共查询到20条相似文献,搜索用时 15 毫秒
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Yannick Baschung Loredana Lupu Adrian Moise Michael Glocker Stephan Rawer Alexander Lazarev Michael Przybylski 《Journal of the American Society for Mass Spectrometry》2018,29(9):1881-1891
Affinity mass spectrometry using selective proteolytic excision and extraction combined with MALDI and ESI mass spectrometry has been applied to the identification of epitope binding sites of lactose, GalNac, and blood group oligosaccharides in two blood group-specific lectins, human galectin-3 and glycine max lectin. The epitope peptides identified comprise all essential amino acids involved in carbohydrate recognition, in complete agreement with available X-ray structures. Tryptic and chymotryptic digestion of lectins for proteolytic extraction/excision-MS was substantially improved by pressure-enhanced digestion using an automated Barocycler procedure (40 kpsi). Both previously established immobilization on affinity microcolumns using divinyl sulfone and coupling of a specific peptide glycoprobe to the gold surface of a biosensor chip were successfully employed for proteolytic excision and extraction of carbohydrate epitopes and affinity measurements. The identified epitope peptides could be differentiated according to the carbohydrate employed, thus demonstrating the specificity of the mass spectrometric approach. The specificities of the epitope ligands for individual carbohydrates were further ascertained by affinity studies using synthetic peptide ligands with immobilized carbohydrates. Binding affinities of the synthetic ligand peptides to lactose, in comparison to the intact full-length lectins, were determined by surface acoustic wave (SAW) biosensor analysis and provided micromolar KD values for the intact lectins, in agreement with results of previous ITC and SPR studies. Binding affinities of the epitope peptides were approximately two orders of magnitude lower, consistent with their smaller size and assembled arrangement in the carbohydrate recognition domains. 相似文献
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Eric B. Monroe Suresh P. Annangudi Andinet A. Wadhams Timothy A. Richmond Ning Yang Bruce R. Southey Elena V. Romanova Liliane Schoofs Geert Baggerman Jonathan V. Sweedler 《Journal of the American Society for Mass Spectrometry》2018,29(5):923-934
Neuropeptides are essential cell-to-cell signaling messengers and serve important regulatory roles in animals. Although remarkable progress has been made in peptide identification across the Metazoa, for some phyla such as Echinodermata, limited neuropeptides are known and even fewer have been verified on the protein level. We employed peptidomic approaches using bioinformatics and mass spectrometry (MS) to experimentally confirm 23 prohormones and to characterize a new prohormone in nervous system tissue from Strongylocentrotus purpuratus, the purple sea urchin. Ninety-three distinct peptides from known and novel prohormones were detected with MS from extracts of the radial nerves, many of which are reported or experimentally confirmed here for the first time, representing a large-scale study of neuropeptides from the phylum Echinodermata. Many of the identified peptides and their precursor proteins have low homology to known prohormones from other species/phyla and are unique to the sea urchin. By pairing bioinformatics with MS, the capacity to characterize novel peptides and annotate prohormone genes is enhanced. 相似文献
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Stefanescu R Born R Moise A Ernst B Przybylski M 《Journal of the American Society for Mass Spectrometry》2011,22(1):148-157
Recent studies suggest that the H1 subunit of the carbohydrate recognition domain (H1CRD) of the asialoglycoprotein receptor
is used as an entry site into hepatocytes by hepatitis A and B viruses and Marburg virus. Thus, molecules binding specifically
to the CRD might exert inhibition towards these diseases by blocking the virus entry site. We report here the identification
of the epitope structure of H1CRD to a monoclonal antibody by proteolytic epitope excision of the immune complex and high-resolution
MALDI-FTICR mass spectrometry. As a prerequisite of the epitope determination, the primary structure of the H1CRD antigen
was characterised by ESI-FTICR-MS of the intact protein and by LC-MS/MS of tryptic digest mixtures. Molecular mass determination
and proteolytic fragments provided the identification of two intramolecular disulfide bridges (seven Cys residues), and a
Cys-mercaptoethanol adduct formed by treatment with β-mercaptoethanol during protein extraction. The H1CRD antigen binds to
the monoclonal antibody in both native and Cys-alkylated form. For identification of the epitope, the antibody was immobilized
on N-hydroxysuccinimide (NHS)-activated Sepharose. Epitope excision and epitope extraction with trypsin and FTICR-MS of affinity-bound
peptides provided the identification of two specific epitope peptides (5–16) and (17–23) that showed high affinity to the
antibody. Affinity studies of the synthetic epitope peptides revealed independent binding of each peptide to the antibody. 相似文献
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Dupré M Cantel S Verdié P Martinez J Enjalbal C 《Journal of the American Society for Mass Spectrometry》2011,22(2):265-279
In this study, we explored the MS/MS behavior of various synthetic peptides that possess a lysine residue at the N-terminal
position. These peptides were designed to mimic peptides produced upon proteolysis by the Lys-N enzyme, a metalloendopeptidase
issued from a Japanese fungus Grifola frondosa that was recently investigated in proteomic studies as an alternative to trypsin digestion, as a specific cleavage at the
amide X-Lys chain is obtained that provides N-terminal lysine peptide fragments. In contrast to tryptic peptides exhibiting
a lysine or arginine residue solely at the C-terminal position, and are thus devoid of such basic amino acids within the sequence,
these Lys-N proteolytic peptides can contain the highly basic arginine residue anywhere within the peptide chain. The fragmentation
patterns of such sequences with the ESI-QqTOF and MALDI-TOF/TOF mass spectrometers commonly used in proteomic bottom-up experiments
were investigated. 相似文献
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Bythell BJ Hendrickson CL Marshall AG 《Journal of the American Society for Mass Spectrometry》2012,23(4):644-654
We report the use of unimolecular dissociation by infrared radiation for gaseous multiphoton energy transfer to determine
relative activation energy (Ea,laser) for dissociation of peptide sequence ions. The sequence ions of interest are mass-isolated; the entire ion cloud is then
irradiated with a continuous wave CO2 laser, and the first order rate constant, kd, is determined for each of a series of laser powers. Provided these conditions are met, a plot of the natural logarithm of
kd versus the natural logarithm of laser power yields a straight line, whose slope provides a measure of Ea,laser. This method reproduces the Ea values from blackbody radiative dissociation (BIRD) for the comparatively large, singly and doubly protonated bradykinin
ions (nominally y
9
and y
9
2+
). The comparatively small sequence ion systems produce Ea,laser values that are systematic underestimates of theoretical barriers calculated with density functional theory (DFT). However,
the relative Ea,laser values are in qualitative agreement with the mobile proton model and available theory. Additionally, novel protonated cyclic-dipeptide
(diketopiperazine) fragmentation reactions are analyzed with DFT. FT-ICR MS provides access to sequence ions generated by
electron capture dissociation, infrared multiphoton dissociation, and collisional activation methods (i.e., b
n
, y
m
, c
n
, z
m
•
ions). 相似文献
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Dr. Xin Yan Ryan M. Bain Prof. R. Graham Cooks 《Angewandte Chemie (International ed. in English)》2016,55(42):12960-12972
The striking finding that reaction acceleration occurs in confined‐volume solutions sets up an apparent conundrum: Microdroplets formed by spray ionization can be used to monitor the course of bulk‐phase reactions and also to accelerate reactions between the reagents in such a reaction. This Minireview introduces droplet and thin‐film acceleration phenomena and summarizes recent methods applied to study accelerated reactions in confined‐volume, high‐surface‐area solutions. Conditions that dictate either simple monitoring or acceleration are reconciled in the occurrence of discontinuous and complete desolvation as the endpoint of droplet evolution. The contrasting features of microdroplet and bulk‐solution reactions are described together with possible mechanisms that drive reaction acceleration in microdroplets. Current applications of droplet microreactors are noted as is reaction acceleration in confined volumes and possible future scale‐up. 相似文献
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小批量制备了电泳纯鲨鱼肝铁蛋白(Liver ferritin of Sphyma zygaena, SZLF). 用透射电子显微镜技术研究SZLF的铁核和蛋白壳亚基解离和重组过程. 用盐酸解离(pH=1.5)和分离膜透析技术制备铁蛋白亚基, 并用酸碱中和方法重组亚基成为脱铁核铁蛋白(apoSZLF), 同时将胰岛素(Insulin, INS)包裹于apoSZLF蛋白壳内, 构建纳米INS核-SZLF. 用电子光谱、MALDI-TOF质谱和SDS-PAGE技术分别揭示了纳米INS核-SZLF分子结构的真实性, 提出铁蛋白亚基包裹INS构建为纳米INS核-铁蛋白的途径. 相似文献
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基体辅助激光解吸/电离质谱(MALDIMS)法自80年代末由Karas,Hilenkamp报道以来,已获得极大的发展.近几年来,其应用范围迅速扩大,各种生物大分子如蛋白质,核酸(DNA),多糖等都已能用MALDIMS法进行分子量测定.1993年,... 相似文献
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Hong‐Xu Na Pei‐Yu Yang Dr. Zheng Yin Yun‐Hong Wang Li‐Xian Chang Prof. Dr. Rui Si Dr. Mohamedally Kurmoo Prof. Dr. Ming‐Hua Zeng 《Chemistry (Weinheim an der Bergstrasse, Germany)》2016,22(51):18404-18411
The square‐planar monomer NiL2 ( Ni1 ), L=2‐ethoxy‐6‐(N‐methyl‐iminomethyl)phenolate, reacts with M(H2O)6(ClO4)2, M=Ni or Co, to form heptanuclear disks [CoxNi7?x(OH)6(L)6](ClO4)2 ? 2 CH3CN ( Co x Ni7?x , x=0–7) and the co‐crystal [CoxNi7?x(OH)6L6][NiL2](ClO4)2 ? 2 CH3CN ( Co x Ni7?x ‐Ni1 ) under ambient conditions. It has proved possible to explore the bottom‐up assembly process of Co x Ni7?x and Co x Ni7?x ‐Ni1 in real time. The final products have been characterized by thermogravimetric analysis, IR, elemental analysis, ICP‐MS, and single‐crystal X‐ray diffraction. Time‐dependent mass spectrometry (MS) revealed the following reaction steps: Ni1→[M2L3]+→[M4(OH)2L4]2+→[M7(OH)6L6]2+. In contrast, the reaction of Ni1 with Zn2+ only reaches halfway, and crystallographic evidence indicates a butterfly structure for [Zn2Ni2(OH)2Cl2] ( Zn2Ni2 ), an intermediate that is difficult to isolate in the above Ni‐Co series. A summation method has been used to analyze the MS of bimetallic clusters with very similar atomic masses, as is the case for Co and Ni. The results provide ample information on the distribution of Co and Ni within each cluster and their statistical distribution within selected crystals. 相似文献
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Fouquet T Humbel S Charles L 《Journal of the American Society for Mass Spectrometry》2011,22(4):649-658
Ammonium adducts of trimethylsilyl-terminated poly(dimethylsiloxane) (CH3-PDMS) produced by electrospray ionization were submitted to collision induced dissociation and revealed a particular MS/MS
behavior: the same three main product ions at m/z 221, 295, and 369 were always generated in very similar relative abundances regardless of the size of the precursor ion.
Combining accurate mass measurements and ab initio calculation allowed very stable cyclic geometries to be obtained for these
ionic species. Dissociation mechanisms were proposed to account for the three targeted ions to be readily generated in a two-step
or a three-step reaction from any CH3-PDMS ammonium adducts. A second set of three product ions was also observed with low abundance at m/z 207, 281, and 355, which were shown in MS3 experiments to be formed in secondary reactions. An alternative dissociation process was shown to consist of a concerted
elimination of ammonia and methane and the need for a methyl of an end-group to be involved in the released methane molecule
would account for this reaction to mainly proceed from the smallest precursor ions. 相似文献
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激光产生的碳原子簇负离子及其质谱研究(II) 总被引:2,自引:0,他引:2
近年来,尽管碳原子簇的合成与研究已成为非常热门的课题,但是对其负离子的研究却相对冷落了一些.两年多前,我们以激光在高真空中直接轰击单质碳样品的方式,产生了n≤441的C_n~-并通过对其质谱的分析确定了它们从链状至环状构型的转化.最近,我们又以同样方式产生了成簇原子数高于100的碳原子簇负离子,记录了它们的飞行时间质谱. 本实验选用的样品是光谱纯的碳粉,直接压入样品托座中的小坑.仪器的构造已有另文详细介绍,实验时仪器的主要工作参数与参考文献[1] 中描述的基本相同,只是激光束 相似文献
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质谱是一种广泛应用于化学、生物医学、药学、环境、农业和能源等各领域的分子结构鉴定技术,这种技术通过准确测定分子离子和碎片离子的质量-电荷比来推导分子结构。如何将试样中待测组分有效气化、离子化,转变为具有不同质-荷比的气态离子是质谱仪器和分析方法研究的关键。基于不同物理化学原理的电离、解离方法各有特点,适合不同分析目的。常见的软电离技术一般产生稳定的偶电子离子,往往需要与其他技术联用才能实现分子离子的进一步解离。除了基于碰撞活化和电子得失的两类常见解离方法,光解离技术利用波长/能量可调控的光辐射来使样品分子电离,并引发特定化学键断裂。本文旨在综述不同电离/解离技术,重点探讨近年来发展的红外和紫外光电离/解离技术基本工作原理、仪器特点及其在生物分子(包括有机小分子、蛋白质、核酸和多糖等)结构鉴定中的应用。 相似文献
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高效液相色谱-串联质谱法同时测定细菌群体感应效应的11种AHLs类信号分子 总被引:1,自引:0,他引:1
建立了高效液相色谱-串联质谱(HPLC-MS/MS)法同时测定细菌群体感应分泌的11种AHLs类信号分子的分析方法.细菌所分泌AHLs类信号分子经乙酸乙酯萃取后简单处理,可直接进样分析.采用Sunfire C18色谱柱(50 mm×2.1 mm, 3.5 μm),甲醇与水(含2 mmoI/L乙酸铵,0.1% 甲酸 )梯度进样,采用电喷雾离子源正离子模式,多反应监测(MRM)模式,外标法进行质谱定量分析.分析时间为12 min.本方法在1~250 μg/L范围内呈良好的线性关系(r>0.996);检出限为0.1~1.0 μg/kg;定量限为0.3~3.0 μg/kg;平均添加回收率为54.4%~128.6%;相对标准偏差为3.5%~13.9%.本方法具有操作简便、快速、准确、灵敏度高等优点,适用于多种AHLs类信号分子化合物的同时定性与定量分析,为微生物群体感应的进一步研究提供了有效的且便利的分析方法. 相似文献
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Krebs Cycle Metabolon: Structural Evidence of Substrate Channeling Revealed by Cross‐Linking and Mass Spectrometry 下载免费PDF全文
It has been hypothesized that the high metabolic flux in the mitochondria is due to the self‐assembly of enzyme supercomplexes (called metabolons) that channel substrates from one enzyme to another, but there has been no experimental confirmation of this structure or the channeling. A structural investigation of enzyme organization within the Krebs cycle metabolon was accomplished by in vivo cross‐linking and mass spectrometry. Eight Krebs cycle enzyme components were isolated upon chemical fixation, and interfacial residues between mitochondrial malate dehydrogenase, citrate synthase, and aconitase were identified. Using constraint protein docking, a low‐resolution structure for the three‐enzyme complex was achieved, as well as the two‐fold symmetric octamer. Surface analysis showed formation of electrostatic channeling upon protein–protein association, which is the first structural evidence of substrate channeling in the Krebs cycle metabolon. 相似文献
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尿毒症被认为是因为患者肾衰而毒素在体内滞留所致^[1]。1972年Babb等^[2]提出“中分子假说”,认为分子量在300-2000范围内的中等分子量的物质是尿毒症的主要毒性物质。从此,人们作了大量的努力去分离和鉴定尿毒症中分子毒物。然而尿毒症中分子毒物的成分极其复杂^[3],从设定的中分子组分中分离得到的大都是些分子量小于800的小分子物质^[4]。因而对中分子假说一直存在争议^[5].我们对尿毒症患者及正常人的血清和尿液进行凝胶色谱分离,从尿毒症血清和尿液及正常人尿液中得到两个中分子峰A,B。将不同来源的A峰中的分子毒物进行离子交换色谱的分离和比较,得到了仅存在于尿毒症血清和正常人尿液的A-3亚峰,经脱盐和飞行时间质谱分析,确定了该组分内含有分子分别为839.69,1007.94,2015.16,16,873.69,1106.67和1680.28的6种化合物。 相似文献
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食品中胆固醇色谱/质谱/质谱的测定 总被引:3,自引:0,他引:3
确立了用色谱/质谱/质谱测定食品中胆固醇的一种新方法,试样经乙酸乙酯提取后,GC/MS/MS测定分析,以胆固醇分子离子为母离了,以其子离子为定量分析的碎片离子。线性好,回收率高,方法可靠。 相似文献