共查询到20条相似文献，搜索用时 15 毫秒
1.
2.
Adam J. Rabalski Jon D. Williams Ryan A. McClure Anil Vasudevan Aleksandra Baranczak 《Proteomics》2019,19(11)
Chemical proteomics enables comprehensive profiling of small molecules in complex proteomes. A critical component to understand the interactome of a small molecule is the precise location on a protein where the interaction takes place. Several approaches have been developed that take advantage of bio‐orthogonal chemistry and subsequent enrichment steps to isolate peptides modified by small molecules. These methods rely on target identification at the level of mass spectrometry making it difficult to interpret an experiment when modified peptides are not identified. Herein, an approach in which fluorescence‐triggered two‐dimensional chromatography enables the isolation of small molecule‐conjugated peptides prior to mass spectrometry analysis is described. In this study, a bromocoumarin moiety has been utilized that fluoresces and generates a distinct isotopic signature to locate and identify modified peptides. Profiling of a cellular cysteinome with the use of a bromocoumarin tag demonstrates that two‐dimensional fluorescence‐based chromatography separation can enable the identification of proteins containing reactive cysteine residues. Moreover, the method facilitates the interrogation of low abundance proteins with greater depth and sensitivity than a previously reported isotope‐targeted approach. Lastly, this workflow enables the identification of small‐molecule modified peptides from a protein‐of‐interest. 相似文献
3.
Schwann cells (SC) are essential for the growth, maintenance, and regeneration of peripheral nerves, but the proteome of normal human SC is poorly defined. Here, a proteomic analysis by LC–MS/MS is performed to define the protein expression profile of primary human SC. A total of 19 557 unique peptides corresponding to 1553 individual proteins are identified. Ingenuity Pathway Analysis (IPA), Gene Ontology (GO), and Database for Annotation, Visualization, and Integrated Discovery (DAVID) are used to assign protein localization and function, and to define enriched pathways. EIF2, mTOR, and integrin signaling are among the most enriched pathways and the most enriched biological function is cell–cell adhesion, which is in agreement with the supportive role of SC in peripheral nerves. In addition, several nociceptors and synaptic proteins are identified and may contribute to the recently discovered role of SC in pain sensation and cancer progression. This proteome analysis of normal human SC constitutes a reference for future molecular explorations of physiological and pathological processes where SC are involved. 相似文献
4.
In this study the analysis and confirmation of flumequine enantiomers in rat plasma by ultra‐fast liquid chromatography coupled with electron spray ionization mass spectrometry (using propranolol as an internal standard [IS]) was developed and validated. Plasma samples were prepared by liquid–liquid extraction using methyl tert‐butyl ether as the extraction solvent. Direct resolution of the R‐ and S‐isomers was performed on a CHIRALCEL OJ‐RH column (4.6 × 150 mm, 5 μm) using acetonitrile / 0.1% formic acid / 1 mM ammonium acetate as the mobile phase. Detection was operated by electron spray ionization in the selected ion monitoring and positive ion mode. The target ions at m/z 262.1 and m/z 260.1 were selected for the quantification of the enantiomers and IS, respectively. The linear range was 0.5–500 ng/mL. The precisions (coefficient of variation, CV%) and recoveries were 1.43–8.68 and 94.24–106.76%, respectively. The lowest quantitation limit for both enantiomers is 0.5 ng/mL, which is sensitive enough to be applied to sample analysis in other related studies. 相似文献
5.
Jiayuan Qu Ju Zhang Lucas Zellmer Yan He Siqi Liu Chenguang Wang Chengfu Yuan Ningzhi xu Hai Huang Dezhong J. Liao 《Protein science : a publication of the Protein Society》2020,29(4):964-976
Most genes in evolutionarily complex genomes are expressed to multiple protein isoforms, but there is not yet any simple high‐throughput approach to identify these isoforms. Using an oversimplified top‐down LC–MS/MS strategy, we detected, around the 26‐kD position of SDS‐PAGE, proteins produced from 782 genes in a Cdk4?/? mouse embryonic fibroblast cell line. Interestingly, only 213 (27.24%, about one‐fourth) of these 782 genes have their proteins with a theoretical molecular mass (TMM) 10% smaller or larger than 26 kD, that is, between 23 and 29 kD, the range set as allowed variation in SDS‐PAGE. These 213 proteins are considered as the wild type (WT). The remaining three‐fourths includes proteins from 66 (9.44%) genes with a TMM smaller than 23 kD and proteins from 503 (64.32%, nearly two‐thirds) genes with a TMM larger than 29 kD; these proteins are categorized into a larger‐group or a smaller‐group, respectively, for their appearance at a higher or lower position of SDS‐PAGE. For instance, at this 26‐kD position we detected proteins from the Rps27a, Snrpf, Hist1h4a, and Rps25 genes whose proteins' TMM is 8.6, 9.7, 11.4, and 13.7 kD, respectively, and detected proteins from the Plelc1 and Prkdc genes, whose largest isoform is 533.9 and 471.1 kD, respectively. We extrapolate that many of those proteins migrating unexpectedly in SDS‐PAGE may be isoforms besides the WT protein. Moreover, we also detected a Cdk4 protein in this Cdk4?/? cell line, thus wondering whether some of other gene‐knockout cells or organisms show similar incompleteness of the knockout. 相似文献
6.
Junko Koyama Atsuko Takeuchi Izumi Morita Yu Nishino Maki Shimizu Munetaka Inoue Norihiro Kobayashi 《Bioorganic & medicinal chemistry》2009,17(21):7493-7499
A rapid, simple, and sensitive liquid chromatography–atmospheric pressure chemical ionization tandem mass spectrometry (LC–APCI-MS/MS) method was developed for the identification and quantification of emodin metabolites in Raji cells, using aloe-emodin as an internal standard. Analyses were performed on an LC system employing a Cosmosil 5C18 AR-II column and a stepwise gradient elution with methanol–20 mM ammonium formate at a flow rate of 1.0 mL/min operating in the negative ion mode. As a result, the starting material emodin and its five metabolites were detected by analyzing extracts of Raji cells that had been cultivated in the presence of emodin. The identification of the metabolites and elucidation of their structures were performed by comparing their retention times and spectral patterns with those of synthetic samples. In addition to the major metabolite 8-O-methylemodin, four other metabolites were assigned as ω-hydroxyemodin, 3-O-methyl-ω-hydroxyemodin, 3-O-methylemodin (physcion), and chrysophanol. 相似文献
7.
A sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is described for the simultaneous identification and quantification of eight endocannabinoid (EC) or related "entourage" compounds in rat brain tissue. Analytes were extracted and purified from rat brain tissue using an ethyl acetate/hexane solvent extraction, followed by a solid phase extraction (SPE) protocol. Chromatographic separation was achieved using a gradient elution, with a mobile phase of acetonitrile, formic acid, and ammonium acetate, at pH 3.6. A Thermo Hypersil C8 HyPurity Advance column (100x2.1 mm i.d., 3 microm) was used with a flow rate of 0.3 ml/min). Anandamide (AEA), 2-arachidonyl glycerol (2-AG), 2-arachidonylglyceryl ether (noladin ether), O-arachidonyl ethanolamide (virodhamine), 2-linoleoyl glycerol (2-LG), arachidonyl glycine, oleoyl ethanolamide (OEA), and palmitoyl ethanolamide (PEA) were quantified by positive ion tandem electrospray ionization mass spectrometry. Internal standards were deuterated AEA, deuterated 2-AG, and heptadecanoyl ethanolamide (HEA). Linearity was proven over the range of 25 fmol to 250 pmol, with a limit of detection of 25 fmol on column for all analytes except 2-AG, noladin ether, and 2-LG (250 fmol). This corresponded to a limit of quantification in biological tissue of 10 pmol/g for all analytes except 2-AG (100 pmol/g). Intra- and interday precision in biological tissue was routinely approximately 20% or lower, and accuracy was between 65% and 155%. This method was used to quantitatively profile regional differences in nine discrete rat brain regions for AEA, 2-AG, 2-LG, OEA, PEA, noladin ether, virodhamine, and arachidonyl glycine. 相似文献
8.
Glassy materials were prepared using two different systems: 50B2O3 – (50 ? x)PbO ? xPbCl2, with x = 0, 2 and 5 in mol % (System BPCl‐I) and 50BO1.5 – (50 ? x)PbO ? xPbCl2 with x = 0, 2, 5 and 7 in cationic % (System BPCl‐II). Structural and optical characterization showed that PbCl when substituted for PbO changed the structure of the glass network by replacing nonbridging oxygens for chlorine ions. This substitution also caused a change in the number of defects responsible for thermoluminescence (TL) emission (electrons and hole trap centres). Thermoluminescence emissions were observed for the first time in lead oxychloroborate glasses after exposure to UV radiation. Sample BPCl‐I‐2 (x = 2 from System I) demonstrated better TL emission compared with other glass samples. One intense peak in the glow curve, centred at ~122°C followed by a shoulder at ~180°C, was highly sensitive to UV radiation. There were also good linear responses at dose range ~0.4 to ~2 J/cm2 for the first peak (low temperature) and ~0.4 to ~4 J/cm2 for the second peak (high temperature). 相似文献
9.
10.
11.
12.
Takumi Takata Seongmin Ha Tamaki Koide Noriko Fujii 《Protein science : a publication of the Protein Society》2020,29(4):941-951
Recent studies have suggested that the isomerization/racemization of aspartate residues in proteins increases in aged tissues. One such residue is Asp151 in lens‐specific αA‐crystallin. Although many isomerization/racemization sites have been reported in various proteins, the factors that lead to those modifications in proteins in vivo remain obscure. Therefore, an in vitro system is needed to assess the mechanisms of modifications of Asp under various conditions. Deamidation of Asn to Asp in proteins occurs more rapidly than isomerization/racemization of Asp, although the reaction passes through the same intermediate in both pathways. Here, therefore, we replaced Asp151 in human lens αA‐crystallin with Asn by using site‐directed mutagenesis. The recombinant protein was expressed in Escherichia coli and used to investigate the deamidation/isomerization/racemization of Asn151 after incubation at 50°C for various durations and under different pH. After incubation, the mutant αA‐crystallin was subjected to enzymatic digestion followed by liquid chromatography–MS/MS to evaluate the ratio of modifications in Asn151‐containing peptides. The Asp151Asn αA‐crystallin mutant showed rapid deamidation to Asp with the formation of specific Asp isomers. In particular, deamidation increased greatly under basic conditions. By contrast, subunit–subunit interactions between αA‐crystallin and αB‐crystallin had little effect on the modification of Asn151. Our findings suggest that the Asp151Asn αA‐crystallin mutant represents a good in vitro model protein to assess deamidation, isomerization, and the racemization intermediates. Furthermore, our in vitro results show a different trend from in vivo data, implying the presence of specific factors that induce racemization from L‐Asp to D‐Asp residues in vivo. 相似文献
13.
A novel type of lipid droplet/lipoprotein (LD/LP) particle from Thermoplasma acidophilum has been identified recently, and based on biochemical evidences, it was named Thermoplasma Quinone Droplet (TaQD). The major components of TaQDs are menaquinones, and to some extent polar lipids, and the 153 amino acid long Ta0547 vitellogenin‐N domain protein. In this paper, the aim is to identify TaQD proteome components with 1D‐SDS‐PAGE/LC–MS/MS and cross reference them with Edman degradation. TaQD samples isolated with three different purification methods—column chromatography, immunoprecipitation, and LD ultracentrifugation—are analyzed. Proteins Ta0093, Ta0182, Ta0337, Ta0437, Ta0438, Ta0547, and Ta1223a are identified as constituents of the TaQD proteome. The majority of these proteins is uncharacterized and has low molecular weight, and none of them is predicted to take part in lipid metabolism. Bioinformatics analyses does not predict any interaction between these proteins, however, there are indications of interactions with proteins taking part in lipid metabolism. Whether if TaQDs provide platform for lipid metabolism and the interactions between TaQD proteins and lipid metabolism proteins occur in the reality remain for further studies. 相似文献
14.
Shengni Li Huafang Jiang Yiya Wang Yinli Liu Xiaohang Shen Wenzhong Liang Zhanying Hong 《Chirality》2016,28(7):569-575
A sensitive and high‐throughput chiral liquid chromatography–tandem mass spectrometry method was developed and validated for the quantification of R‐pantoprazole and S‐pantoprazole in human plasma. Sample extraction was carried out by using ethyl acetate liquid–liquid extraction in 96‐well plate format. The separation of pantoprazole enantiomers was performed on a CHIRALCEL OJ‐RH column and an overlapping injection mode was used to achieve a run time of 5.0 min/sample. The mobile phase consisted of 1) 10 mM ammonium acetate in methanol: acetonitrile (1:1, v/v) and 2) 20 mM ammonium acetate in water. Isocratic elution was used with flow rate at 500 μL/min. The enantiomers were quantified on a triple‐quadrupole mass spectrometer under multiple reaction monitoring (MRM) mode with m/z 382.1/230.0 for pantoprazole and m/z 388.4/230.1 for pantoprazole‐d7. Linearity from 20.0 to 5000 ng/mL was established for each enantiomer (r2 > 0.99). Extraction recovery ranged from 91.7% to 96.4% for R‐pantoprazole and from 92.5% to 96.5% for S‐pantoprazole and the IS‐normalized matrix factor was 0.98 to 1.07 for R‐pantoprazole and S‐pantoprazole, respectively. The method was demonstrated with acceptable accuracy, precision, selectivity, and stability and the method was applied to support a pharmacokinetic study of a phase I clinical trial of racemic pantoprazole in healthy Chinese subjects. Chirality 28:569–575, 2016. © 2016 Wiley Periodicals, Inc. 相似文献
15.
An efficient, sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) chiral analysis method was established for determination of chloroquine and hydroxychloroquine enantiomers in rat liver microsomes. Effects of polysaccharide chiral stationary phases and basic additives on chiral separations of two analytes were discussed in detail. Amylose tris(3, 5-dimethylphenylcarbamate)-coated chiral stationary phase showed the best separation performance for them with acetonitrile-diethylamine-ethanol-diethylamine mixture (90:0.1:10:0.1, v/v/v/v) among four chiral stationary phases. Then, multiple reaction monitoring mode was selected as the data acquisition for determination of two pairs of enantiomers. The proposed LC–MS/MS chiral analysis method was validated in terms of linearity, accuracy, precision, and specificity. Good linearity with correlation coefficient over 0.998 was obtained in the concentration range of 0.05–5 μM. Limits of quantification for chloroquine and hydroxychloroquine enantiomers were 5.0 and 1.0 nM, respectively. The recoveries ranged from 81.14% to 111.09%. The intra-day and inter-day relative standard deviation were less than 6.5%. Moreover, concentrations of chloroquine and hydroxychloroquine enantiomers in rat liver microsomes were determined through the proposed LC–MS/MS analysis method. After incubated with rat liver microsomes for 10 min, the enantiomeric factor of hydroxychloroquine decreased from 0.50 to 0.45 (p < 0.001). In brief, our developed determination method for chloroquine and hydroxychloroquine enantiomers through LC–MS/MS spectrometry showed the characteristics of high-efficiency, fast speed, and very low detection limit, and would be greatly beneficial for screening and quantitation of them in biological matrices. 相似文献
16.
Melissa R. Pergande Fidel Serna‐Perez Sheher Banu Mohsin Jonathon Hanek Stephanie M. Cologna 《Proteomics》2019,19(18)
Niemann–Pick disease, type C1 (NPC1) is a fatal, autosomal recessive, neurodegenerative disorder caused by mutations in the NPC1 gene. As a result of the genetic defect, there is accumulation of unesterified cholesterol and sphingolipids in the late endosomal/lysosomal system causing both visceral and neurological defects. These manifest clinically as hepatosplenomegaly, liver dysfunction, and neurodegeneration. While significant progress has been made to better understand NPC1, the downstream effects of cholesterol storage and the major mechanisms that drive these pathologies remains less understood. In this study, it is sought to investigate free fatty acid levels in Npc1?/? mice with focus on the polyunsaturated ω‐3 and ω‐6 fatty acids. Since fatty acids are the main constituents of numerous lipids species, a discovery based lipidomic study of liver tissue in Npc1?/? mice is also performed. To this end, alterations in fatty acid synthesis, including the ω‐3 and 6 fatty acids, are reported. Further, alterations in enzymes that regulate the synthesis of ω‐3 and 6 fatty acids are reported. Analysis of the liver lipidome reveals alterations in both storage and membrane lipids including ceramides, fatty acids, phosphatidylcholamines, phosphatidylglycerols, phosphatidylethanolamines, sphingomyelins, and triacylglycerols in Npc1?/? mice at a late stage of disease. 相似文献
17.
18.
I. Mazzeo E. Giorgini G. Gioacchini F. Maradonna M. C. Vílchez S. Baloche S. Dufour L. Pérez O. Carnevali J. F. Asturiano 《Journal of fish biology》2016,89(4):2055-2069
A multi‐technique approach was used to study the changes occurring in European eel Anguilla anguilla ovaries during hormonally‐induced vitellogenesis. Aside from classic techniques used to monitor the vitellogenic process, such as ovary histology, fat content analysis, sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and vitellogenin enzyme linked immunosorbent assay (ELISA), a new technique, Fourier‐transform infrared (FT‐IR) microspectroscopy, was used to analyse A. anguilla ovaries. The results from the different techniques provided different ways of approaching the same process. Although it is considered a time consuming approach, of all the employed techniques, histology provided the most direct evidences about vitellogenesis. SDS–PAGE and ELISA were also useful for studying vitellogenesis, whereas fat analysis cannot be used for this purpose. The FT‐IR analysis provided a representative IR spectrum for each ovarian stage (previtellogenic stage, early vitellogenic stage, mid‐vitellogenic stage and late vitellogenic stage), demonstrating that it is a valid method able to illustrate the distribution of the oocytes within the ovary slices. The chemical maps obtained confirmed changes in lipid concentrations and revealed their distribution within the oocytes at different maturational stages. When the results and the accuracy of the FT‐IR analysis were compared with those of the traditional techniques commonly used to establish the vitellogenic stage, it became evident that FT‐IR is a useful and reliable tool, with many advantages, including the fact that it requires little biological material, the costs involved are low, analysis times are short and last but not least, the fact that it offers the possibility of simultaneously analysing various biocomponents of the same oocyte. 相似文献
19.
20.
Daniel Paul Zolg Mathias Wilhelm Peng Yu Tobias Knaute Johannes Zerweck Holger Wenschuh Ulf Reimer Karsten Schnatbaum Bernhard Kuster 《Proteomics》2017,17(21)
Beyond specific applications, such as the relative or absolute quantification of peptides in targeted proteomic experiments, synthetic spike‐in peptides are not yet systematically used as internal standards in bottom‐up proteomics. A number of retention time standards have been reported that enable chromatographic aligning of multiple LC–MS/MS experiments. However, only few peptides are typically included in such sets limiting the analytical parameters that can be monitored. Here, we describe PROCAL (ProteomeTools Calibration Standard), a set of 40 synthetic peptides that span the entire hydrophobicity range of tryptic digests, enabling not only accurate determination of retention time indices but also monitoring of chromatographic separation performance over time. The fragmentation characteristics of the peptides can also be used to calibrate and compare collision energies between mass spectrometers. The sequences of all selected peptides do not occur in any natural protein, thus eliminating the need for stable isotope labeling. We anticipate that this set of peptides will be useful for multiple purposes in individual laboratories but also aiding the transfer of data acquisition and analysis methods between laboratories, notably the use of spectral libraries. 相似文献