Screening and Structural Characterization of alpha-Glucosidase Inhibitors from Hawthorn Leaf Flavonoids Extract by Ultrafiltration LC-DAD-MSn and SORI-CID FTICR MS

In vitro a-glucosidase inhibition assays and ultrafiltration liquid chromatography with photodiode array detection coupled to electrospray ionization tandem mass spectrometry (ultrafiltration LC-DAD-ESI-MSn) were combined to screen a-glucosidase inhibitors from hawthorn leaf flavonoids extract (HLFE). As a result, four compounds were identified as alpha-glucosidase inhibitors in the HLFE, and their structures were confirmed to be quercetin-3-O-rha-(1-4)-glc-rha and C-glycosylflavones (vitexin-2 ''-O-glucoside, vitexin-2 ''-O-rhamnoside and vitexin) by high-resolution sustained off resonance irradiation collision-induced dissociation (SORI-CID) data obtained by Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS).

Bimolecular quadruplexes and their transitions to higher-order molecular structures detected by ESI-FTICR-MS

Four individual quadruplexes, which are self-assembled in ammonium acetate solution from telomeric sequences of closely related DNA strands - d(G(4)T(4)G(4)), d(G(3)T(4)G(4)), d(G(3)T(4)G(3)), and d(G(4)T(4)G(3)) - have been detected in the gas phase using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS). The bimolecular quadruplexes associate with the same number of NH4+ in the gas phase as NMR shows that they do in solution. The quadruplex structures formed in solution are maintained in the gas phase. Furthermore, the mass spectra show that the bimolecular quadruplexes generated by the strands d(G(3)T(4)G(3)) and d(G(4)T(4)G(3)) are unstable, being converted into trimolecular and tetramolecular structures with increasing concentrations of NH4+ in the solution. Circular dichroism (CD) spectra reveal structural changes during the process of strand stoichiometric transitions, in which the relative orientation of strands in the quadruplexes changes from an antiparallel to a parallel arrangement. Such changes were observed for the strand d(G(4)T(4)G(3)), but not for the strand d(G(3)T(4)G(3)). The present work provides a significant insight into the formation of various DNA quadruplexes, especially the higher-order species.

NADW Photodegradation FTICR-MS

Duplicate, filtered samples of North Atlantic Deep Water (NADW) were irradiated for 28 days in a solar simulator. Duplicate dark controls were placed alongside the irradiated samples. After 28 days, samples were extensively photo-degraded based upon colored dissolved organic matter (CDOM) photo-bleaching (> 95%). Samples were solid phase extracted using PPL resin to isolate, concentrate and desalt the dissolved organic matter (DOM) in the samples. Ultrahigh resolution electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) enabled 3024 molecular formulas to be identified in the dark control. Photo-degradation decreased molecular diversity, with 2402 formulas assigned post-irradiation. Molecular formulas were classified based upon their photo-lability as 1) photo-resistant; 2) photo-labile; and, 3) photo-produced. Photo-resistant DOM made up 73% of all formulas and was dominated by highly unsaturated molecular signatures consistent with carboxylic-rich alicyclic molecules, suggesting that these apparently bio-refractory compounds may also survive multiple passages through sunlit surface waters and thus accumulate for timeframes exceeding ocean ventilation. Photo-labile DOM was enriched in low molecular weight formulas, aromatic molecular formulas, and sulfur and phosphorous containing formulas. Compounds containing both sulfur and nitrogen were particularly photo-labile and may represent an underappreciated component of the photo-reactive CDOM pool. Photo-produced DOM was enriched in higher molecular weight formulas, as well as aliphatic and peptide formulas. Molecular formulas are indexed by their photo-lability classification in the supplementary information.

傅立叶变换离子回旋共振质谱在含糖小分子分析中的应用

傅立叶变换离子回旋共振质谱是一种近些年来逐渐发展起来的新型质谱仪器，由于该类质谱检测器的设计和检测原理与传统质谱有着根本的区别，通过它获取的数据具有高分辨和高质量测量精度的特点。通常，超过100000以上的分辨本领能够对质谱中非常临近的质谱峰进行区分，并结合串联质谱中的高质谱测量精度数据，可以给出明确的串联质谱碎裂途径。 本论文选择4对具有同分异构特点的二糖黄酮进行了系统研究，实验采取负离子模式的电喷雾傅立叶变换离子回旋共振质谱，结合持续非共振辐照碰撞诱导解离模式，对同分异构体的区分进行了研究。在实验过程中，建立了一种全新的质量校正方法，使得质谱测量平均误差小于1.00 ppm1。首次直接利用子离子的结构信息，确定了负离子模式下二糖黄酮的去质子化位点。实验中还发现，RDA解离途径仅仅当二糖黄酮的苷元是黄烷酮并且B环上没有过多的富电子基团的情况下才能发生，同时，具有α1→2糖连接的二糖黄酮在串联质谱中能够发生多键解离，并采用Gaussian 03 程序利用 B3LYP/6-31G方法对其进行了理论计算。为了进一步讨论α1→2糖连接二糖黄酮的串联质谱特点，在温和实验条件下，对上述化合物进行了氢氘交换实验。实验中首次发现温和条件下，黄酮的氢氘交换位点依赖于苷元结构，除了糖链上羟基和苷元上的酚羟基能够发生氢氘交换外，苷元为黄烷酮的二糖黄酮中，C（3）、C（6）和C（8）上的氢能够被直接交换掉，而苷元为黄酮骨架的二糖黄酮则在此位点不发生氢氘交换反应，并依据高质量测量精度数据对其子离子产生途径进行研究。 论文还系统研究了由葡萄糖缩合而成的二糖，4对二糖异构体负离子模式电喷雾傅立叶变换离子回旋共振质谱研究表明，其离子化过程中，生成的去质子化的二聚体是主要气相离子，依据单糖的实验和计算化学结果，论文中提出二糖化合物的离子化模型。计算化学的结果还证实，构成二聚体的单体直接具有强烈的相互作用，能够在串联质谱中产生共价键解离的子离子，而不是简单的单体解离。利用SORI CID还对二糖化合物的糖连接位点和糖苷键构型进行了区分研究。 论文的最后一部分研究了人工合成类肝素类化合物DHα、THα 和 THβ的结构表征，在极其温和的负离子模式电喷雾质谱条件下，类肝素化合物仍然容易发生多个SO3中性丢失。串联质谱中的子离子通过傅立叶变换离子回旋共振质谱高质量精度测量数据进行了确认。实验对研究类肝素类化合物的质谱表征提供了借鉴。

天然产物及相关复方配伍的质谱及液相色谱研究

质谱技术以其灵敏度高、样品需求量少、快速准确的特点在生物大分子体系和天然产物的化学成分研究中起着极为重要的作用。特别是软电离质谱技术的发展，大大简化了化合物的测定过程，提高了分析研究速度，为分析植物及食物中的结构信息提供了快速便捷的方法。HPLC-ESI-MSn是90年代发展成熟的分析技术，它集液相色谱的高分离能力与质谱的高灵敏度和高专属性于一体，已成为包括药物微量杂质、药物降解产物、药代代谢动力学研究、组合化学合成产物高通量分析以及天然产物的化学筛选在内的现代药学研究领域最强有力的分析工具之一。本文首先采用电喷雾多级串联质谱技术，系统地研究了负离子条件下黄酮普元、黄酮醇普元以及二氢黄酮普元等化合物的特征质谱行为，并对中药黄芬中三种黄酮昔化合物进行了研究。利用这些特征碎片离子，可以简便快捷的区分结构类似的黄酮类化合物及其同分异构体。通过对黄酮C一昔类化合物的电喷雾串联质谱（ESI-MSn）研究，提出黄酮C-苷类化合物的特征碎裂规律，证明〔M-H-60］-，〔M-H-90］-，〔M-H-120〕-为黄酮C-苷类化合物的特征离子。为鉴定植物粗提物中黄酮类化合物奠定了基础。之后我们进一步采用高效液相色谱与电喷雾质谱联用技术，在线区分混合物中的黄酮类化合物的同分异构体。根据黄酮类化合物的电喷雾质谱规律以及化合物的色谱保留时间分析鉴定了黄答中的七种黄酮类化合物。建立了一种有效检测黄芩（Scutellariabaicalensis Georgi）中黄酮类化合物的快速灵敏的分析方法，为建立黄答药材的质量控制标准提供了借鉴。对于黄酮类化合物灵敏、快速和准确的分析方法的建立，不仅为黄酮类化合物的结构的快速鉴定提供了一定的依据，而且对生药的鉴别和制剂的质控起到重要的作用。利用电喷雾多级串联质谱技术，对化学结构相似的黄酮营、二氢黄酮营和黄酮醇昔类化合物进行了对比研究。并且首次利用高分辨质谱FTICR-MS及SORI-CID技术对naringin进行了质谱研究。利用其超高分辩率、准确质量确证了中性碎片丢失，进一步证明了我们对其碎裂机理的推断。之后，我们通过金属离子与黄酮昔溶液混合后，采用电喷雾串联质潜法进行测定。实验表明通过人为加入某些金属离子如Li+，Na+和K+，特别是Li＋可以提高黄酮昔类化合物分析灵敏度，从而提供较多结构信息。并且在串联质谱中表现不同的碎裂行为，为黄酮类化合物结构分析提供补充信息。研究表明结合〔M-H］-，〔M＋Na〕+，〔M＋Li〕+离子串联质潜提供的信息，能够有效的鉴定黄酮普的结构。本文通过电喷雾串联质谱（ESI-MSn）和液质联机技术（HPLC-ESI-MS／MS）研究了原小聚碱型生物碱。首次利用高分辨质谱FTICR-MS和SORI-CTD技术对原小桨碱型生物碱进行了高分辨质谱研究，提出了各种碎片离子的碎裂途径，并总结了原小聚碱型生物碱的质谱碎裂规律，为鉴定此类型的生物碱提供了依据。之后通过电喷雾串联质谱研究了四种生物碱化合物质谱行为。最后我们利用液质联机技术（HPLC-ESI-MSn），通过与标准品的液相保留时间对照及原小聚碱型生物碱的质谱碎裂规律，建立了分析鉴定常用中药中原小聚碱型生物碱的快捷方法。并通过选择离子监控技术（SIM）提高了对于同分异构体的分离鉴定。通过色谱UV吸收峰定量研究了这四种药用植物中小璧碱和巴马汀的含量，为临床应用提供一定的化学基础，为建立中药质量控制标准及植物分类学提供一定的依据。采用HPLC法考察了常见的黄连药对组合煎煮对黄连中小聚碱、巴马丁煎出量的影响。为制药工艺的优化奠定了基础。之后我们以中药理论为指导，按药物性味对改良半夏泻心汤中各药进行分组，利用高效液相色谱分析了不同配伍条件下小璧碱、巴马丁煎出量的变化。为组方的合理性和科学性提供了一定的化学物质基础依据。

Studies of the intermolecular DNA triplexes of C+center dot GC and T center dot AT triplets by electrospray ionization Fourier-transform ion cyclotron resonance mass spectrometry

Formation and stabilities of four 14-mer intermolecular DNA triplexes, consisting of third strands with repeating sequence CTCT, CCTT, CTT, or TTT, were studied by electrospray ionization Fourier-transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) in the gas phase. The gas-phase stabilities of the triplexes were compared with their CD spectra and melting behaviors in solution, and parallel correlation between two phases were obtained. In the presence of 20 mm NH4+ (pH 5.5), the formation of the TTT triplex was not detected in both solution and the gas phase.

马钱苷的电喷雾串联质谱及傅立叶变换离子回旋共振质谱研究

利用电喷雾串联质谱(ESI-MSn)技术研究马钱苷的质谱碎裂机理,探讨其碎裂方式,并利用傅立叶变换离子回旋共振质谱(FTICR-MS)及SORI-CID高分辨率、高质量准确度对其碎裂机理进行验证研究。采用H/D交换和串联质谱的方法,发现马钱苷羟基上的活泼氢在质谱中性碎片丢失过程中较易被转移,表现出较大的化学活性。通过加入特定的Na+和Li+等碱金属离子,极大地提高了马钱苷化合物质谱分析的灵敏度,获得了更多的结构信息,建立了马钱苷化合物质谱鉴定和分析的新方法。

类肝素化合物的傅立叶变换离子回旋共振-电喷雾质谱研究

采用负离子模式的电喷雾傅立叶变换离子回旋共振质谱(ESI-FTICR-MS)对3个合成的肝素类化合物DHα、THα和THβ进行了全扫描一级谱和持续非共振辐照碰撞诱导解离(SORI-CID)串联质谱分析。样品浓度10μmol/L,流动泵注射直接进样。化合物中的磺酸胺和磺酸酯结构容易在一般电喷雾条件下发生分解,失去一个或者多个SO3,一级全扫描谱图中得到的谱峰均是带有单电荷或者双电荷的准分子离子峰。

Interactions of mitoxantrone with duplex and triplex DNA studied by electrospray ionization mass spectrometry

We have examined interactions between mitoxantrone (MXT) and DNA duplexes or triplexes with different base compositions by using electrospray ionization mass spectrometry (ESI-MS), respectively. MXT interacts preferentially with DNA duplexes compared to the triplexes. In the mass spectrum of the duplex-MXT mixture, the complex peaks dominated in the ratios of duplex/MXT of 1:1, 1:2 and 1:3, and the 1:2 duplex/MXT peak was the most abundant. In contrast, only 1:1 triplex-MXT complexes were observed in the mass spectrum of the triplex-MXT mixture, and the most intensive peak was a free triplex ion without MXT.

米托蒽醌与双链及三链核酸相互作用的电喷雾质谱研究

The interaction of mitoxantrone (MXT) with duplex and triplex DNA, contain repeating sequence CTCT, CCTT and CTT were studied by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS). The 1:3 specific complexes of mitoxantrone and duplex DNA and 1:2 specific complexes of mitoxantrone and triplex DNA were observed. The results show that mitoxantrone has no remarkable sequence selectivity, however it has distinct structure selectivity, and destabilization the triplex.

Structural analysis of monoterpene glycosides extracted from Paeonia lactiflora Pall. using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry and high-performance liquid chromatography/electrospray ionization tandem mass spectrometry

Paeoniflorin standard was first investigated by electrospray ionization Fourier transform ion cyclotron resonance tandem mass spectrometry (ESI-FTICR-MS/MS) using a sustained off-resonance irradiation (SORI) collision-induced dissociation (CID) method at high mass resolution. The experimental results demonstrated that the unambiguous elemental composition of product ions can be obtained at high mass resolution. Comparing MS/MS spectra and the experimental methods of hydrogen and deuterium exchange, the logical fragmentation pathways of paeoniflorin have been proposed. Then, the extracts of the traditional Chinese medicine Paeonia lactiflora Pall. were analyzed by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS). By comparison with the ESI-FTICR-MS/MS data of paeoniflorin, the isomers paeoniflorin and albiflorin in Paeonia lactiflora Pall. have been identified using HPLC/MS with CID in an ion trap and in-source CID. Furthermore, using the characteristic fragmentation pathways, the retention times (t(R)) in HPLC and MS/MS spectra, the structures of three other kinds of monoterpene glycoside compounds have been identified on-line without time-consuming isolation.

Investigation of heptakis(2,6-di-O-methyl)-beta-cyclodextrin inclusion complexes with flavonoid glycosides by electrospray ionization mass spectrometry

The non-covalent complexes between three flavonoid glycosides (quercitrin, hyperoside and rutin) and heptakis(2,6-di-O-methyl)-beta-cyclodextrin (DM-beta-CD) were investigated by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS). The 1:1 complexation of each flavonoid glycoside (guest) to the DM-beta-CD (host) was monitored in the negative ion mode by mixing each guest with an up to 30-fold molar excess of the host. The binding constants for all complexes were calculated by a linear equation in the order: DM-beta-CD:quercitrin > DM-beta-CD:rutin > DM-beta-CD:hyperoside. A binding model for the complexes has also been proposed based on the binding constants and tandem mass spectrometric data of these complexes.

A study of isomeric diglycosyl flavonoids by SORI CID of Fourier transform ion cyclotron mass Spectrometry in negative ion mode

High-resolution Sustained off resonance irradiation (SORI) CID was employed to distinguish four pairs of isomeric diglycosyl flavonoids in the negative mode using the electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FTICR MS). All of these isomers can be distinguished via MS/MS data. For these diglycosyl flavones and flavanones, the deprotonated alpha 1-->6 linkage diglycosyl flavonoids produce fewer fragments than the alpha 1-->2 linkage type compounds and the Retro-Diels-Alder (RDA) reaction in MS/MS only takes place when the aglycone is a flavanone and glycosylated with an alpha 1-->2 intersaccharide linkage disaccharide. The deprotonation sites after collisional activation are discussed according to the high mass accuracy and high-resolution data of tandem spectrometry. Some of these high-resolution SORI CID product ions from alpha 1-->2 linkage diglycosyl flavonoids involve multibond cleavages; the possible mechanism is discussed based on the computer modeling using Gaussian 03 program package at the B3LYP/6-31G level of theory. Unambiguous elementary composition data provides fragmentation information that has not been reported previously.

Analysis of strychnos alkaloids using electrospray ionization Fourier transform ion cyclotron resonance multi-stage tandem mass spectrometry

The fragmentations of four strychnos alkaloids have been investigated by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) in the positive ion mode. Experiments using multi-stage tandem mass spectrometry (ESI-FT-ICR-MSn) allowed us to obtain precise elemental compositions of product ions at high mass resolution. The experimental data demonstrated that the nitrogen bridge and the coordinated oxygen atom on the nitrogen bridge in the alkaloid compounds were the active sites in the MS2 fragmentations. The loss of CH3 or the OCH3 group in those alkaloids, which have an OCH3 substituent, was the dominant fragmentation mode in the MS3 fragmentations. Logical fragmentation schemes for strychnos alkaloids have been proposed and these should be useful for the identification of these compounds.

Comparison of bottom-up protein identification methods

With the rapid development of proteomics, a number of different methods appeared for the basic task of protein identification. We made a simple comparison between a common liquid chromatography-tandem mass spectrometry (LC-MS/MS) workflow using an ion trap mass spectrometer and a combined LC-MS and LC-MS/MS method using Fourier transform ion cyclotron resonance (FTICR) mass spectrometry and accurate peptide masses. To compare the two methods for protein identification, we grew and extracted proteins from E. coli using established protocols. Cystines were reduced and alkylated, and proteins digested by trypsin. The resulting peptide mixtures were separated by reversed-phase liquid chromatography using a 4 h gradient from 0 to 50% acetonitrile over a C18 reversed-phase column. The LC separation was coupled on-line to either a Bruker Esquire HCT ion trap or a Bruker 7 tesla APEX-Qe Qh-FTICR hybrid mass spectrometer. Data-dependent Qh-FTICR-MS/MS spectra were acquired using the quadrupole mass filter and collisionally induced dissociation into the external hexapole trap. Proteins were in both schemes identified by Mascot MS/MS ion searches and the peptides identified from these proteins in the FTICR MS/MS data were used for automatic internal calibration of the FTICR-MS data, together with ambient polydimethylcyclosiloxane ions.