Publications by authors named "Fuquan Yang"

130 Publications

Comprehensive analysis of glycosphingolipid glycans by lectin microarrays and MALDI-TOF mass spectrometry.

Nat Protoc 2021 Jun 7. Epub 2021 Jun 7.

Laboratory for Functional Glycomics, College of Life Sciences, Northwest University, Xi'an, China.

Glycosphingolipids (GSLs) are ubiquitous glycoconjugates present on the cell membrane; they play significant roles in many bioprocesses such as cell adhesion, embryonic development, signal transduction and carcinogenesis. Analyzing such amphiphilic molecules is a major challenge in the field of glycosphingolipidomics. We provide a step-by-step protocol that uses a lectin microarray to analyze GSL glycans from cultured cells. The procedure describes (i) extraction of GSLs from cell pellets, (ii) N-monodeacylation using sphingolipid ceramide N-deacylase digestion to form lyso-GSLs, (iii) fluorescence labeling at the newly exposed amine group, (iv) preparation of a lectin microarray, (v) GSL-glycan analysis by a lectin microarray, (vi) complementary mass spectrometry analysis and (vii) data acquisition and analysis. This method is high-throughput, low cost and easy to conduct, and it provides detailed information about glycan linkages. This protocol takes ~10 d.
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http://dx.doi.org/10.1038/s41596-021-00544-yDOI Listing
June 2021

PHF8-promoted TOPBP1 demethylation drives ATR activation and preserves genome stability.

Sci Adv 2021 05 5;7(19). Epub 2021 May 5.

State Key Laboratory of Experimental Hematology, 2011 Collaborative Innovation Center of Tianjin for Medical Epigenetics, Key Laboratory of Breast Cancer Prevention and Therapy (Ministry of Education), Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University Cancer Institute and Hospital, Tianjin Medical University General Hospital, Tianjin Medical University, Tianjin 300070, China.

The checkpoint kinase ATR [ATM (ataxia-telangiectasia mutated) and rad3-related] is a master regulator of DNA damage response. Yet, how ATR activity is regulated remains to be investigated. We report here that histone demethylase PHF8 (plant homeodomain finger protein 8) plays a key role in ATR activation and replication stress response. Mechanistically, PHF8 interacts with and demethylates TOPBP1 (DNA topoisomerase 2-binding protein 1), an essential allosteric activator of ATR, under unperturbed conditions, but replication stress results in PHF8 phosphorylation and dissociation from TOPBP1. Consequently, hypomethylated TOPBP1 facilitates RAD9 (RADiation sensitive 9) binding and chromatin loading of the TOPBP1-RAD9 complex to fully activate ATR and thus safeguard the genome and protect cells against replication stress. Our study uncovers a demethylation and phosphorylation code that controls the assembly of TOPBP1-scaffolded protein complex, and provides molecular insight into non-histone methylation switch in ATR activation.
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http://dx.doi.org/10.1126/sciadv.abf7684DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8099190PMC
May 2021

Proteomics reveals the function reverse of MPSSS-treated prostate cancer-associated fibroblasts to suppress PC-3 cell viability via the FoxO pathway.

Cancer Med 2021 04 11;10(7):2509-2522. Epub 2021 Mar 11.

Key Laboratory of Protein and Peptide Pharmaceuticals & Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.

Prostate cancer-associated fibroblasts (prostate CAFs) are essential components of the tumor microenvironment and can promote tumor progression through their immunosuppressive functions. MPSSS, a novel polysaccharide purified from Lentinus edodes, has been reported to have anti-tumor activity. MPSSS could also inhibit the immunosuppressive function of prostate CAFs, which has been demonstrated through that the secretome of MPSSS-treated prostate CAFs could inhibit the proliferation of T cells. However, how the secretome of MPSSS-treated prostate CAFs influence prostate cancer progression is still unclear. Interestingly, we found that the low molecular weight (3-100kD) secretome of prostate CAFs (lmwCAFS) could promote the growth of PC-3 cells, while that of MPSSS-treated prostate CAFs (MT-lmwCAFS) could inhibit their growth. We carried out comparative secretomic analysis of lmwCAFS and MT-lmwCAFS to identify functional molecules that inhibit the growth of PC-3 cells, and proteomic analysis of lmwCAFS-treated PC-3 cells and MT-lmwCAFS-treated PC-3 cells to investigate the underlying molecular mechanism. These analyses suggest that TGF-β3 from MT-lmwCAFS may inhibit the growth of PC-3 cells. The validated experiments revealed that TGF-β3 from MT-lmwCAFS activated p21 expression in PC-3 cells by regulating the FoxO pathway thereby inducing G0/G1 cell cycle arrest of PC-3 cells. Overall, our data demonstrated that MPSSS reversed the ability of prostate CAFs to suppress the cell viability of PC-3 cells, which might provide a potential therapeutic strategy to prevent prostate cancer progression.
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http://dx.doi.org/10.1002/cam4.3825DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7982613PMC
April 2021

Oleic Acid and Eicosapentaenoic Acid Reverse Palmitic Acid-induced Insulin Resistance in Human HepG2 Cells via the Reactive Oxygen Species / JUN Pathway.

Genomics Proteomics Bioinformatics 2021 Feb 22. Epub 2021 Feb 22.

Key Laboratory of Protein and Peptide Pharmaceuticals & Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China. Electronic address:

The monounsaturated fatty acid (MUFA) oleic acid (OA) has previously been shown to reverse saturated fatty acid palmitic acid (PA)-induced hepatic insulin resistance (IR). However, its underlying molecular mechanism is unclear. In addition, previous studies have also shown that the ω-3 polyunsaturated fatty acid (PUFA) eicosapentaenoic acid (EPA) reverses PA-induced muscle IR, but whether EPA plays the same role in hepatic IR and its possible mechanism involved need to be further clarified. Here, we confirmed that EPA reversed PA-induced IR in HepG2 cells and compared the proteomic changes after treatment with different free fatty acids (FFAs). A total of 234 proteins were determined to be differentially expressed after MUFA treatment. Their functions were mainly related to responses to stress and endogenous stimuli, lipid metabolic process, and protein binding. For PUFA treatment, the PA-induced expression changes of 1326 proteins could be reversed by EPA treatment, 415 of which were mitochondrial proteins, with most of the functional proteins involved in oxidative phosphorylation (OXPHOS) and tricarboxylic acid (TCA) cycle. Mechanistic studies revealed that the protein encoded by JUN and reactive oxygen species (ROS) play a role in OA- and EPA-reversed PA-induced IR, respectively. EPA or OA alleviated PA-induced abnormal adenosine triphosphate (ATP) production, ROS generation, and calcium (Ca) content. Importantly, HO-activated production of ROS increased the protein expression of JUN, further resulting in IR in HepG2 cells. Taken together, we demonstrate that ROS/JUN is a common response pathway employed by HepG2 cells toward FFA-regulated IR.
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http://dx.doi.org/10.1016/j.gpb.2019.06.005DOI Listing
February 2021

Antidiabetic Effects of Gegen Qinlian Decoction via the Gut Microbiota Are Attributable to Its Key Ingredient Berberine.

Genomics Proteomics Bioinformatics 2020 Dec 24. Epub 2020 Dec 24.

CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China. Electronic address:

Gegen Qinlian Decoction (GQD), a traditional Chinese medicine (TCM) formula, has long been used for the treatment of common metabolic diseases, including type 2 diabetes mellitus. However, the main limitation of its wider application is ingredient complexity of this formula. Thus, it is critically important to identify the major active ingredients of GQD and to illustrate mechanisms underlying its action. Here, we compared the effects of GQD and berberine, a hypothetical key active pharmaceutical ingredient of GQD, on a diabetic rat model by comprehensive analyses of gut microbiota, short-chain fatty acids, proinflammatory cytokines, and ileum transcriptomics. Our results show that berberine and GQD had similar effects on lowering blood glucose levels, modulating gut microbiota, inducing ileal gene expression, as well as relieving systemic and local inflammation. As expected, both berberine and GQD treatment significantly altered the overall gut microbiota structure and enriched many butyrate-producing bacteria, including Faecalibacterium and Roseburia, thereby attenuating intestinal inflammation and lowering glucose. Levels of short-chain fatty acids in rat feces were also significantly elevated after treatment with berberine or GQD. Moreover, concentration of serum proinflammatory cytokines and expression of immune-related genes, including Nfkb1, Stat1, and Ifnrg1, in pancreatic islets were significantly reduced after treatment. Our study demonstrates that the main effects of GQD can be attributed to berberine via modulating gut microbiota. The strategy employed would facilitate further standardization and widespread application of TCM in many diseases.
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http://dx.doi.org/10.1016/j.gpb.2019.09.007DOI Listing
December 2020

Comprehensive Analysis of the Proteome and PTMomes of C2C12 Myoblasts Reveals that Sialylation Plays a Role in the Differentiation of Skeletal Muscle Cells.

J Proteome Res 2021 01 20;20(1):222-235. Epub 2020 Nov 20.

Key Laboratory of Protein and Peptide Pharmaceuticals & Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.

The C2C12 myoblast is a model that has been used extensively to study the process of skeletal muscle differentiation. Proteomics has advanced our understanding of skeletal muscle biology and also the differentiation process of skeletal muscle cells. However, there is still no comprehensive analysis of C2C12 myoblast proteomes, which is important for the understanding of key drivers for the differentiation of skeletal muscle cells. Here, we conducted multidimensional proteome profiling to get a comprehensive analysis of proteomes and PTMomes of C2C12 myoblasts with a TiSH strategy. A total of 8313 protein groups were identified, including 7827 protein groups from nonmodified peptides, 3803 phosphoproteins, and 977 formerly sialylated N-linked glycoproteins. Integrated analysis of proteomic and PTMomic data showed that almost all of the kinases and transcription factors in the muscle cell differentiation pathway were phosphorylated. Further analysis indicated that sialylation might play a role in the differentiation of C2C12 myoblasts. Further functional analysis demonstrated that C2C12 myoblasts showed a decreased level of sialylation during skeletal muscle cell differentiation. Inhibition of sialylation with the sialyltransferase inhibitor 3Fax-Neu5Ac resulted in the lower expression of MHC and suppression of myoblast fusion. In all, these results indicate that sialylation has an effect on the differentiation of skeletal muscle cells.
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http://dx.doi.org/10.1021/acs.jproteome.0c00353DOI Listing
January 2021

The future of Extracellular Vesicles as Theranostics - an ISEV meeting report.

J Extracell Vesicles 2020 Sep 4;9(1):1809766. Epub 2020 Sep 4.

Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, China.

The utilization of extracellular vesicles (EVs) in clinical theranostics has rapidly advanced in the past decade. In November 2018, the International Society for Extracellular Vesicles (ISEV) held a workshop on "EVs in Clinical Theranostic". Here, we report the conclusions of roundtable discussions on the current advancement in the analysis technologies and we provide some guidelines to researchers in the field to consider the use of EVs in clinical application. The main challenges and the requirements for EV separation and characterization strategies, quality control and clinical investigation were discussed to promote the application of EVs in future clinical studies.
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http://dx.doi.org/10.1080/20013078.2020.1809766DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7580849PMC
September 2020

Separation and characterization of extracellular vesicles from human plasma by asymmetrical flow field-flow fractionation.

Anal Chim Acta 2020 Aug 11;1127:234-245. Epub 2020 Jul 11.

Laboratory of Protein and Peptide Pharmaceuticals & Proteomics Laboratory, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China; University of Chinese Academy of Sciences, Beijing, 100049, China. Electronic address:

It is a big challenge to isolate extracellular vesicles (EVs) from human plasma because of the contamination from high abundant lipoproteins, such as high density lipoprotein (HDL) and low density lipoprotein particles (LDL). In this study, the parameters of asymmetrical flow field-flow fractionation (AF4) technology and sample preparation, including cross flow gradient, focusing time, ultrafiltration condition, sample amount and injection volume have been optimized and successfully utilized for the separation and characterization of EVs from human plasma. This study demonstrated that the great potential of AF4 in the separation of EVs from HDL and LDL in human plasma with high reproducibility and purity. This study indicated excessive focusing time in the AF4 separation and 100-300 kDa MWCO membrane based ultrafiltration in the pre-preparation will cause loss of EVs. A total of 1038 proteins have been identified in seven replicates of purified EVs from pooled human plasma sample. They are mainly enriched in extracellular exosomes, involved in extracellular matrix structural constituent, and associated with extracellular matrix-receptor interaction pathway. This study also indicated that human plasma contains more EVs than the paired serum at the same volume, and showed age- and gender-independent individual variability of the amount of EVs in human plasma. This study displayed that AF4 technique can serve as a powerful platform for the separation of EVs from human plasma, serum or human body fluids and this technology will promote the studies on EVs, such as proteomics, biomarker discovery and functions.
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http://dx.doi.org/10.1016/j.aca.2020.06.071DOI Listing
August 2020

Sequential Precipitation and Delipidation Enables Efficient Enrichment of Low-Molecular Weight Proteins and Peptides from Human Plasma.

J Proteome Res 2020 08 3;19(8):3340-3351. Epub 2020 Jul 3.

Key Laboratory of Protein and Peptide Pharmaceuticals & Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.

Low-molecular weight proteins and peptides (LMWPs, <30 kDa) in human plasma serve as potential biomarkers or drug targets and are endowed with desirable traits for biological and clinical studies. However, the identification of LMWPs from plasma is retarded by high-abundance proteins, high-molecular weight proteins, and lipids. Here, we present a sequential precipitation and delipidation (SPD) method for the efficient enrichment of LMWPs based on methyl--butyl ether/methanol/water systems. The enriched LMWP sample was analyzed by single-shot liquid chromatography-tandem mass spectrometry employing both HCD and EThcD without tryptic digestion, and 725 peptides were identified on average. The LMWP sample was also digested and analyzed using a bottom-up proteomics pipeline, and 289 proteins were identified, of which 129 (44.6%) proteins were less than 30 kDa and lipoprotein-associated proteins were significantly enriched. Additionally, 25 neuropeptides and 19 long noncoding RNA-encoded polypeptides were identified. Taken together, the SPD method shows good sensitivity and reproducibility when compared with other enrichment methods and has great potential for clinical biomarker discovery and application.
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http://dx.doi.org/10.1021/acs.jproteome.0c00232DOI Listing
August 2020

Proteomic profiling of MIN6 cell-derived exosomes.

J Proteomics 2020 07 24;224:103841. Epub 2020 May 24.

State Key Laboratory of Pharmaceutical Biotechnology, Collaborative Innovation Center of Chemistry for Life Sciences, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute for Life Sciences (NAILS), School of Life Sciences, Nanjing University, 163 Xianlin Road, Nanjing, Jiangsu 210023, China. Electronic address:

Exosomes have been widely used in research on the early clinical diagnosis, prognosis and treatment of various cancers due to their features of small size (30-120 nm), non-immunogenicity and ability to cross biological barriers. However, few studies have investigated exosomes involved in metabolic diseases. Early studies have found that adipose tissue can be a source of exosomes regulating metabolism, but the related functions of exosomes secreted by other tissues in the regulation of metabolic diseases have not been determined. In addition, islets were found to be able to secrete miRNA via exosomes, suggesting that islet exosomes may be among the sources of exosomes involved in the regulation of metabolic diseases and that the relevant protein profiles have not been characterized to date. Therefore, identifying the protein contents of pancreatic β cell-derived exosomes would benefit further research investigating the protein functions and mechanisms associated with diabetes-related metabolic diseases. SIGNIFICANCE: Exosomes are emerging tools for investigating metabolic diseases in recent years, but little research has been done. In our work, functional identification of MIN6 cell-derived exosomal proteins and comparative analysis of islet β cell exosomal protein data from different cell lines or from different species revealed that exosomes secreted by islet β cells may be involved in the regulation of glucose metabolism. These results may suggest that intercellular communication induced by exosome transfer among tissues may account for the major reason of diabetic metabolic disorder. In addition, these results may provide a theoretical basis for the study of the physiological and pathological functions of islet β cell exosomes for the future studies.
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http://dx.doi.org/10.1016/j.jprot.2020.103841DOI Listing
July 2020

Proteomic Changes of Klebsiella pneumoniae in Response to Colistin Treatment and Mutation-Mediated Colistin Resistance.

Antimicrob Agents Chemother 2020 05 21;64(6). Epub 2020 May 21.

Key Laboratory of Protein and Peptide Pharmaceuticals and Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

Polymyxins are increasingly used as the critical last-resort therapeutic options for multidrug-resistant Gram-negative bacteria. Unfortunately, polymyxin resistance has increased gradually over the past few years. Although studies on polymyxin mechanisms are expanding, systemwide analyses of the underlying mechanism for polymyxin resistance and stress response are still lacking. To understand how adapts to colistin (polymyxin E) pressure, we carried out proteomic analysis of a strain cultured with different concentrations of colistin. Our results showed that the proteomic responses to colistin treatment in involve several pathways, including (i) gluconeogenesis and the tricarboxylic acid (TCA) cycle, (ii) arginine biosynthesis, (iii) porphyrin and chlorophyll metabolism, and (iv) enterobactin biosynthesis. Interestingly, decreased abundances of class A β-lactamases, including TEM, SHV-11, and SHV-4, were observed in cells treated with colistin. Moreover, we present comprehensive proteome atlases of paired polymyxin-susceptible and -resistant strains. The polymyxin-resistant strain Ci, a mutant of ATCC BAA 2146, showed a missense mutation in This mutant, which displayed lipid A modification with 4-amino-4-deoxy-l-arabinose (l-Ara4N) and palmitoylation, showed striking increases in the expression of CrrAB, PmrAB, PhoPQ, ArnBCADT, and PagP. We hypothesize that mutations induce elevated expression of the operon and via PmrAB and PhoPQ. Moreover, the multidrug efflux pump KexD, which was induced by mutation, also contributed to colistin resistance. Overall, our results demonstrated proteomic responses to colistin treatment and the mechanism of CrrB-mediated colistin resistance, which may offer valuable information on the management of polymyxin resistance.
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http://dx.doi.org/10.1128/AAC.02200-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7269499PMC
May 2020

Abnormal Galactosylated-Glycans recognized by Bandeiraea Simplicifolia Lectin I in saliva of patients with breast Cancer.

Glycoconj J 2020 Jun 27;37(3):373-394. Epub 2020 Feb 27.

Laboratory for Functional Glycomics, College of Life Sciences, Northwest University, Xi'an, 710069, China.

Currently, the definitive diagnosis in breast cancer requires biopsy and histopathology, such the most effective markers are tissue-based. However, the advantages of saliva in collection and storage make it possible for assessing human pathology and contributing to the development of cancer-related biomarkers for clinical application. The present study validated alteration of salivary protein glycopatterns recognized by Bandeiraea simplicifolia lectin I (BS-I) in the saliva of patients with breast diseases using saliva microarrays, and the N/O-glycan profiles of their salivary glycoproteins isolated by the BS-I-magnetic particle conjugates from 259 female subjects (66 healthy volunteers (HV), 65 benign breast cyst or tumor patients (BB), 66 patients with breast cancer in stage I (BC-I) and 62 patients with breast cancer in stage II (BC-II)) were analyzed by MALDI-TOF/TOF-MS. The results showed that the expression level of galactosylated glycans recognized by BS-I was significantly increased in patients with breast cancer compared with HV (p < 0.05). Totally, there were 11/10, 10/19, 7/24 and 7/9 galactosylated N-/O-linked glycans were identified and annotated from the pooled salivary samples of HV, BB, BC-I and BC-II, respectively. One galactosylated N-glycan peak (m/z 2773.977), and 4 galactosylated O-glycan peaks (m/z 868.295, 882.243, 884.270 and 1030.348) were found only in BC-I. These findings could provide pivotal information on galactosylated N/O-linked glycans related to breast cancer, and promote the study of biomarkers for early-stage breast cancer based on precise alterations of galactosylated N/O-glycans in saliva.
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http://dx.doi.org/10.1007/s10719-020-09910-6DOI Listing
June 2020

Large-scale Identification of N-linked Intact Glycopeptides in Human Serum using HILIC Enrichment and Spectral Library Search.

Mol Cell Proteomics 2020 04 26;19(4):672-689. Epub 2020 Feb 26.

Laboratory of Protein and Peptide Pharmaceuticals & Proteomics Laboratory, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China. Electronic address:

Large-scale identification of -linked intact glycopeptides by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) in human serum is challenging because of the wide dynamic range of serum protein abundances, the lack of a complete serum N-glycan database and the existence of proteoforms. In this regard, a spectral library search method was presented for the identification of -linked intact glycopeptides from -linked glycoproteins in human serum with target-decoy and motif-specific false discovery rate (FDR) control. Serum proteins were firstly separated into low-abundance and high-abundance proteins by acetonitrile (ACN) precipitation. After digestion, the -linked intact glycopeptides were enriched by hydrophilic interaction liquid chromatography (HILIC) and a portion of the enriched -linked intact glycopeptides were processed by Peptide-N-Glycosidase F (PNGase F) to generate -linked deglycopeptides. Both -linked intact glycopeptides and deglycopeptides were analyzed by LC-MS/MS. From -linked deglycopeptides data sets, 764 -linked glycoproteins, 1699 -linked glycosites and 3328 unique -linked deglycopeptides were identified. Four types of -linked glycosylation motifs (NXS/T/C/V, X≠P) were used to recognize the -linked deglycopeptides. The spectra of these -linked deglycopeptides were utilized for -linked deglycopeptides library construction and identification of -linked intact glycopeptides. A database containing 739 N-glycan masses was constructed and utilized during spectral library search for the identification of -linked intact glycopeptides. In total, 526 -linked glycoproteins, 1036 -linked glycosites, 22,677 -linked intact glycopeptides and 738 N-glycan masses were identified under 1% FDR, representing the most in-depth serum N-glycoproteome identified by LC-MS/MS at -linked intact glycopeptide level.
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http://dx.doi.org/10.1074/mcp.RA119.001791DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7124471PMC
April 2020

Negative auto-regulation of sulfur dioxide generation in vascular endothelial cells: AAT1 S-sulfenylation.

Biochem Biophys Res Commun 2020 Feb 19. Epub 2020 Feb 19.

Department of Pediatrics, Peking University First Hospital, Beijing, 100034, China. Electronic address:

Recently, endogenous sulfur dioxide (SO) has been found to exert an important function in the cardiovascular system. However, the regulatory mechanism for SO generation has not been entirely clarified. Hence, we aimed to explore the possible auto-regulation of endogenous SO generation and its mechanisms in vascular endothelial cells. We showed that SO did not affect the protein expression of aspartate aminotransferase 1 (AAT1), a major SO synthesis enzyme, but significantly inhibited AAT activity in primary human umbilical vein endothelial cells (HUVECs) and porcine purified AAT1 protein. An AAT1 enzymatic kinetic study showed that SO reduced the Vmax (1.89 ± 0.10 vs 2.55 ± 0.12, μmol/mg/min, P < 0.05) and increased the Km (35.97 ± 9.54 vs 19.33 ± 1.76 μmol/L, P < 0.05) values. Furthermore, SO induced S-sulfenylation of AAT1 in primary HUVECs and purified AAT1 protein. LC-MS/MS analysis indicated that SO sulfenylated AAT1 at Cys192. Mechanistically, thiol reductant DTT treatment or C192S mutation prevented SO-induced AAT1 sulfenylation and the subsequent inhibition of AAT activity in purified AAT1 protein and primary HUVECs. Our findings reveal, for the first time, a mechanism of auto-regulation of SO generation through sulfenylation of AAT1 at Cys192 to suppress AAT activity in vascular endothelial cells. These findings will greatly deepen the understanding of regulatory mechanisms in the cardiovascular homeostasis.
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http://dx.doi.org/10.1016/j.bbrc.2020.02.040DOI Listing
February 2020

Author Correction: H2A.Z facilitates licensing and activation of early replication origins.

Nature 2020 02;578(7793):E8

National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41586-020-1948-yDOI Listing
February 2020

H2A.Z facilitates licensing and activation of early replication origins.

Nature 2020 01 25;577(7791):576-581. Epub 2019 Dec 25.

National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.

DNA replication is a tightly regulated process that ensures the precise duplication of the genome during the cell cycle. In eukaryotes, the licensing and activation of replication origins are regulated by both DNA sequence and chromatin features. However, the chromatin-based regulatory mechanisms remain largely uncharacterized. Here we show that, in HeLa cells, nucleosomes containing the histone variant H2A.Z are enriched with histone H4 that is dimethylated on its lysine 20 residue (H4K20me2) and with bound origin-recognition complex (ORC). In vitro studies show that H2A.Z-containing nucleosomes bind directly to the histone lysine methyltransferase enzyme SUV420H1, promoting H4K20me2 deposition, which is in turn required for ORC1 binding. Genome-wide studies show that signals from H4K20me2, ORC1 and nascent DNA strands co-localize with H2A.Z, and that depletion of H2A.Z results in decreased H4K20me2, ORC1 and nascent-strand signals throughout the genome. H2A.Z-regulated replication origins have a higher firing efficiency and early replication timing compared with other origins. Our results suggest that the histone variant H2A.Z epigenetically regulates the licensing and activation of early replication origins and maintains replication timing through the SUV420H1-H4K20me2-ORC1 axis.
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http://dx.doi.org/10.1038/s41586-019-1877-9DOI Listing
January 2020

Screening and identification of non-inflammatory specific protein markers in Wilms' tumor tissues.

Arch Biochem Biophys 2019 11 21;676:108112. Epub 2019 Sep 21.

Department of Surgery, the First Affiliated Hospital, Zhengzhou University, Zhengzhou, 450052, Henan, China. Electronic address:

Wilms' tumor is one of the most common malignancies in children, and early diagnosis is critical for its subsequent treatment and prognosis. Our previous study employed proteomics to investigate protein markers in the serum of Wilms' tumor children. The present study aimed to identify specific protein markers in Wilms' tumor. Proteomic comparison of Wilms' tumor with normal kidney tissues and the sera of systemic inflammatory response syndrome (SIRS) controls was performed. Surface-enhanced laser desorption ionization time-of-flight (SELDI-TOF-MS) identified a protein with m/z 8350 as specific to Wilms' tumor. The target protein was purified using sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and identified as profilin-1 by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF). Its expression was validated using real-time polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Our data identify profilin-1 as a potential protein marker for Wilms' tumor and demonstrate the feasibility of the above procedures for screening and identification of tumor-specific protein markers.
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http://dx.doi.org/10.1016/j.abb.2019.108112DOI Listing
November 2019

Glycopatterns and Glycoproteins Changes in MCN and SCN: A Prospective Cohort Study.

Biomed Res Int 2019 31;2019:2871289. Epub 2019 Jul 31.

Department of Gastroenterology, Chinese PLA General Hospital, Beijing 100853, China.

Advances in imaging improve the detection of malignant pancreatic cystic including mucinous cystic neoplasm (MCN), intraductal papillary mucinous neoplasm (IPMN), and mucinous cystic adenocarcinoma (MCA), but the distinction between benign and malignant lesions remains a problem. In an effort to establish glycopatterns as potential biomarkers for differential diagnosis between MCN and SCN, we systematically investigated the alterations of glycopatterns in cystic fluids for both SCN and MCN. Among the 75 patients enrolled, 37 were diagnosed as MCN and 38 as SCN based on histology. Lectin microarray analysis was performed on each sample, and the fluorescence intensity was used to obtain the fold-change. Then, mixed cyst fluids of MCN group and SCN group were cross bonded with magnetic particles coupled by Lectin STL and WGA, respectively. Hydrophilic interaction liquid chromatography (HILIC) enrichment was performed, liquid chromatography (LC)/mass spectrometry (MS) analysis and bioinformatical analysis was conducted to find the differential glycoproteins between MCNs and SCNs. . Through analysis of lectin microarray between MCNs and SCNs, stronger lectin signal patterns were assigned to Lectin WFA, DBA, STL, WGA, and BPL; and weaker signal patterns were assigned to Lectin PTL-I, Con A, ACA, and MAL-I. The glycoproteins were enriched by STL or WGA-coupled magnetic particles. Furthermore, the 10 identified correspondding genes were found to be significantly elevated in the mucinous cystadenoma: CLU, A2M, FGA, FGB, FGG, PLG, SERPINA1, SERPING1, C5, C8A, and C9. Bioinformatics analysis revealed that the above genes may activate the KEGG pathway: immune complement system. . This study shows changes in glycopatterns and glycoproteins are associated with MCNs and SCNs.
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http://dx.doi.org/10.1155/2019/2871289DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6699316PMC
January 2020

Analysis of Glycosphingolipid Glycans by Lectin Microarrays.

Anal Chem 2019 08 9;91(16):10663-10671. Epub 2019 Aug 9.

Laboratory for Functional Glycomics, College of Life Sciences , Northwest University , Xi'an , China.

Glycosphingolipids (GSLs) are ubiquitous glycoconjugates of cell membranes. Identification of unknown GSL-glycan structures is still a major challenge. To address this challenge, we developed a novel strategy for analysis of GSL-glycans from cultured cells based on a lectin microarray that can directly detect and reveal glycopatterns of GSL extracts without the need for glycan release. There were six steps to perform the analysis of GSL-glycans: (i) extraction of GSLs from cell pellets, (ii) quantification of GSL-glycans using orcinol-sulfuric acid reaction, (iii) preparation of lyso-GSLs by using sphingolipid ceramide N-deacylase, (iv) fluorescence labeling of lyso-GSLs, (v) detection by a lectin microarray, (vi) data acquisition and analysis. Simultaneously, a supplementary verification analysis for GSL-glycans was performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Optimized experimental conditions, which consisted of the blocking buffer, incubation buffer, and appropriate GSL concentration, were investigated by analyzing the glycopatterns of a standard ganglioside (GM1a) via lectin microarray. The analysis of GSL-glycans from human hepatocarcinoma cell lines (MHCC97L, MHCC97H, and HCCLM3) showed that there were 27 lectins (e.g., WFA, MAL-II, and LTL) to give significantly different signals compared with a normal human liver cell line (HL-7702), indicating up- and/or down-regulations of corresponding glycopatterns such as α1-2 fucosylation and α2-3 sialylation, and changes of certain glycostructures such as Galβ1-3GalNAcβ1-4(NeuAcα2-3)Galβ1-4Glc:Cer and GalNAcα1-3(Fucα1-2)Galβ1-3GlcNAcβ1-3Galβ1-4Glc:Cer. The lectin microarray analysis of lyso-GSLs labeled by fluorescence has proven to be credible, which can provide the glycopatterns and detailed linkage information on GSL-glycans.
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http://dx.doi.org/10.1021/acs.analchem.9b01945DOI Listing
August 2019

Alphabet Projection of Spectra.

J Proteome Res 2019 09 29;18(9):3268-3281. Epub 2019 Jul 29.

Department of Computer Science , University of Montana , Missoula , Montana 59801 , United States.

In the metabolomics, glycomics, and mass spectrometry of structured small molecules, the combinatoric nature of the problem renders a database impossibly large, and thus de novo analysis is necessary. De novo analysis requires an alphabet of mass difference values used to link peaks in fragmentation spectra when they are different by a mass in the alphabet divided by a charge. Often, this alphabet is not known, prohibiting de novo analysis. A method is proposed that, given fragmentation mass spectra, identifies an alphabet of / differences that can build large connected graphs from many intense peaks in each spectrum from a collection. We then introduce a novel approach to efficiently find recurring substructures in the de novo graph results.
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http://dx.doi.org/10.1021/acs.jproteome.9b00216DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7520079PMC
September 2019

Comprehensive characterization and proteoform analysis of the hydrophobic surfactant proteins B and C in calf pulmonary surfactant.

J Pharm Biomed Anal 2019 Sep 21;174:625-632. Epub 2019 Jun 21.

Key Laboratory of Protein and Peptide Pharmaceuticals & Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China. Electronic address:

Calf pulmonary surfactant (CPS), which contains about 98% lipids and 2% hydrophobic surfactant proteins B (SP-B) and C (SP-C), has been used as a surfactant preparation for the clinical replacement therapy of respiratory distress syndrome (RDS). Characterization of SP-B and SP-C in CPS is informative for quality control and the evaluation of their biological activities. However, analysis of SP-B and SP-C is impeded by the high content of lipids in CPS. Here, we describe an integrated method by combining size exclusion chromatography (SEC)-based delipidation, SDS-PAGE separation, in-gel digestion and mass spectrometric analysis for comprehensive characterization and proteoform analysis of the extremely hydrophobic SP-B and SP-C in CPS. This study has shown that 30 proteoforms of SP-C with different truncations and modifications were identified and SP-B was found to be existed as a dimer form in the CPS.
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http://dx.doi.org/10.1016/j.jpba.2019.06.027DOI Listing
September 2019

M2 macrophages promote NSCLC metastasis by upregulating CRYAB.

Cell Death Dis 2019 05 16;10(6):377. Epub 2019 May 16.

Department of Respiratory Medicine, The Second Hospital, Dalian Medical University, Dalian, China.

The mechanism by which tumor-associated macrophages (TAMs) affect cancer progression is not fully understood. This study developed a microfluidic-based co-culture device to mimic the tumor microenvironment to assess TAM effects on invasion and metastasis in NSCLC. The results showed lung carcinoma cells could cause macrophages to show the M2 (a TAM-like) phenotype, and these M2 macrophages promoted lung cancer cell EMT and invasion. Proteomic analysis by the iTRAQ quantitation strategy and GO ontology of the cancer cells indicated that αB-Crystallin (CRYAB) might be involved in this process. Further, we confirmed the role of CRYAB in cancer invasion and metastasis through cell and animal experiments, as well as human cancer tissue assessment. Overall, we demonstrated that M2 macrophages promote malignancy in lung cancer through the EMT by upregulating CRYAB expression and activating the ERK1/2/Fra-1/slug signaling pathway.
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http://dx.doi.org/10.1038/s41419-019-1618-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6522541PMC
May 2019

Ferroptosis as a target for protection against cardiomyopathy.

Proc Natl Acad Sci U S A 2019 02 28;116(7):2672-2680. Epub 2019 Jan 28.

The First Affiliated Hospital, School of Public Health, Institute of Translational Medicine, Zhejiang University School of Medicine, 310058 Hangzhou, China;

Heart disease is the leading cause of death worldwide. A key pathogenic factor in the development of lethal heart failure is loss of terminally differentiated cardiomyocytes. However, mechanisms of cardiomyocyte death remain unclear. Here, we discovered and demonstrated that ferroptosis, a programmed iron-dependent cell death, as a mechanism in murine models of doxorubicin (DOX)- and ischemia/reperfusion (I/R)-induced cardiomyopathy. In canonical apoptosis and/or necroptosis-defective , , or mice, DOX-treated cardiomyocytes showed features of typical ferroptotic cell death. Consistently, compared with dexrazoxane, the only FDA-approved drug for treating DOX-induced cardiotoxicity, inhibition of ferroptosis by ferrostatin-1 significantly reduced DOX cardiomyopathy. RNA-sequencing results revealed that heme oxygenase-1 () was significantly up-regulated in DOX-treated murine hearts. Administering DOX to mice induced cardiomyopathy with a rapid, systemic accumulation of nonheme iron via heme degradation by Nrf2-mediated up-regulation of Hmox1, which effect was abolished in -deficent mice. Conversely, zinc protoporphyrin IX, an Hmox1 antagonist, protected the DOX-treated mice, suggesting free iron released on heme degradation is necessary and sufficient to induce cardiac injury. Given that ferroptosis is driven by damage to lipid membranes, we further investigated and found that excess free iron accumulated in mitochondria and caused lipid peroxidation on its membrane. Mitochondria-targeted antioxidant MitoTEMPO significantly rescued DOX cardiomyopathy, supporting oxidative damage of mitochondria as a major mechanism in ferroptosis-induced heart damage. Importantly, ferrostatin-1 and iron chelation also ameliorated heart failure induced by both acute and chronic I/R in mice. These findings highlight that targeting ferroptosis serves as a cardioprotective strategy for cardiomyopathy prevention.
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http://dx.doi.org/10.1073/pnas.1821022116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6377499PMC
February 2019

Regulation of HIV-1 Gag-Pol Expression by Shiftless, an Inhibitor of Programmed -1 Ribosomal Frameshifting.

Cell 2019 01;176(3):625-635.e14

CAS Key Laboratory of Infection and Immunity, CAS Centre for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China. Electronic address:

Programmed -1 ribosomal frameshifting (-1PRF) is a widely used translation recoding mechanism. HIV-1 expresses Gag-Pol protein from the Gag-coding mRNA through -1PRF, and the ratio of Gag to Gag-Pol is strictly maintained for efficient viral replication. Here, we report that the interferon-stimulated gene product C19orf66 (herein named Shiftless) is a host factor that inhibits the -1PRF of HIV-1. Shiftless (SFL) also inhibited the -1PRF of a variety of mRNAs from both viruses and cellular genes. SFL interacted with the -1PRF signal of target mRNA and translating ribosomes and caused premature translation termination at the frameshifting site. Downregulation of translation release factor eRF3 or eRF1 reduced SFL-mediated premature translation termination. We propose that SFL binding to target mRNA and the translating ribosome interferes with the frameshifting process. These findings identify SFL as a broad-spectrum inhibitor of -1PRF and help to further elucidate the mechanisms of -1PRF.
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http://dx.doi.org/10.1016/j.cell.2018.12.030DOI Listing
January 2019

TMT-Based Quantitative Proteomics Analysis Reveals Airborne PM-Induced Pulmonary Fibrosis.

Int J Environ Res Public Health 2018 12 31;16(1). Epub 2018 Dec 31.

Laboratory of Environment and Health, College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China.

Epidemiological and experimental studies have documented that long-term exposure to fine particulate matter (PM) increases the risk of respiratory diseases. However, the details of the underlying mechanism remain unclear. In this study, male C57BL/6 mice were exposed to ambient PM (mean daily concentration ~64 µg/m³) for 12 weeks through a "real-world" airborne PM exposure system. We found that PM caused severe lung injury in mice as evidenced by histopathological examination. Then, tandem mass tag (TMT) labeling quantitative proteomic technology was performed to analyze protein expression profiling in the lungs from control and PM-exposed mice. A total of 32 proteins were differentially expressed in PM-exposed lungs versus the controls. Among these proteins, 24 and 8 proteins were up- and down-regulated, respectively. Gene ontology analysis indicated that PM exerts a toxic effect on lungs by affecting multiple biological processes, including oxidoreductase activity, receptor activity, and protein binding. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that extracellular matrix (ECM)⁻receptor interaction, phagosome, small cell lung cancer, and phosphatidylinositol 3-kinase(PI3K)-protein kinase B (Akt) signaling pathways contribute to PM-induced pulmonary fibrosis. Taken together, these results provide a comprehensive proteomics analysis to further understanding of the molecular mechanisms underlying PM-elicited pulmonary disease.
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http://dx.doi.org/10.3390/ijerph16010098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6339163PMC
December 2018

Identification of abnormal fucosylated-glycans recognized by LTL in saliva of HBV-induced chronic hepatitis, cirrhosis, and hepatocellular carcinoma.

Glycobiology 2019 03;29(3):242-259

Laboratory for Functional Glycomics, College of Life Sciences, Northwest University, Xi'an, China.

The hepatitis B virus (HBV)-induced chronic liver diseases are serious health threats worldwide. There is evidence to display the alterations of salivary N-linked glycans related to the development of HBV-infected liver diseases. Here, we further investigated the alterations of fucosylated N/O-glycans recognized by LTL in saliva from 120 subjects (30 healthy volunteers (HV), 30 patients with hepatitis B (HB), 30 patients with hepatic cirrhosis (HC), and 30 patients with hepatocellular carcinoma (HCC)) using salivary microarrys and MALDI-TOF/TOF-MS. The results showed that the expression level of fucosylated glycans recognized by LTL was significantly increased in HCC compared with other subjects (P < 0.0001). Besides, the fucosylated glycoproteins were isolated from pooled saliva of HV, HB, HC, and HCC by LTL-magnetic particle conjugates. Then, N/O- glycans were released from the isolated glycoproteins with PNGase F and NaClO, and were identified by MALDI-TOF-MS, respectively. Totally, there were 21/20, 25/18, 29/19, and 28/24 N/O-glycan peaks that were identified and annotated with proposed structures in saliva of HV, HB, HC, and HCC. Among the total, there were 8 N-glycan peaks (e.g., m/z 1905.634, 2158.777 and 2905.036) and 15 O-glycan peaks (e.g., 1177.407, 1308.444 and 1322.444) that only presented in patients with HBV-induced liver diseases. One N-glycan peak (m/z 2205.766) was unique in HC, and 9 O-glycan peaks (e.g., m/z 1157.420, 1163.417 and 1193.402) were unique in HCC. This study could facilitate the discovery of biomarkers for HC and HCC based on precise alterations of fucosylated N/O-glycans in saliva.
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http://dx.doi.org/10.1093/glycob/cwy108DOI Listing
March 2019

Deposition Mapping of Polycyclic Aromatic Compounds in the Oil Sands Region of Alberta, Canada and Linkages to Ecosystem Impacts.

Environ Sci Technol 2018 11 22;52(21):12456-12464. Epub 2018 Oct 22.

Air Quality Research Division, Science and Technology Branch , Environment and Climate Change Canada , Toronto , Ontario M3H 5T4 Canada.

This study produced gridded deposition estimates of polycyclic aromatic compounds (PACs), including 17 polycyclic aromatic hydrocarbons (PAHs), 21 alkylated PAHs (alk-PAHs), and 5 dibenzothiophenes (DBTs), over the oil sands region of Alberta, Canada and surrounding communities. Gridded annual total deposition of PACs in 2011 ranged from 55 to 175 000 μg m yr and the mean and median fluxes were 1700 and 760 μg m yr, respectively. The domain-wide mean dry and wet deposition were 600 and 1100 μg m yr. PAHs, alk-PAHs and DBTs contributed 19%, 74%, and 7% to the total dry deposition, and 42%, 49%, and 9% to the total wet deposition. Dominant chemical species contributing to total deposition were naphthalene, retene and phenanthrene for PAHs and C2-benz[a]anthracene/triphenylene/chrysene, C2-fluoranthene/pyrene and C2-fluorene for alk-PAHs. The highest PAC deposition was found over the surface mineable area, which received 9 times the deposition flux of outlying areas. Additional deposition hotspots were also observed south of the surface mineable area notably over in situ bitumen production sites. The deposition of alk-PAHs impacted a more extensive area than that of PAHs or DBTs. This result suggests that atmospheric deposition is a key process in wildlife exposure to PACs across the region.
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http://dx.doi.org/10.1021/acs.est.8b02486DOI Listing
November 2018

SIRT5 deacylates metabolism-related proteins and attenuates hepatic steatosis in ob/ob mice.

EBioMedicine 2018 Oct 29;36:347-357. Epub 2018 Sep 29.

National Laboratory of Biomacromolecules, Institute of Biophysics, Cthe Strategic Priority Research Programses, Beijing 100101, China. Electronic address:

Background: Sirtuin 5 (SIRT5) is a NAD-dependent lysine deacylase. The SIRT5 deficiency mouse model shows that it is dispensable for metabolic homeostasis under normal conditions. However, the biological role of SIRT5 and acylation in pathological states such as obesity and type 2 diabetes (T2D) remains elusive.

Methods: The hepatic SIRT5-overexpressing ob/ob mouse model (ob/ob-SIRT5 OE) was established by CRISPR/Cas9 gene editing tool Protein malonylation and succinylation lysine sites were identified by immunoprecipitation coupled lipid chromatography - tandem mass spectrometry (LC-MS/MS) methods.

Findings: The ob/ob-SIRT5 OE mice showed decreased malonylation and succinylation, improved cellular glycolysis, suppressed gluconeogenesis, enhanced fatty acid oxidation, and attenuated hepatic steatosis. A total of 955 malonylation sites on 434 proteins and 1377 succinylation sites on 429 proteins were identified and quantitated. Bioinformatics analysis revealed that malonylation was the major SIRT5 target in the glycolysis/gluconeogenesis pathway, whereas succinylation was the preferred SIRT5 target in the oxidative phosphorylation pathway.

Interpretation: Hepatic overexpression of SIRT5 ameliorated the metabolic abnormalities of ob/ob mice, probably through demalonylating and desuccinylating proteins in the main metabolic pathways. SIRT5 and related acylation might be potential targets for metabolic disorders. FUND: National Key R&D Program of China, the National Natural Science Foundation of China, the Strategic Priority Research Programs (Category A) of the Chinese Academy of Sciences, the Interdisciplinary Medicine Seed Fund of Peking University and the National Laboratory of Biomacromolecules.
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http://dx.doi.org/10.1016/j.ebiom.2018.09.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6197389PMC
October 2018

N-Linked Glycopeptide Identification Based on Open Mass Spectral Library Search.

Biomed Res Int 2018 14;2018:1564136. Epub 2018 Aug 14.

National Center for Mathematics and Interdisciplinary Sciences, Key Laboratory of Random Complex Structures and Data Science, Academy of Mathematics and Systems Science, Chinese Academy of Sciences, Beijing 100101, China.

Confident characterization of intact glycopeptides is a challenging task in mass spectrometry-based glycoproteomics due to microheterogeneity of glycosylation, complexity of glycans, and insufficient fragmentation of peptide bones. Open mass spectral library search is a promising computational approach to peptide identification, but its potential in the identification of glycopeptides has not been fully explored. Here we present pMatchGlyco, a new spectral library search tool for intact N-linked glycopeptide identification using high-energy collisional dissociation (HCD) tandem mass spectrometry (MS/MS) data. In pMatchGlyco, (1) MS/MS spectra of deglycopeptides are used to create spectral library, (2) MS/MS spectra of glycopeptides are matched to the spectra in library in an open (precursor tolerant) manner and the glycans are inferred, and (3) a false discovery rate is estimated for top-scored matches above a threshold. The efficiency and reliability of pMatchGlyco were demonstrated on a data set of mixture sample of six standard glycoproteins and a complex glycoprotein data set generated from human cancer cell line OVCAR3.
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http://dx.doi.org/10.1155/2018/1564136DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6112209PMC
December 2018