分子间非共价相互作用的质谱分析方法研究进展

总结了各种软电离质谱技术在研究分子间非共价相互作用中的应用及发展,尤其是在生物大分子非共价复合物结构和功能研究方面的进展。     

超滤质谱技术在药物小分子与生物靶分子相互作用研究中的应用*

药物小分子与生物体内靶分子之间的相互作用,是药物发挥其药理活性的重要途径之一。因此,高通量地筛选出能够与生物靶分子相互作用且具有生物活性的药物小分子,对于新药开发研究具有重要的理论意义和实际应用价值。超滤质谱技术是超滤装置与质谱技术结合后形成的一种新的分析方法,它能够在液相条件下快速地识别并鉴定出与生物靶分子结合的药物小分子配体。与其它分析方法相比,超滤质谱技术具有快速、灵敏、高通量的特点,因而被广泛应用于研究药物小分子和生物靶分子相互作用。本文综述了超滤质谱技术的原理、特点和实验操作中的一些关键问题。重点介绍了超滤质谱技术在药物小分子与生物靶分子相互作用研究中的应用进展,并对其未来的发展方向进行了展望。     

氢氘交换质谱技术在蛋白质和蛋白复合物结构研究中的应用进展

蛋白质是生命功能的执行者,其功能的发挥受自身结构动态变化、与其他生物分子的相互作用及修饰等因素的调节。因此,对蛋白质及蛋白复合物结构的研究有助于揭示重要生命过程中的分子机理与机制。氢氘交换质谱(Hydrogen deuterium exchange mass spectrometry,HDX-MS)是研究蛋白质结构、动态变化和相互作用的强有力工具,也是传统生物物理手段的重要补充。该文综述了HDX-MS的基本原理、机制、实验方法和研究最新进展,并从蛋白质自身动态变化、蛋白质-小分子相互作用、蛋白质-蛋白质相互作用3个方面介绍了近年来HDX-MS在蛋白及蛋白复合物研究中的应用进展。     

N-甲基吡咯类聚酰胺的合成及其与DNA的相互作用

本研究主要是设计并合成了3个含有4个N-甲基吡咯的聚酰胺化合物(PyPyPyPyβDp,PyPyPyPyγDp, NO₂PyPyPyPyβDp),并使用ESI-MS法研究了其与DNA的相互作用.     

黄酮与溶菌酶相互作用的强度衰减-基质辅助激光解吸离子化-质谱研究

建立了黄酮与溶菌酶相互作用研究的强度衰减-基质辅助激光解吸离子化-质谱(Intensity fading matrix-assisted laser desorption/ionization mass spectrometry,IF-MALDI-MS)分析方法.在优化的基质DHB条件下,分别研究了木犀草素、染料木素、芹菜素、槲皮素和大豆黄素与溶菌酶相互作用,比较了溶菌酶加入前后黄酮的相对丰度的变化,并通过竞争实验的方法研究了5种黄酮与溶菌酶结合的亲和性大小.结果表明,5种黄酮与溶菌酶均存在相互作用,亲和性强弱为:木犀草素＞芹菜素,染料木素＞槲皮素＞大豆黄素.结合5种黄酮的结构特征,讨论了黄酮的结构及其与溶菌酶亲和性的关系,发现C5位和C3’位的羟基有利于黄酮与溶菌酶的结合,C3位的羟基不利于黄酮与溶菌酶的结合.本方法具有简便,快速,高效等优点,也可以应用于其它天然产物与蛋白质的相互作用的研究.     

质谱技术在核酸研究领域的应用

质谱技术具有快速、准确、灵敏度高等优点,近年来在生物分析方面得到了广泛的应用.核酸作为生命的基本物质,一直是生命领域的研究热点,进展日新月异.而质谱方法也成为了科学家研究核酸的强有力工具,具有广阔的发展前景.本文介绍了核酸检测的质谱技术,并简要综述了质谱在核酸的高级结构研究、与小分子相互作用、DNA损伤与修饰等领域的应用,重点介绍了中国学者的研究成果.     

砷与蛋白相互作用研究进展

本文综述了砷与不同蛋白相互作用的研究,比较了不同形态砷与蛋白结合能力的差异,进而讨论了砷的毒性与形态的关系.砷摄入体内后首先会进入血液,部分与血液中的蛋白结合,结合态和游离态的砷化合物随着血液循环到达各个器官、组织,甚至进入细胞内部与其他功能蛋白、受体、酶等发生直接或间接的相互作用,从而影响各种蛋白等活性分子的正常生理活动.在这过程中,不同形态砷的毒性也会体现在与蛋白相互作用能力的强弱上.同时,砷的解毒过程也在进行着,人们在ars操纵子中发现的两种去毒性蛋白与砷的相互作用被认为是将砷从细胞内排出,从而去除砷毒性的有效机制.     

基于先进紫外光源的生物质谱技术的最新进展及展望

生物大分子质谱是研究复杂生命体系中分子的序列组成、动态结构、相互作用和空间分布的前沿科学领域.先进紫外光源特别是同步辐射、自由电子激光等大科学装置的发展,为光解离-串联质谱和光电离-质谱成像等生物大分子质谱研究尖端技术的进步提供了新的机遇.本综述中,我们总结了不同波长先进光源包括X射线、极紫外激光等在蛋白质、脂质等生物分子高效光解离和序列、结构、相互作用表征,光化学氧化标记和动态结构分析,高效光电离和高灵敏度质谱检测及空间分布成像等领域的研究进展;进一步展望了新一代先进紫外光源特别是具有更高亮度、更短脉冲、更高重频的极紫外自由电子激光在生物大分子质谱研究中的应用前景和可能带来的技术突破.     

ATP与氨基酸弱相互作用的质谱与理论计算研究

利用电喷雾质谱、荧光、核磁和理论计算研究了ATP与19种氨基酸的弱相互作用.在质谱中发现除甘氨酸(Gly)、丙氨酸(Ala)、缬氨酸(Val)外,其它氨基酸均可观测到与ATP因弱相互作用形成的复合物离子.利用不同质谱锥孔电压下复合物稳定性的不同,分析了侧链基团对ATP与19种氨基酸弱相互作用的影响.并利用荧光光谱和核磁共振波谱法研究了芳香性氨基酸与ATP的弱相互作用.结果表明,氨基酸与ATP的弱相互作用强弱顺序为:色氨酸(Trp)＞苯丙氨酸(Phe)＞具有R‖C-NH2的氨基酸＞具有-RCOOH、-R-NH2的氨基酸＞具有-RSH、-ROH的氨基酸＞R为长链的氨基酸＞R为短链的氨基酸.不同官能团的氨基酸与ATP的弱相互作用的模拟计算也证实了此结论,并发现氨基酸的主侧链基团与ATP分子基团间的多个分子间因氢键作用使复合物能稳定存在.这一结果将为预测蛋白与ATP结合位点及研究ATP的识别机理提供依据.     

肌红蛋白的电喷雾质谱研究——推荐一个化学生物学教学实验

在原有实验的基础上,设计、实践了一个利用电喷雾质谱研究生物大分子与小分子相互作用的更为合理、简单、易行的实验方法.讨论了该实验在化学生物学专业本科生教学工作中的实践效果及经验.     

Chiral recognition of borneol by association with zinc(II) and l-tryptophan in the gas phase

Chiral recognition of the racemic borneol was obtained in the gas phase by using electrospray ionization time-of-flight mass spectrometry (ESI-TOFMS) and electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTMS). Both single stage MS and tandem MS were employed for investigating the chiral recognition depending strongly on the stereochemistry of the ligands in the zinc(II)-l-tryptophan-borneol multimeric cluster ions. It was found that the type of acid which was used for adjusting the pH of the mixed solution played an important role in the chiral recognition, which was obtained when acetic acid or propanoic acid was used as an additive. No chiral recognition was observed using hydrochloric acid or formic acid. Furthermore, the nozzle potential was an important parameter for optimizing the discrimination of chirality. In tandem MS, the difference in intensity between two diastereomeric complex ions showed chiral recognition behavior. Such a difference could be determined by two approaches. One was using the fragment ion as an internal standard, the other was a new approach of using the isotopic form of parent complex ion as an internal standard.     

Structural characterization of hexoses and pentoses using lead cationization. An electrospray ionization and tandem mass spectrometric study

The analytical potential of the complexation of isomeric underivatized hexoses (D-glucose, D-galactose, D-mannose, D-talose, D-fructose), methylglycosides (1-O-methyl-alpha-D-glucose and 1-O-methyl-beta-D-glucose) and pentoses (D-ribose, D-xylose, D-arabinose and D-lyxose) by Pb(2+) ions, was investigated by electrospray ionization and tandem mass spectrometry (MS/MS). Pb(2+) ions react mainly with monosaccharides by proton abstraction to generate [Pb(monosaccharide)(m) - H](+) ions (m = 1-3). At low cone voltage, a less abundant series of doubly charged ions of general formula [Pb(monosaccharide)(n)](2+) is also observed. The maximum number n of monosaccharides surrounding a single Pb(2+) ion depends on the metal : monosaccharide ratio. Our study shows that MS/MS experiments have to be performed to differentiate Pb(2+)-coordinated monosaccharides. Upon collision, [Pb(monosaccharide) - H](+) species mainly dissociate according to cross-ring cleavages, leading to the elimination of C(n)H(2n)O(n) neutrals. The various fragmentation processes observed allow the C(1), C(2) and C(4) stereocenters of aldohexoses to be characterized, and also a clear distinction aldoses and fructose. Furthermore, careful analysis of tandem mass spectra also leads to successful aldopentose distinction. Lead cationization combined with MS/MS therefore appears particularly useful to identify underivatized monosaccharides.     

A carbonyl oxygen migration in electrospray ionization mass spectrometry and its application in differentiating alpha- and beta-alanyl peptides

A novel rearrangement reaction with a carbonyl oxygen migration was observed in the electrospray ionization tandem mass spectra of N-diisopropyloxyphosphoryl dipeptides and their analogues. A possible mechanism was proposed and supported by the MS/MS study, derivatization of different functional groups and deuterium labeling experiments. It was found that metal ions could catalyze the rearrangement through a five-membered ring intermediate. A strong affinity between the phosphoryl group and oxygen atom in the gas phase was proposed to result in this kind of rearrangement reaction, which might provide some basic information on the nature of phosphorylation in biochemistry. The replacement of N-terminal alpha-alanine by beta-alanine stopped the migration, which provides a simple method for differentiating the alpha- and beta-alanine residues at the N-terminus of peptides.     

Ca2+‐complex stability of GAPAGPLIVPY peptide in gas and aqueous phase,investigated by affinity capillary electrophoresis and molecular dynamics simulations and compared to mass spectrometric results

Strong, sequence‐specific gas‐phase bindings between proline‐rich peptides and alkaline earth metal ions in nanoESI‐MS experiments were reported by Lehmann et al. (Rapid Commun. Mass Spectrom. 2006, 20, 2404–2410), however its relevance for physiological‐like aqueous phase is uncertain. Therefore, the complexes should also be studied in aqueous solution and the relevance of the MS method for binding studies be evaluated. A mobility shift ACE method was used for determining the binding between the small peptide GAPAGPLIVPY and various metal ions in aqueous solution. The findings were compared to the MS results and further explained using computational methods. While the MS data showed a strong alkaline earth ion binding, the ACE results showed nonsignificant binding. The proposed vacuum state complex also decomposed during a molecular dynamic simulation in aqueous solution. This study shows that the formed stable peptide–metal ion adducts in the gas phase by ESI‐MS does not imply the existence of analogous adducts in the aqueous phase. Comparing peptide–metal ion interaction under the gaseous MS and aqueous ACE conditions showed huge difference in binding behavior.     

Tandem mass spectra of ammonium ion,metal ion and ligated metal ion adducts of acyclic sugar derivatives

The tandem mass (MS/MS) spectra of ammonium ion, metal ion and ligated metal ion adducts of chain-extended acyclic nitro-containing deoxyglucose and deoxygalactose derivatives have been studied. The ammonium adducts fragment primarily by elimination of ammonia followed by acetic acid, thus not giving much structural information. In contrast, cationization of these compounds by metal ions and ligated metal ions gave structurally informative and useful fragment ions on MS/MS. The metal ions and ligated metal ions play an important role in controlling and directing fragmentation. Retro-aldol fragmentation is facilitated by metal ions such as Li(+), Na(+), Ag(+) and Cu(+), whereas the adducts with higher alkali metal ions such as Rb(+) and Cs(+) fragment to give only the corresponding metal ions. The divalent metal ions such as Cu(2+) and Ba(2+) also induce retro-aldol fragmentation. However, the charge is carried by the aldehyde fragment in the case of Cu(2+) adducts, whereas the nitroalkane fragment carries the charge in the case of Ba(2+) adducts. Ligated metal ions such as ZnCl(+), CuCl(+), InCl(2) (+) and BaCl(+) also behave similarly and induce retro-aldol fragmentation in these acyclic sugars. Both the metal ion and ligated metal ion adducts can fragment by elimination of metal-containing neutral molecules.     

Mass spectrometric study of the ionized C60: (gamma-Cyclodextrin)2 inclusion complex by collision induced dissociation

The water soluble inclusion complex [C(60):(gamma-cyclodextrin)(2)] has been characterized using electrospray tandem mass spectrometry and collision induced dissociation. [C(60):(gamma-cyclodextrin)(2)] ions were detected in the gas phase as doubly deprotonated, doubly protonated and doubly sodiated ions. The absence of monocharged complex ions following electronebulization is a likely consequence of the dimeric nature and structural symmetry of the inclusion complex. The collision induced dissociation of positive ions led exclusively to the observation of the protonated and sodiated cyclodextrin ions as well as their fragments. In negative ion mode the closed shell anion C(60)H(-) was the dominant fragment detected at low collision energies whereas at higher collision energies the signal corresponding to deprotonated cyclodextrin units becomes significant. Since C(60) (2-) has been reported to have a nonnegligible basicity compared to C(60) and C(60) (-), it is likely that the proton transfer involved in the formation of the C(60)H(-) anion occurs following transfer of the two electrons from the deprotonated gamma-cyclodextrins to the fullerene. Finally, the charge state of the inclusion complex ions is also shown to affect the interaction strengths between its subunits. The relative stabilities of the three ionic species studied in gas phase following electronebulization are as follows: [C(60):(gamma-cyclodextrin)(2) + 2H](2+) < [C(60):(gamma-cyclodextrin)(2)- 2H](2-) < [C(60):(gamma-cyclodextrin)(2) + 2Na](2+).     

Gas Phase Ion Chemistry of Transition Metal Clusters: Production, Reactivity, and Catalysis

This review focuses on the use of mass spectrometry to examine the gas phase ion chemistry of metal clusters. Ways of forming gas phase clusters are briefly overviewed and then the gas phase chemistry of silver clusters is discussed to illustrate the concepts of magic numbers and how reactivity can be size dependent. The chemistry of other bare and ligated metal clusters is examined, including mixed metal dimer ions as models for microalloys. Metal clusters that catalyze gas phase chemical reactions such as the oxidation of CO and organic substrates are reviewed. Finally the interface between nanotechnology and mass spectrometry is also considered.     

Structural characterization of glycoporphyrins by electrospray tandem mass spectrometry

Porphyrin derivatives having a galactose or a bis(isopropylidene)galactose structural unit, linked by ester or ether bonds, were characterized by electrospray tandem mass spectrometry (ES-MS/MS). The electrospray mass spectra of these glycoporphyrins show the corresponding [M + H](+) ions. For the glycoporphyrins with pyridyl substituents and those having a tetrafluorophenyl spacer, the doubly charged ions [M + 2H](2+) were also observed in ES-MS with high relative abundance. The fragmentation of both [M + H](+) and [M + 2H](2+) ions exhibited common fragmentation pathways for porphyrins with the same sugar residue, independently of the porphyrin structural unit and type of linkage. ES-MS/MS of the [M + H](+) ions of the galactose-substituted porphyrins gave the fragment ions [M + H - C(2)H(4)O(2)](+), [M + H - C(3)H(6)O(3)](+), [M + H - C(4)H(8)O(4)](+) and [M + H - galactose residue](+). The fragmentation of the [M + 2H](2+) ions of the porphyrins with galactose shows the common doubly charged fragment ions [porphyrin + H](2+), [M + 2H - C(2)H(4)O(2)](2+), [M + 2H - C(4)H(8)O(4)](2+), [M + 2H - galactose residue](2+) and the singly charged fragment ions [M + H - C(3)H(6)O(3)](+) and [M + H - galactose residue](+). The fragmentation of the [M + H](+) ions of glycoporphyrins with a protected galactosyl residue leads mainly to the ions [M + H - CO(CH(3))(2)](+), [M + H - 2CO(CH(3))(2)](+), [M + H - 2CO(CH(3))(2) - CO](+), [M + H - C(10)H(16)O(4)](+) and [M + H - protected galactose](+). The doubly charged ions [M + 2H](2+) fragment to give the doubly charged ions [porphyrin + H](2+) and the singly charged ions [M + H - protected galactose residue](+) and [M + H - CO(CH(3))(2)](+). For the porphyrins where the sugar structural unit is linked by an ester bond, [M + 2H](2+), ES-MS/MS showed a major and typical fragmentation corresponding to combined loss of a sugar structural unit and further loss of water, leading to the ion [M + 2H - sugar residue - H(2)O](2+), independently of the structure of the sugar structural unit. These results show that ES-MS/MS can be a powerful tool for the characterization of the sugar structural unit of glycoporphyrins, without the need for chemical hydrolysis.     

Observation of an unusually facile fragmentation pathway of gas-phase peptide ions: a study on the gas-phase fragmentation mechanism and energetics of tryptic peptides modified with 4-sulfophenyl isothiocyanate (SPITC) and 4-chlorosulfophenyl isocyanate (SPC) and their 18-crown-6 complexes

Various peptide modifications have been explored recently to facilitate the acquisition of sequence information. N-terminal sulfonation is an interesting modification because it allows unambiguous de novo sequencing of peptides, especially in conjunction with MALDI-PSD-TOF analysis; such modified peptide ions undergo fragmentation at energies lower than those required conventionally for unmodified peptide ions. In this study, we systematically investigated the fragmentation mechanisms of N-terminal sulfonated peptide ions prepared using two different N-terminal sulfonation reagents: 4-sulfophenyl isothiocyanate (SPITC) and 4-chlorosulfophenyl isocyanate (SPC). Collision-induced dissociation (CID) of the SPC-modified peptide ions produced a set of y-series ions that were more evenly distributed relative to those observed for the SPITC-modified peptides; y(n-1) ion peaks were consistently and significantly larger than the signals of the other y-ions. We experimentally investigated the differences between the dissociation energies of the SPITC- and SPC-modified peptide ions by comparing the MS/MS spectra of the complexes formed between the crown ether 18-crown-6 (CE) and the modified peptides. Upon CID, the complexes formed between 18-crown-6 ether and the protonated amino groups of C-terminal lysine residues underwent either peptide backbone fragmentation or complex dissociation. Although the crown ether complexes of the unmodified ([M + CE + 2H]2+) and SPC-modified ([M* + CE + 2H]2+) peptides underwent predominantly noncovalent complex dissociation upon CID, the low-energy dissociations of the crown ether complexes of the SPITC-modified peptides ([M' + CE + 2H]2+) unexpectedly resulted in peptide backbone fragmentations, along with a degree of complex dissociation. We performed quantum mechanical calculations to address the energetics of fragmentations observed for the modified peptides.     

Aryl–Phenyl Scrambling in Intermediate Organopalladium Complexes: A Gas‐Phase Study of the Mizoroki–Heck Reaction

The intramolecular aryl–phenyl scrambling reaction within palladium–DPPP–aryl complex (DPPP=1,3‐bis(diphenylphosphino)propane) ions was analyzed by state‐of‐the‐art tandem MS, including gas‐phase ion/molecule reactions. The Mizoroki–Heck cross‐coupling reaction was performed in the gas phase, and the intrinsic reactivity of important intermediates could be examined. Moreover, linear free‐energy correlations were applied, and a mechanism for the scrambling reaction proceeding via phosphonium cations was assumed.