pFind Studio: a computational solution for mass spectrometry-based proteomics
2018
Analytical Chemistry2018. Zhang, XQ et al.
Fudan Univ, Dept Chem, Shanghai 200433, Peoples R China.
ABSTRACT:Palmitoylation is one of the most important protein translational modifications and plays vital roles in many key biological processes. Aberrant palmitoylation has been associated with a variety of human diseases. So it is of great significance to profile the palmitoylated proteomes qualitatively and quantitatively. Here, we described a novel method based on the cysteine-stable isotope labeling in cell culture (cysteine-SILAC) to facilitate the quantitation of palmitoylated proteins by mass spectrometry (MS), in which "light" or "heavy" samples could be pooled and subjected to the subsequent analysis procedures simultaneously, minimizing systematic errors caused by parallel operations and improving quantitative accuracy and precision. The mass tags lay on the cysteine residues, which were the potential palmitoylated sites, indicating that all the putative modified sites/peptides could be quantified, including the C-terminal peptide of one protein. Due to the isotopically labeled cysteine, the nonspecifically adsorbed peptide without cysteine was singlet in MS spectra, whereas pair peaks should be the signals of putative palmitoylated peptides, which could reduce spectral complexity and achieve double verification for the putative palmitoylated peptides. Finally, the palmitoylome in hepatocellular carcinoma (HCC) cells with different metastasis potentials (MHCC-97L and HCC-LM3 cells) were analyzed for the first time. Totally, 151 proteins were found to be differentially palmitoylated with high confidence, including many important proteins involved in a variety of biological processes, such as protein palmitoylation, cell proliferation, signal transduction, regulation of cell migration, and so on.
Use: pFind
International Journal of Molecular Sciences2018. Zhao, Q et al.
Liaoning Tradit Chinese Med Univ, Chinese Mat Med Proc Engn Technol Res Ctr Liaonin, Key Lab Chinese Mat Med Proc Principle Anal, State Adm Tradit Chinese Med,Pharmaceut Coll, Dalian 110060, Peoples R China.
ABSTRACT:The traditional Chinese drug Bombyx Batryticatus (BB), which is also named the white stiff silkworm, has been widely used in Chinese clinics for thousands of years. It is famous for its antispasmodic and blood circulation-promoting effects. Cardiomyocyte hypertrophy, interstitial cell hyperplasia, and myocardial fibrosis are closely related to the N-glycosylation of key proteins. To examine the alterations of N-glycosylation that occur in diabetic myocardium during the early stage of the disease, and to clarify the therapeutic effect of 1-Deoxynojirimycin (1-DNJ) extracted from BB, we used the db/db (diabetic) mouse model and an approach based on hydrophilic chromatography solid-phase extraction integrated with an liquid Chromatograph Mass Spectrometer (LC-MS) identification strategy to perform a site-specific N-glycosylation analysis of left ventricular cardiomyocyte proteins. Advanced glycation end products (AGEs), hydroxyproline, connective tissue growth factor (CTGF), and other serum biochemical indicators were measured with enzyme-linked immunosorbent assays (ELISA). In addition, the -1,6-fucosylation of N-glycans was profiled with lens culinaris agglutinin (LCA) lectin blots and fluorescein isothiocyanate (FITC)-labelled lectin affinity histochemistry. The results indicated that 1-DNJ administration obviously downregulated myocardium protein N-glycosylation in db/db mice. The expression levels of serum indicators and fibrosis-related cytokines were reduced significantly by 1-DNJ in a dose-dependent manner. The glycan -1,6-fucosylation level of the db/db mouse myocardium was elevated, and the intervention effect of 1-DNJ administration on N-glycan -1,6-fucosylation was significant. To verify this result, the well-known transforming growth factor- (TGF-)/Smad2/3 pathway was selected, and core -1,6-fucosylated TGF- receptor II (TGFR-II) was analysed semi-quantitatively with western blotting. The result supported the conclusions obtained from LCA lectin affinity histochemistry and lectin blot analysis. The expression level of -1,6-fucosyltransferase (FUT8) mRNA was also detected, and the results showed that 1-DNJ administration did not cause obvious inhibitory effects on FUT8 expression. Therefore, the mechanism of 1-DNJ for relieving diabetic cardiomyopathy (DCM)-associated fibrosis can be concluded as the inhibition of N-acetylglucosamine (N-GlcNAc) formation and the reduction of substrate concentration.
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Journal of Proteome Research2018. He, CT et al.
Anhui Med Univ, Hefei 230032, Anhui, Peoples R China.
ABSTRACT:Microproteins are peptides composed of 100 amino acids (AA) or fewer, encoded by small open reading frames (smORFs). It has been demonstrated that microproteins participate in and regulate a wide range of functions in cells. However, the annotation and identification of microproteins is challenging in part owing to their low molecular weight, low abundancy, and hydrophobicity. These factors have led to the unannotation of smORFs in genome processing and have made their identification at the protein level difficult. Large-scale enrichment of microproteins in proteogenomics has made it possible to efficiently identify microproteins and discover unannotated smORFs in Saccharomyces cerevisiae. We integrated four microprotein-specific enrichment strategies to enhance coverage. We identified 117 microproteins, verified 31 missing proteins (MPs), and discovered 3 novel smORFs. In total, 31 proteins were confirmed as MPs by spectrum quality checking. Three novel smORFs (YKL104W-A, YHR052C-B, and YHR054C-B) were reserved after spectrum quality checking, peptide synthesizing, homologue matching, and so on. This study not only demonstrates that there are potential smORF candidates to be annotated in an extensively studied organism but also presents an efficient strategy for the discovery of small MPs. All MS data sets have been deposited to the ProteomeXchange with identifier PXD008586.
Use: pFind
EUROPEAN JOURNAL OF MEDICINAL CHEMISTRY2018. Yang, CR et al.
Capital Normal Univ, Dept Chem, Beijing 100048, Peoples R China.
ABSTRACT:Two series of thieno[2,3-d]pyrimidine derivatives bearing a dithiocarbamate side chain at the C2 position were synthesized and evaluated for cytotoxic activity in human lung cancer A549 and colon cancer HCT-116 cell lines. Compound 3n exhibited the most cytotoxic effect on A549 cells with an IC50 value of 4.87 mu M inducing a cell cycle arrest at G2/M phase and activating the spindle assembly checkpoint (SAC). To identify the target protein(s) of 3n, we incorporated biotin with 3n through a three-carbon chain and an amide bond to synthesize probe 10. The targeted proteins were pulled down from the A549 total cell lysate by biotin-streptavidin affinity purification and analyzed by mass spectrometry. Tubulin was the only protein identified, which is related to the SAC and directly binds to probe 10 both in vivo and in vitro. Furthermore, compound 3n inhibited tubulin polymerization in vitro in a dose-dependent manner, competed with taxol in binding to tubulin, exerting cytotoxic activity toward taxol-resistant A549 cells. These results demonstrate that thieno[2,3-d]pyrimidine derivative 3n exhibits cytotoxicity in cancer cells by targeting tubulin to activate the SAC and potentially acts as a therapeutic lead compound for taxol-resistant cancers. (C) 2018 Elsevier Masson SAS. All rights reserved.
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Analytical chemistry2018. Ma, Fengfei et al.
Univ Wisconsin, Sch Pharm, Madison, WI 53705 USA; Univ Wisconsin, Dept Chem, 1101 Univ Ave, Madison, WI 53706 USA
ABSTRACT:3- and 4-Hydroxyprolines (HyP) are regioisomers that play different roles in various species and organs. Despite their distinct functions inside cells, they are generally considered indistinguishable using mass spectrometry due to their identical masses. Here, we demonstrate, for the first time, that characteristic w ions can be produced by electron-transfer/higher energy collision dissociation (EThcD) dual fragmentation technique to confidently discriminate 3-HyP/4-HyP isomers. An integrated and high throughput strategy was developed which combined online LC separation with EThcD for large-scale differentiation of 3-HyP/4-HyP in complex samples. An automated algorithm was developed for charge state dependent characterization of 3-HyP/4-HyP isomers. Using this combined discrimination approach, we identified 108 3-HyP sites and 530 4-HyP sites from decellularized pancreas, allowing more than 5-fold increase of both 3-HyP and 4-HyP identifications compared to previous reports. This approach outperformed ETD and HCD in the analysis of HyP-containing peptides with unique capacity to generate w ions for HyP discrimination, improved fragmentation of precursor ions, as well as unambiguous localization of modifications. A high content of 3-HyP was observed in the C-terminal (GPP)(n) domain of human CO1A1, which was previously only identified in vertebrate fibrillar collagens from tendon. Unexpectedly, some unusual HyP sites at Xaa position in Gly-HyP-Ala, Gly-HyP-Val, Gly-HyP-Gln, Gly-HyP-Ser, and Gly-HyP-Arg were also confirmed to be 3-hydroxylated, whose functions and enzymes are yet to be discovered. Overall, this novel discrimination strategy can be readily implemented into de novo sequencing or other proteomic search engines.
Use: pGlyco; pFind
Electrophoresis2018. Yin, H et al.
Hong Kong Polytech Univ, Shenzhen Res Inst, Shenzhen, Peoples R China.
ABSTRACT:A liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based methodology has been developed to differentiate core- and antennary-fucosylated glycosylation of glycopeptides. Both the glycosylation sites (heterogeneity) and multiple possible glycan occupancy at each site (microheterogeneity) can be resolved via intact glycopeptide analysis. The serum glycoprotein alpha-1-antitrypsin (A1AT) which contains both core- and antennary-fucosylated glycosites was used in this study. Sialidase was used to remove the sialic acids in order to simplify the glycosylation microheterogeneity and to enhance the MS signal of glycopeptides with similar glycan structures. beta 1-3,4 galactosidase was used to differentiate core- and antennary-fucosylation. In-source dissociation was found to severely affect the identification and quantification of glycopeptides with low abundance glycan modification. The settings of the mass spectrometer were therefore optimized to minimize the in-source dissociation. A three-step mass spectrometry fragmentation strategy was used for glycopeptide identification, facilitated by pGlyco software annotation and manual checking. The collision energy used for initial glycopeptide fragmentation was found to be crucial for improved detection of oxonium ions and better selection of Y1 ion (peptide+GlcNAc). Structural assignments revealed that all three glycosylation sites of A1AT glycopeptides contain complex N-glycan structures: site Asn70 contains biantennary glycans without fucosylation; site Asn107 contains bi-, tri- and tetra-antennary glycans with both core- and antennary-fucosylation; site Asn271 contains bi- and tri-antennary glycans with both coreand antennary-fucosylation. The relative intensity of core- and antennary-fucosylation on Asn107 was similar to that of the A1AT protein indicating that the glycosylation level of Asn107 is much larger than the other two sites.
Use: pFind; pGlyco
Cancer research2018. Gou, QH et al.
Sichuan Univ, West China Hosp, Chengdu 610041, Sichuan, Peoples R China.
ABSTRACT:Long noncoding RNAs (lncRNA) are emerging as a novel class of regulators in gene expression associated with tumorigenesis. However, the role of lncRNAs in papillary thyroid carcinoma (PTC) is poorly understood. Here, we conducted global lncRNA profiling and identified lncRNA AB074169 (lncAB) as significantly downregulated in PTC. Decreased expression of lncAB in PTC was caused by CpG hypermethylation within its gene promoter. Functional studies showed that lncAB overexpression led to cell-cycle arrest and tumor growth inhibition in vitro and in vivo, whereas lncAB knockdown promoted cell proliferation. Mechanistic analyses revealed that lncAB bound KH-type splicing regulatory protein (KHSRP) and also decreased expression of KHSRP, thus increasing CDKN1a (p21) expression and decreasing CDK2 expression to repress cell proliferation. Taken together, these findings demonstrate that lncAB functions as a tumor suppressor during PTC tumorigenesis. Significance: These findings identify a tumor-suppressive long noncoding RNA in papillary thyroid carcinoma. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/15/4163/F1.large.jpg. Cancer Res; 78(15); 4163-74. (C) 2018 AACR.
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Journal of Biological Chemistry2018. Kaasboll, OJ et al.
Oslo Univ Hosp, Inst Surg Res, A3-1057,POB 4950 Nydalen, NO-0424 Oslo, Norway.
ABSTRACT:Connective tissue growth factor (CTGF; now often referred to as CCN2) is a secreted protein predominantly expressed during development, in various pathological conditions that involve enhanced fibrogenesis and tissue fibrosis, and in several cancers and is currently an emerging target in several early-phase clinical trials. Tissues containing high CCN2 activities often display smaller degradation products of full-length CCN2 (FL-CCN2). Interpretation of these observations is complicated by the fact that a uniform protein structure that defines biologically active CCN2 has not yet been resolved. Here, using DG44 CHO cells engineered to produce and secrete FL-CCN2 and cell signaling and cell physiological activity assays, we demonstrate that FL-CCN2 is itself an inactive precursor and that a proteolytic fragment comprising domains III (thrombospondin type 1 repeat) and IV (cystine knot) appears to convey all biologically relevant activities of CCN2. In congruence with these findings, purified FL-CCN2 could be cleaved and activated following incubation with matrix metalloproteinase activities. Furthermore, the C-terminal fragment of CCN2 (domains III and IV) also formed homodimers that were approximate to 20-fold more potent than the monomeric form in activating intracellular phosphokinase cascades. The homodimer elicited activation of fibroblast migration, stimulated assembly of focal adhesion complexes, enhanced RANKL-induced osteoclast differentiation of RAW264.7 cells, and promoted mammosphere formation of MCF-7 mammary cancer cells. In conclusion, CCN2 is synthesized and secreted as a preproprotein that is autoinhibited by its two N-terminal domains and requires proteolytic processing and homodimerization to become fully biologically active.
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Biophysics Reports2018. Lu, Shan et al.
Key Lab of Intelligent Information Processing of Chinese Academy of Sciences (CAS), University of CAS, Institute of Computing Technology, CAS, Beijing 100190, China; National Institute of Biological Sciences, Beijing, Beijing 102206, China
ABSTRACT:Disulfide bonds are vital for protein functions, but locating the linkage sites has been a challenge in protein chemistry, especially when the quantity of a sample is small or the complexity is high. In 2015, our laboratory developed a sensitive and efficient method for mapping protein disulfide bonds from simple or complex samples (Lu et al. in Nat Methods 12:329, 2015). This method is based on liquid chromatography-mass spectrometry (LC-MS) and a powerful data analysis software tool named pLink. To facilitate application of this method, we present step-by-step disulfide mapping protocols for three types of samples-purified proteins in solution, proteins in SDS-PAGE gels, and complex protein mixtures in solution. The minimum amount of protein required for this method can be as low as several hundred nanograms for purified proteins, or tens of micrograms for a mixture of hundreds of proteins. The entire workflow-from sample preparation to LC-MS and data analysis-is described in great detail. We believe that this protocol can be easily implemented in any laboratory with access to a fast-scanning, high-resolution, and accurate-mass LC-MS system.
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Scientific Reports2018. Yanting Wang, Wenxian Lan et al.
State Key Laboratory of Bioorganic and Natural Product Chemistry, Center for Excellence in Molecular Synthesis,Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences (CAS), 345 Lingling Road, Shanghai, 200032, China
ABSTRACT:Large-conductance Ca2+- and voltage-dependent K+ (BK) channels display diversebiologicalfunctions while their pore-forming asubunitis coded by a single Slo1 gene. The varietyofBKchannels is correlated with the effectsofBKalpha coexpression with auxiliary beta (beta 1-beta 4) subunits, as well as newly defined. subunits. Charybdotoxin (ChTX) blocksBKchannelthrough physically occluding the K+-conduction pore.Humanbrain enriched beta 4subunit(h beta 4) alters the conductance-voltage curve, slows activation and deactivation time coursesofBKchannels. Itsextracellularloop(h beta 4-loop) specifically impedesChTXto bindBKchannelpore. However, thestructureofbeta 4subunit'sextracellularloopand the molecular mechanism for gating kinetics, toxinsensitivityofBKchannels regulated by beta 4 are still unclear. To address them, here, we first identified four disulfide bonds in h beta 4-loopby mass spectroscopy and NMR techniques. Then we determineditsthree-dimensionalsolutionstructure, performed NMR titration and electrophysiological analysis, and found that residue Asn123ofbeta 4subunitregulated the gating and pharmacological characteristicsofBKchannel. Finally, by constructingstructuremodelsofBKalpha/beta 4 and thermodynamic double-mutant cycle analysis, we proposed that BKasubunitmight interact with beta 4subunitthrough the conserved residue Glu264(BKalpha) coupling with residue Asn123(beta 4).
Use: pLink