ying Wang, Yuan Dong, Guoquan Yan, and Weiming Gu
- Subjects
Microbiology (medical) ,Membrane permeability ,Quantitative proteomics ,lcsh:QR1-502 ,ATP-binding cassette transporter ,Biology ,urologic and male genital diseases ,medicine.disease_cause ,Proteomics ,Microbiology ,lcsh:Microbiology ,comparative proteomics ,03 medical and health sciences ,Antibiotic resistance ,extended-spectrum cephalosporin resistance ,medicine ,030304 developmental biology ,0303 health sciences ,030306 microbiology ,Translation (biology) ,Ribosomal RNA ,Neisseria gonorrhoeae ,female genital diseases and pregnancy complications ,iTRAQ ,membrane permeability ,metabolism - Abstract
Neisseria gonorrhoeae isolates exhibit resistance to extended-spectrum cephalosporins (ESCs), the last remaining option for first-line empirical monotherapy. Here, we investigated the proteomic profiles of N. gonorrhoeae clinical isolates with ESC-resistance to support exploration of the antimicrobial resistance mechanisms for N. gonorrhoeae. We used comparative iTRAQ quantitative proteomics to investigate differential protein expression of three ESC-resistant N. gonorrhoeae clinical isolates using N. gonorrhoeae ATCC49226 as a reference strain. The expression of 40 proteins was downregulated and expression of 56 proteins was upregulated in all three ESC-resistant N. gonorrhoeae isolates. Proteins with predicted function of translation, ribosomal structure and biogenesis, as well as components of the Type IV secretory systems, were significantly upregulated. Two differentially expressed proteins of ABC transporters were also reported by other teams in proteomics studies of N. gonorrhoeae isolates under antimicrobial stress conditions. Differentially expressed proteins are involved in energy production and metabolism of carbohydrates and amino acids. Our results indicated that amino acid and carbohydrate metabolism, cell membrane structure, interbacterial DNA transfer, and ribosome components might be involved in mediating ESC-resistance in N. gonorrhoeae. These findings facilitate a better understanding of the mechanisms of ESC-resistance in N. gonorrhoeae and provide useful information for identifying novel targets in the development of antimicrobials against N. gonorrhoeae.
- Published
- 2020
43. Multilayered N-Glycoproteome Profiling Reveals Highly Heterogeneous and Dysregulated Protein N-Glycosylation Related to Alzheimer's Disease
- Author
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Mingqi Liu, Yang Zhang, Guoquan Yan, Zhong-Feng Wang, Lei Zhang, Jun Yao, Yi-Teng Xu, Shao-Ming Sang, Xing Gao, Juanjuan Xie, Huali Shen, Lujie Yang, Pengyuan Yang, Wen-Jing Qian, and Pan Fang
- Subjects
Proteomics ,Glycan ,Glycosylation ,Proteome ,Transgene ,Mice, Transgenic ,Disease ,Computational biology ,010402 general chemistry ,01 natural sciences ,Analytical Chemistry ,Cell Line ,Pathogenesis ,chemistry.chemical_compound ,Mice ,N-linked glycosylation ,Alzheimer Disease ,Polysaccharides ,Tandem Mass Spectrometry ,Animals ,Humans ,Glycoproteins ,Brain Chemistry ,biology ,010401 analytical chemistry ,Glutamate receptor ,Glycopeptides ,Brain ,0104 chemical sciences ,carbohydrates (lipids) ,chemistry ,biology.protein ,Protein Processing, Post-Translational - Abstract
Protein N-glycosylation is ubiquitous in the brain and is closely related to cognition and memory. Alzheimer's disease (AD) is a multifactorial disorder that lacks a clear pathogenesis and treatment. Aberrant N-glycosylation has been suggested to be involved in AD pathology. However, the systematic variations in protein N-glycosylation and their roles in AD have not been thoroughly investigated due to technical challenges. Here, we applied multilayered N-glycoproteomics to quantify the global protein expression levels, N-glycosylation sites, N-glycans, and site-specific N-glycopeptides in AD (APP/PS1 transgenic) and wild-type mouse brains. The N-glycoproteomic landscape exhibited highly complex site-specific heterogeneity in AD mouse brains. The generally dysregulated N-glycosylation in AD, which involved proteins such as glutamate receptors as well as fucosylated and oligomannose glycans, were explored by quantitative analyses. Furthermore, functional studies revealed the crucial effects of N-glycosylation on proteins and neurons. Our work provides a systematic multilayered N-glycoproteomic strategy for AD and can be applied to diverse biological systems.
- Published
- 2019
44. Comparative Proteomics of Extended-Spectrum Cephalosporin-Resistant
- Author
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Nannan, Diao, Guoquan, Yan, Yang, Yang, Yuan, Dong, Ying, Wang, and Weiming, Gu
- Subjects
comparative proteomics ,transportation ,iTRAQ ,membrane permeability ,protein synthesis ,extended-spectrum cephalosporin resistance ,urologic and male genital diseases ,Microbiology ,metabolism ,female genital diseases and pregnancy complications ,Neisseria gonorrhoeae ,Original Research - Abstract
Neisseria gonorrhoeae isolates exhibit resistance to extended-spectrum cephalosporins (ESCs), the last remaining option for first-line empirical monotherapy. Here, we investigated the proteomic profiles of N. gonorrhoeae clinical isolates with ESC-resistance to support exploration of the antimicrobial resistance mechanisms for N. gonorrhoeae. We used comparative iTRAQ quantitative proteomics to investigate differential protein expression of three ESC-resistant N. gonorrhoeae clinical isolates using N. gonorrhoeae ATCC49226 as a reference strain. The expression of 40 proteins was downregulated and expression of 56 proteins was upregulated in all three ESC-resistant N. gonorrhoeae isolates. Proteins with predicted function of translation, ribosomal structure and biogenesis, as well as components of the Type IV secretory systems, were significantly upregulated. Two differentially expressed proteins of ABC transporters were also reported by other teams in proteomics studies of N. gonorrhoeae isolates under antimicrobial stress conditions. Differentially expressed proteins are involved in energy production and metabolism of carbohydrates and amino acids. Our results indicated that amino acid and carbohydrate metabolism, cell membrane structure, interbacterial DNA transfer, and ribosome components might be involved in mediating ESC-resistance in N. gonorrhoeae. These findings facilitate a better understanding of the mechanisms of ESC-resistance in N. gonorrhoeae and provide useful information for identifying novel targets in the development of antimicrobials against N. gonorrhoeae.
- Published
- 2019
45. Magnetic capture of polydopamine-encapsulated Hela cells for the analysis of cell surface proteins
- Author
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Mingxia Gao, Yiying Liu, Guoquan Yan, and Xiangmin Zhang
- Subjects
Proteomics ,0301 basic medicine ,Indoles ,Polymers ,Cell ,Biophysics ,Biocompatible Materials ,Cell Surface Proteins ,010402 general chemistry ,01 natural sciences ,Biochemistry ,HeLa ,Magnetics ,03 medical and health sciences ,Membrane Microdomains ,Coated Materials, Biocompatible ,medicine ,Humans ,Protein Interaction Domains and Motifs ,Epidermal growth factor receptor ,STAT3 ,biology ,Membrane Proteins ,biology.organism_classification ,Molecular biology ,0104 chemical sciences ,030104 developmental biology ,medicine.anatomical_structure ,Membrane ,Membrane protein ,biology.protein ,Magnetic nanoparticles ,HeLa Cells - Abstract
A novel method to characterize cell surface proteins and complexes has been developed. Polydopamine (PDA)-encapsulated Hela cells were prepared for plasma membrane proteome research. Since the PDA protection, the encapsulated cells could be maintained for more than two weeks. Amino groups functionalized magnetic nanoparticles were also used for cell capture by the reaction with the PDA coatings. Plasma membrane fragments were isolated and enriched with assistance of an external magnetic field after disruption of the coated cells by ultrasonic treatment. Plasma membrane proteins (PMPs) and complexes were well preserved on the fragments and identified by shot-gun proteomic analytical strategy. 385 PMPs and 1411 non-PMPs were identified using the method. 85.2% of these PMPs were lipid-raft associated proteins. Ingenuity Pathway Analysis was employed for bio-information extraction from the identified proteins. It was found that 653 non-PMPs had interactions with 140 PMPs. Among them, epidermal growth factor receptor and its complexes, and a series of important pathways including STAT3 pathway were observed. All these results demonstrated that the new approach is of great importance in applying to the research of physiological function and mechanism of the plasma membrane proteins. Significance This work developed a novel strategy for the proteomic analysis of cell surface proteins. According to the results, 73.3% of total identified proteins were lipid-raft associated proteins, which imply that the proposed method is of great potential in the identification of lipid-raft associated proteins. In addition, a series of protein-protein interactions and pathways related to Hela cells were pointed out. All these results demonstrated that our proposed approach is of great importance and could well be applied to the physiological function and mechanism research of plasma membrane proteins.
- Published
- 2018
46. Highly efficient enrichment of low-abundance intact proteins by core-shell structured Fe3O4-chitosan@graphene composites
- Author
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Mingxia Gao, Xiaoni Fang, Guoquan Yan, Peng Zhang, and Xiangmin Zhang
- Subjects
Detection limit ,Graphene ,Composite number ,02 engineering and technology ,010402 general chemistry ,021001 nanoscience & nanotechnology ,Proteomics ,01 natural sciences ,Blood proteins ,0104 chemical sciences ,Analytical Chemistry ,law.invention ,Chitosan ,chemistry.chemical_compound ,Adsorption ,chemistry ,law ,Graphite ,Composite material ,0210 nano-technology - Abstract
In proteomics research, the screening and monitoring of disease biomarkers is still a major challenge, mainly due to their low concentration in biological samples. However, the universal enrichment of intact proteins has not been further studied. In this work, we developed a Fe3O4-chitosan@graphene (Fe3O4-CS@G) core-shell composite to enrich low-abundance proteins from biological samples. Fe3O4-CS@G composite holds chitosan layer decorated Fe3O4 core, which improves the hydrophilicity of materials greatly. Meanwhile, the graphene nanosheets shell formed via electrostatic assembly endows the composite with huge surface area (178m2/g). The good water dispersibility ensures the sufficient contact opportunities between graphene composites and proteins, and the large surface area provides enough adsorption sites for the enrichment of proteins. Using Fe3O4-CS@G, four standard proteins Cyt-c, BSA, Myo and OVA were enriched with better adsorption capacity and recovery rate, compared with previously reported magnetic graphene composites. Additionally, the mechanism of compared to" is corrected into "compared with". proteins adsorption on Fe3O4-CS@G was further studied, which indicates that hydrophobic and electrostatic interaction work together to facilitate the universal and efficient enrichment of proteins. Human plasma sample was employed to further evaluate the enrichment performance of Fe3O4-CS@G. Eventually, 123 proteins were identified from one of SAX fractions of human plasma, which is much better than commercial Sep-pak C18 enrichment column (39 proteins). All these outstanding performances suggest that Fe3O4-CS@G is an ideal platform for the enrichment of low-abundance intact proteins and thus holds great potential to facilitate the identification of biomarkers from biological samples in proteomics research.
- Published
- 2017
47. Site-Specific Quantification of Protein Ubiquitination on MS2 Fragment Ion Level via Isobaric Peptide Labeling
- Author
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Guoquan Yan, Ting Cao, Huimin Bao, Ying Zhang, Caiyun Fang, Haojie Lu, and Lei Zhang
- Subjects
0301 basic medicine ,chemistry.chemical_classification ,Binding Sites ,Staining and Labeling ,biology ,Chemistry ,Quantitative proteomics ,Ubiquitination ,Peptide ,Peptide Fragments ,Protein ubiquitination ,Analytical Chemistry ,03 medical and health sciences ,030104 developmental biology ,Ubiquitin ,Biochemistry ,MCF-7 Cells ,biology.protein ,Humans ,Isobaric process ,Amino Acid Sequence ,Binding site ,Peptide sequence ,Quantitative analysis (chemistry) - Abstract
Proteome-wide quantitative analysis of protein ubiquitination is important to gain insight into its various cellular functions. However, it is still challenging to monitor how ubiquitination at each individual lysine residue is independently regulated, especially the whereabouts of peptides containing more than one ubiquitination site. In recent years, isobaric peptide termini labeling has been considered a promising strategy in quantitative proteomics, benefiting from its high accuracy by quantifying with a series of b, y fragment ion pairs. Herein, we extended the concept of isobaric peptide termini labeling to large-scale quantitative analysis of protein ubiquitination. A novel MS2 fragment ion based quantitative approach was developed, allowing the quantification of ubiquitination at site level via isobaric K-ε-GG peptide labeling, which combined metabolic labeling, K-ε-GG immunoaffinity enrichment, and site-selective N-terminus dimethylation. The feasibility of this proposed strategy was demonstrated through the ubiquitin proteome analysis of differently labeled MCF-7 cell digests. As a result, 2970 unique K-ε-GG peptides of 1383 proteins containing 2874 ubiquitinated sites were confidently quantified with high accuracy and sensitivity. In addition, we demonstrated that quantification on MS2 fragment ion level makes it possible to precisely quantify each individual ubiquitinated lysine residue in 39 K-ε-GG peptides bearing two ubiquitination sites by the use of specific ubiquitinated b, y ion pairs. It is expected that this proposed approach will serve as a powerful tool to quantify ubiquitination at the site level, especially for those multiubiquitinated peptides.
- Published
- 2017
48. Strategy for high-throughput identification of protein complexes by array-based multi-dimensional liquid chromatography-mass spectrometry
- Author
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Xuantang Wang, Haoyang Zheng, Mingxia Gao, Xiangmin Zhang, and Guoquan Yan
- Subjects
Chromatography ,Elution ,Chemistry ,010401 analytical chemistry ,Organic Chemistry ,General Medicine ,010402 general chemistry ,Mass spectrometry ,Proteomics ,01 natural sciences ,Biochemistry ,0104 chemical sciences ,Analytical Chemistry ,Protein–protein interaction ,Identification (information) ,Tandem Mass Spectrometry ,Liquid chromatography–mass spectrometry ,Multiprotein Complexes ,Protein Interaction Mapping ,Multi dimensional ,Humans ,Throughput (business) ,Chromatography, Liquid ,HeLa Cells - Abstract
Comprehensive elucidation of the composition of multiprotein complexes in model organisms is essential to understand conserved biological systems, but large-scale mapping physical association networks is still challenging due to limited throughput of present methods. In this work, a strategy coupling array-based online two-dimensional liquid chromatography (array-based 2D-LC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was demonstrated for high throughput and in-depth identification of protein complexes from cultured human HeLa cell extracts. Mixed-bed ion-exchange column was employed as the first dimensional (1stD) separating mode and an array consisting of eight reversed phase columns was developed as the second dimensional (2ndD) mode. Taking advantage of array parallel strategy, this online system showed an 8-fold increase in throughput. After array-based online 2D-LC separation, altogether 256 × 2ndD fractions were collected for further LC-MS/MS analysis. Public databases of protein-protein interaction (PPI) and co-elution curves identified by LC-MS were applied to reconstruct the protein complexes. A rigorous inspection was operated by cataloging the protein complexes into chromatographic fractions to minimize the number of false positives. As result, a total number of 4,436 proteins were identified and 26,092 elution curves were graphed. A network consisting of 47,745 PPIs was established among 2,201 proteins and presented 1,530 putative protein complexes with high confidence. Most of the identified PPIs were linked to diverse biological processes and may reveal further disease mechanism and therapeutic strategy.
- Published
- 2021
49. pGlyco 2.0 enables precision N-glycoproteomics with comprehensive quality control and one-step mass spectrometry for intact glycopeptide identification
- Author
-
Guoquan Yan, Zhang Xiaojin, Catherine C. L. Wong, Wen-Feng Zeng, Meng-Qiu Dong, Yong Cao, Pengyuan Yang, Chao Peng, Si-Min He, Huali Shen, Mingqi Liu, Chao Liu, Pan Fang, Jianqiang Wu, Weiqian Cao, Yang Zhang, Hui-Jun Tu, Hao Chi, Rui-Xiang Sun, Biyun Jiang, and Jiangming Huang
- Subjects
0301 basic medicine ,False discovery rate ,Male ,Proteomics ,Quality Control ,Glycan ,Glycosylation ,Science ,General Physics and Astronomy ,Computational biology ,Biology ,Bioinformatics ,Mass spectrometry ,General Biochemistry, Genetics and Molecular Biology ,Article ,Workflow ,03 medical and health sciences ,chemistry.chemical_compound ,Search engine ,Polysaccharides ,Tandem Mass Spectrometry ,Animals ,Humans ,lcsh:Science ,Carbon Isotopes ,Multidisciplinary ,Nitrogen Isotopes ,Glycopeptides ,General Chemistry ,Glycopeptide ,Glycoproteomics ,High-Throughput Screening Assays ,Mice, Inbred C57BL ,Search Engine ,030104 developmental biology ,chemistry ,Proteome ,biology.protein ,lcsh:Q ,Protein Processing, Post-Translational ,Software - Abstract
The precise and large-scale identification of intact glycopeptides is a critical step in glycoproteomics. Owing to the complexity of glycosylation, the current overall throughput, data quality and accessibility of intact glycopeptide identification lack behind those in routine proteomic analyses. Here, we propose a workflow for the precise high-throughput identification of intact N-glycopeptides at the proteome scale using stepped-energy fragmentation and a dedicated search engine. pGlyco 2.0 conducts comprehensive quality control including false discovery rate evaluation at all three levels of matches to glycans, peptides and glycopeptides, improving the current level of accuracy of intact glycopeptide identification. The N-glycoproteome of samples metabolically labeled with 15N/13C were analyzed quantitatively and utilized to validate the glycopeptide identification, which could be used as a novel benchmark pipeline to compare different search engines. Finally, we report a large-scale glycoproteome dataset consisting of 10,009 distinct site-specific N-glycans on 1988 glycosylation sites from 955 glycoproteins in five mouse tissues., Protein glycosylation is a heterogeneous post-translational modification that generates greater proteomic diversity that is difficult to analyze. Here the authors describe pGlyco 2.0, a workflow for the precise one step identification of intact N-glycopeptides at the proteome scale.
- Published
- 2017
50. Selective enrichment of glycopeptides/phosphopeptides using Fe 3 O 4 @Au-B(OH) 2 @mTiO 2 core-shell microspheres
- Author
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Chunhui Deng, Dongpo Xu, Mingxia Gao, Xiangmin Zhang, and Guoquan Yan
- Subjects
chemistry.chemical_classification ,Chromatography ,biology ,Chemistry ,010401 analytical chemistry ,Peptide ,010402 general chemistry ,01 natural sciences ,Horseradish peroxidase ,Glycopeptide ,0104 chemical sciences ,Analytical Chemistry ,chemistry.chemical_compound ,Phosphoprotein ,biology.protein ,Bovine serum albumin ,Bifunctional ,Selectivity ,Boronic acid - Abstract
In this work, the bifunctional Fe3O4@Au-B(OH)2@mTiO2 core-shell core-shell microspheres were designed and synthesized for the selective enrichment of glycopeptides/ phosphopeptides. Due to the bifunctional property of the titanium dioxide and the boronic acid group, the microspheres were successfully applied to the enrichment of phosphopeptides and glycopeptides, evaluated by capturing phosphopeptides from tryptic digestion of model phosphoprotein bovine β-casein diluted to 2.0pgμL-1 (8.0×10-17molμL-1) and glycopeptides from tryptic digestion of model glycoprotein horseradish peroxidase (HRP) diluted to 80pgμL-1 (2.0×10-15molμL-1). The enrichment selectivity of the bifunctional microspheres was evaluated by capturing phosphopeptides from a peptide mixture of β-casein and bovine serum albumin (BSA) with the molar ratio of 1:1000 (4.2×10-12mol of β-casein and 4.2×10-9mol of BSA in 100μL) and glycopeptides from a peptide mixture of HRP and BSA up to the ratio of 1:100 (5.0×10-12mol of HRP and 5.0×10-10mol of BSA in 100μL).
- Published
- 2017
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