电泳/质谱技术研究鲨鱼肝铁蛋白及亚基多聚体特性

改良鲨鱼肝铁蛋白(liver ferritin ofsphyrna zygaena,SZLF)分离技术,并结合非变性梯度聚丙烯酰胺凝胶电泳(NGPAGE)制备质谱纯SZLF,以维系铁蛋白分子和它的亚基之间的作用类型,供研究铁蛋白结构与功能。实验结果表明,SZLF由分子量约为20kDa的单类型亚基组成。SZLF和它的亚基均组成不同的聚合体。聚合体类型和聚合数目与铁蛋白亚基和分离介质有关。MALDI-TO F质谱仪的激光和基质协同能有效解吸SZLF中的亚基成为准分子离子,并供质量分析,其亚基特征质谱峰m/z值分别为10889.35和22030.45,确定为带双电荷[M 2H]2 和单电荷[M H] 的SZLF亚基分子量。SDS-PAGE和变性IEF技术研究指出,形成不同聚合态的SZLF,其分子之间的相互作用强度高于SZLF亚基自身,难以通过MALDI-TOF质谱技术测定其亚基的结构信息。尽管SZLF由单类型亚基组成,但它能通过聚合体和自身亚基之间的相互作用差异性发挥极其重要的生理功能。     

棕色固氮菌细菌铁蛋白捕获能力、稳定性和亚基相互作用的强度

选用柱层析、电泳和反相高效液相色谱(RP-HPLC)技术制备质谱纯棕色固氮菌细菌铁蛋白(Bacteri-al ferritin ofAzotobacter vinelandii,AVBF),并采用释放铁动力学和肽质量指纹图谱(Peptide mass fingerprint-ing,PMF)技术分别鉴定AVBF。基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)和电泳技术揭示AVBF亚基之间相互作用强度、稳定性和聚合态。AVBF可直接捕获有机小分子亚甲蓝(MB),其捕获率为15.0±2.0MB/AVBF,认为介于AVBF亚基单体之间的血红素参与捕获MB。较高浓度(40%~50%)的乙腈和丙酮均能使AVBF和鲨鱼肝铁蛋白(Liver ferritin of shark,SLF)释放不稳定亚基,但在较低浓度(20%~30%)的乙腈条件下,却需要借助来源于质谱仪的激光才能使AVBF或SLF释放不稳定亚基,并供质谱分析。AVBF亚基之间的相互作用强度明显低于SLF。铁蛋白亚基之间的相互作用强度高低与铁蛋白执行释放和储存铁的速率有关。     

MALDI-TOF质谱、电泳和透射电子显微镜技术研究魟鱼肝铁蛋白H和L亚基特性

小批量制备质谱纯魟鱼肝铁蛋白(liver ferritin of Dasyatis akajei, DALF),以透射电子显微镜术(TEM)测定DALF、蛋白壳和铁核的分子尺寸.SDS-PAGE技术指出,DALF由H 和L两种不同类型的亚基组成.采用肽质量指纹图谱(PMF)技术进一步佐证H和L亚基类型和同源性.氧化还原剂中性红、劳氏紫、甲基紫精和 pH 1.5酸度均无法削弱DALF的H-L和L-L亚基之间的相互作用强度、并解吸出L亚基离子,供MALDI-TOF质谱仪分析.通过增加激光强度和降低基质pH途径,可直接分析DALF的H和L亚基的分子量,指出DALF中H-L和L-L亚基之间的相互作用强度明显高于H-H亚基类型.     

蛋白质辅助基质提高激光解吸/电离多肽离子化率和绝对强度的研究

人血清转铁蛋白(Human serum transferrin, HTF)、牛血清白蛋白(Bovine serum albumin, BSA)、鲨鱼肝铁蛋白(Sphyrna zygaena liver ferritin, SZLF)、马脾铁蛋白(Horse spleen ferritin, HSF)均能辅助基质提高激光解吸/电离胰岛素(Insulin, INS)和海兔酸性多肽(Aplysia acidic peptide, AP)离子化率和质谱峰的绝对强度(简称为绝对强度), 其中绝对强度分别提高10和4倍, 这一现象不依赖于INS浓度, 而与蛋白质类型和结构有关. SZLF和脱铁核SZLF(apoSZLF)辅助基质提高INS绝对强度的能力几乎相同, 蛋白质中的金属离子含量对这种效应无明显影响, 主要取决于蛋白质的氨基酸组成与结构. 采用胶内酶解和肽指纹技术(PMF)鉴定HTF过程中, 发现基质中分别添加SZLF, apoSZLF和HSF后, HTF肽片段质谱检测的质谱峰数目及绝对强度均有明显增加, 进一步提高了数据库鉴定HTF的可信度.     

基质辅助激光解吸/电离质谱法研究海兔卵多肽内切酶组成、结构与功能

选择DEAE-52纤维素和Sephadex G-150为分离介质,采用柱层析法分离纯化海兔卵多肽酶(En-dopeptidease of Aplysiaegg,AEE)。选用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)法测定AEE纯度和亚基分子量(约39kDa)。基质辅助激光解吸/电离(MALDI-TOF)质谱技术测定AEE由单类型亚基(M)组成,其m/z比值分别为12738.17、18108.79和38221.42,即分子式为[M3 ]、[M2 ]和[M ]。其中AEE分子量为38221.42Da(称为AEE39),是一种含金属锌的多肽酶。以胰岛素(INS)为探针,研究AEE39酶切INS能力,其酶切产物m/z比值为1449.51、2085.84、4080.41和4165.42。自行设计INS序列分析软件,鉴定AEE39酶切INS的位点,发现易酶切位点是Leu-X(氨基酸残基),其次是Glu-X,部分Phen-X、Asn-X和Ser-X结构也可以发生酶切。通过INS和海兔吸引素的分子结构比对后,指出海兔卵中的AEE39主要功能之一是负责酶切海兔吸引素,起着海兔之间信息交往、召唤、识别和交配的重要作用。AEE39具有酶切酸性多肽(acidic pep-tide,AP)中Leu-Leu的能力,属于一种广谱性多肽内切酶。     

鲨鱼肝铁蛋白亚基解离与组装机理的研究

小批量制备质谱纯鲨鱼肝铁蛋白(Liver Ferritin of Sphyrna zygaena,SZLF)。在弱酸介质(pH1.0)中,天然电泳结果显示,SZLF蛋白质亚基20 min后开始解离。选用透射电子显微镜跟踪SZLF亚基解离与重组装全过程和蛋白壳与铁核尺寸变化,发现SZLF在亚基酸解离过程中,随着pH值的降低,铁核和蛋白壳的尺寸呈现相同的变化趋势,这种变化趋势可能与铁核内层铁的释放和蛋白壳的解离与去折叠有关。SZLF蛋白壳的重组装过程则是一个快速过程,并且是由松散熔球态向紧密态转变的过程。SZLF由单类型亚基组成,而马脾铁蛋白(Horse Spleen Ferritin,HSF)由H和L两种亚基类型组成。在基质pH3.0条件和激光辅助下,混合HSF和SZLF仍然可释放各自的亚基且形成准亚基离子,供基质辅助激光解析电离飞行时间质谱分析,说明此时SZLF的亚基间相互作用强度减弱但并没有去折叠。TEM技术在铁蛋白解离和重组装过程中的应用,为进一步研究铁蛋白纳米包装的过程和机理提供新颖的、可行的和更加直观的研究手段。     

电泳和质谱技术研究4种胰脏铁蛋白的亚基类型和等电点特性

采用电泳和质谱技术对所制备的鸡、鸭、牛和猪胰脏铁蛋白的亚基类型和等电点特性进行了研究。采用天然聚丙烯酰胺凝胶电泳(PAGE)技术研究的结果表明,上述4种铁蛋白呈现不同的迁移率,据此可知鸡胰铁蛋白的相对分子质量(Mr)>鸭胰铁蛋白的Mr>黄牛胰铁蛋白的Mr>猪胰铁蛋白的Mr,而且均大于马脾铁蛋白(HSF)的Mr。采用十二烷基硫酸钠(SDS)-PAGE技术研究的结果表明,上述4种铁蛋白均由H(heavy chain)和L(light chain)类型的亚基组成,但H和L亚基的相对数量（即H/L亚基数量的比值）有差别。采用肽指纹图谱技术分别鉴定各铁蛋白的H和L亚基。选用变性等电聚焦方法研究发现,上述4种铁蛋白分别由3～6种不同等电点的亚基聚合体组成,说明铁蛋白的H和L亚基之间呈现复杂的相互作用和不同的聚合体。不同陆生动物胰脏铁蛋白亚基之间相互作用的强度和聚合态存在着差异,这一差异特性可能与调控铁蛋白释放铁的速率有关,也与动物对铁的需求和铁解毒速率有关。     

苯二酚异构体双电荷离子和单电荷离子的动能谱研究

本文利用质量分析离子动能谱(MIKES)、碰撞诱导解离(CID)技术和电子捕获诱导解离(ECID)技术, 研究了邻、间、对苯二酚分子在电子轰击质谱(EIMS)中产生的双电荷离子[C6H6O2]^2^+, [C6H4O]^2^+和单电荷离子[C6H6O2]^+。根据测定的电荷分离反应动能释放值T和由此计算出的两电荷间距R, 推测出过渡态的结构。有趣的是, 可利用单电荷离子[C6H6O2]^+的MIKES/CID谱区分苯二酚异构体。     

气相中二氯苯的双电荷离子[C6H4CI2]^2+和[C6H3CI]^2+的动能谱研究

本文利用质量分析离子动能(MIKES)和碰撞诱导解离(CID)技术, 研究了邻、间、对二氯苯分子在电子轰击质谱(EIMS)中产生的[C6H4CI2]^2+和[C6H4CI]^2+双电荷离子的单分子电荷分离(CS)反应。根据测定和CS反应的动能释放值T和由此估算的反应过渡态的电荷间距的最小值R, 推测过渡态的结构。有趣的是, 可以利用双电荷离子[C6H4CI2]^2+的分解反应区分二氯苯的位置异构体。     

用MALDI-TOF质谱和电子光谱研究纳米胰岛素核-铁蛋白的构建技术和特性

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.     

LC‐MS/MS methods for the determination of edaravone and/or taurine in rat plasma and its application to a pharmacokinetic study

Three liquid chromatography–tandem mass spectrometry (LC‐MS/MS) methods were respectively developed and validated for the simultaneous or independent determination of taurine and edaravone in rat plasma using 3‐methyl‐1‐p‐tolyl‐5‐pyrazolone and sulfanilic acid as the internal standards (IS). Chromatographic separations were achieved on an Agilent Zorbax SB‐Aq (100 × 2.1 mm, 3.5 µm) column. Gradient 0.03% formic acid–methanol, isocratic 0.1% formic acid–methanol (90:10) and 0.02% formic acid–methanol (40:60) were respectively selected as the mobile phase for the simultaneous determination of two analytes, taurine or edaravone alone. The MS acquisition was performed in multiple reaction monitoring mode with a positive and negative electrospray ionization source. The mass transitions monitored were m/z [M + H]⁺ 175.1 → 133.0 and [M + H]⁺ 189.2 → 147.0 for edaravone and its IS, m/z [M ? H]^? 124.1 → 80.0 and [M ? H]^? 172.0 → 80.0 for taurine and its IS, respectively. The validated methods were successfully applied to study the pharmacokinetic interaction of taurine and edaravone in rats after independent intravenous administration and co‐administration with a single dose. Our collective results showed that there were no significant alterations on the main pharmacokinetic parameters (area under concentration–time curve, mean residence time, half‐life and clearance) of taurine and edaravone, implying that the proposed combination therapy was pharmacologically feasible. Copyright © 2014 John Wiley & Sons, Ltd.     

A mechanistic study of the H/D exchange reactions of protonated arginine and arginine-containing Di- and tripeptides

The gas-phase H/D exchange reactions of arginine (R) and arginine-containing di- and tri-peptide (gly-arg (GR), arg-gly (RG), gly-gly-arg (GGR), gly-arg-gly (GRG) and arg-gly-gly (RGG)) [M+H]⁺ ions with deuterated ammonia (ND₃) were investigated by using Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR), ion mobility-mass spectrometry (IM-MS), ab initio and density functional theory-based molecular orbital calculations and molecular modeling. Three exchanges are observed for arginine and arginine-containing tri-peptide [M+H]⁺ ions, whereas the di-peptide [M+H]⁺ ions undergo a single H/D exchange. In addition, C-terminal methylation blocks H/D exchange of arginine and the arginine-containing peptide [M+H]⁺ ions, and a single H/D exchange is observed for N-terminal acetylated arginine [M+H]⁺ ions. A general mechanism for H/D exchange involving a collision complex that is best described as a “solvated salt-bridge” structure is proposed.     

MS/MS in structural analysis of an oviposition-deterring pheromone

A recently characterized oviposition-deterring pheromone (ODP, structure 1) of the European cherry fruit fly was used as a test case for probing the potential of tandem mass spectrometry (MS/MS) in structure elucidation as a stand-alone technique. The glycolipid-taurinate 1 was subjected to MS/MS analyses under a variety of conditions with and without preceding chemical degradation. Acidic methanolysis of 1 and subsequent in-batch derivatization (trideuterioacetylation) yielded methyl 2,3,4,6-tetrakis-O-trideuterioacetyl-glucopyranoside (2), methyl 8,15-bis-trideuterioacetoxy-palmitate (3), and taurine (4) as suitable target compounds for direct mixture analysis.Low energy collision induced dissociation (CID) on selected precursor ions (MS/MS on [M + H – CH₃OH]⁺ and [M + H]⁺ produced by fast atom bombardment (FAB)) allowed direct identification of 2 and 4, respectively, by comparison with appropriate reference ions. In the case of 3, low energy CID (desorption chemical ionization (DCI) instead of FAB, MS/MS on [M + H]⁺) permitted deduction of gross molecular structure, but failed to provide positional detail. In sharp contrast,high energy CID of trideuterioacetylated intact 1 (FAB-MS/MS on [M – H]^– ions of la) clearly revealed a linear 8,15-hydroxylated palmitic acid backbone. Less certain was assignment of 15-O-glucosylation by this approach.     

Ion‐pairing high‐performance liquid chromatography‐mass spectrometry of impurities and reduction products of sulphonated azodyes

An HPLC separation method with triethylammonium acetate mobile phase additive developed for the analysis of impurities in polysulphonated azo dyes provides good separation selectivity and compatibility with electrospray ionisation (ESI) mass spectrometry. The negative‐ion ESI mass spectra containing only peaks of deprotonated molecules [M–H]^– for monosulphonic acids, [M–xH]^x–, and sodiated adducts [M–(x + y)H + yNa]^x– for polysulphonic acids allow easy molecular mass determination of unknown impurities. Based on the knowledge of the molecular masses and of the fragment ions in the MS/MS spectra, probable structures of trace impurities in commercial dye samples are proposed. To assist in the interpretation of the mass spectra of complex polysulphonated azodyes, additional information can be obtained after chemical reduction of azodyes to aromatic amines. The structures of the non‐sulphonated reduction products can be determined by reversed‐phase HPLC/MS with positive‐ion atmospheric pressure chemical ionisation and of the sulphonated products by ion‐pairing HPLC/MS with negative‐ion ESI.     

HPLC‐DAD and HPLC‐ESI‐MS separation,determination and identification of the spin‐labeled diastereoisomers of podophyllotoxin

Spin‐labeled nitroxide derivatives of podophyllotoxin had better antitumor activity and less toxicity than that of the parent compounds. However, the 2‐H configurations of these spin‐labeled derivatives cannot be determined by nuclear magnetic resonance (NMR) methods. In the present paper, a high‐performance liquid chromatography‐diode array detection (HPLC‐DAD) and a high‐performance liquid chromatography‐electrospray ionization tandem mass spectrometry (HPLC‐ESI/MS/MS) method were developed and validated for the separation, identification of four pairs of diastereoisomers of spin‐labeled derivatives of podophyllotoxin at C‐2 position. In the HPLC‐ESI/MS spectra, each pair of diastereoisomers of the spin‐labeled derivatives in the mixture was directly confirmed and identified by [M+H]⁺ ions and ion ratios of relative abundance of [M‐ROH+H]⁺ (ion 397) to [M+H]⁺. When the [M‐ROH+H]⁺ ions (at m/z 397) were selected as the precursor ions to perform the MS/MS product ion scan. The product ions at m/z 313, 282, and 229 were the common diagnostic ions. The ion ratios of relative abundance of the [M‐ROH+H]⁺ (ion 397) to [M+H]⁺, [A+H]⁺ (ion 313) to [M‐ROH+H]⁺, [A+H‐OCH₃]⁺ (ion 282) to [M‐ROH+H]⁺ and [M‐ROH‐ArH+H]⁺ (ion 229) to [M‐ROH+H]⁺ of each pair of diastereoisomers of the derivatives specifically exhibited a stereochemical effect. Thus, by using identical chromatographic conditions, the combination of DAD and MS/MS data permitted the separation and identification of the four pairs of diastereoisomers of spin‐labeled derivatives of podophyllotoxin at C‐2 in the mixture.     

Use of liquid chromatography/tandem mass spectrometry and online databases for identification of phosphocholines and lysophosphatidylcholines in human red blood cells

In this work a systematic strategy integrating liquid chromatography/tandem mass spectrometry (LC/MS/MS) and online databases was developed to identify phosphocholines (PC) and lysophosphatidylcholines (LPC) in human red blood cells (RBCs). First of all, the neutral loss scan of 59 and the precursor ion scan of m/z 184 were performed to find out the possible lipids with phosphocholine head‐group structure in RBCs. The acquired [M+H]⁺ and [M+Na]⁺ adduct ions were then identified online using the Human Metabolome Database (HMDB) and the LIPID MAPS, which were then further confirmed by their MS/MS fragmentation. Based on the comparison of chemical structures of the detected PC and LPC with their corresponding MS/MS fragmentation pathways, several new diagnostic fragments or fragmentation pathway were found. These include, (1) the neutral losses of 183 could be used as a diagnostic fragmentation to discriminate PC and LPC; (2) product ions at m/z 104 could be used to distinguish LPC and their sn‐2 isomers; (3) fragment ions at m/z 184 are characteristic fragmentation that could be used for discrimination of sodiated ions [M+Na]⁺ and protonated ions [M+H]⁺, especially for co‐eluted PC or LPC with a molecular weight difference of 22. The structures of the above‐mentioned fragment ions were confirmed by quadrupole time‐of‐flight (Q‐TOF) MS. Furthermore, a PC and LPC focused LC/MS semi‐quantification approach was also developed and validated. This approach could be useful for future lipidomic study. Copyright © 2009 John Wiley & Sons, Ltd.     

Direct analysis in real time mass spectrometry (DART‐MS) of highly non‐polar low molecular weight polyisobutylenes

Low molecular weight polyisobutylenes (PIB) with chlorine, olefin and succinic acid end‐groups were studied using direct analysis in real time mass spectrometry (DART‐MS). To facilitate the adduct ion formation under DART conditions, NH₄Cl as an auxiliary reagent was deposited onto the PIB surface. It was found that chlorinated adduct ions of olefin and chlorine telechelic PIBs, i.e. [M + Cl]^? up to m/z 1100, and the deprotonated polyisobutylene succinic acid [M? H]^? were formed as observed in the negative ion mode. In the positive ion mode formation of [M + NH₄]⁺, adduct ions were detected. In the tandem mass (MS/MS) spectra of [M + Cl]^?, product ions were absent, suggesting a simple dissociation of the precursor [M + Cl]^? into a Cl^? ion and a neutral M without fragmentation of the PIB backbones. However, structurally important product ions were produced from the corresponding [M + NH₄]⁺ ions, allowing us to obtain valuable information on the arm‐length distributions of the PIBs containing aromatic initiator moiety. In addition, a model was developed to interpret the oligomer distributions and the number average molecular weights observed in DART‐MS for PIBs and other polymers of low molecular weight. Copyright © 2015 John Wiley & Sons, Ltd.     

Quantification of heteroclitin D in rat plasma: validation of an LC/MS/MS method and its application in a preclinical pharmacokinetic study

A rapid, sensitive and specific liquid chromatography tandem mass spectrometry (LC/MS/MS) method was developed and validated for the quantification of heteroclitin D in rat plasma after using gambogic acid as internal standard (IS). Chromatographic separation was done on a Thermo Hypersil GOLD column (30 × 2.1 mm, 3 µm) using a mobile phase consisting of methanol–water–formic acid (80:20:0.1, v/v/v). The mass spectrometer worked with positive electrospray ionization in multiple reaction monitoring mode, using target ions at [M + H]⁺ m/z 483.3 for heteroclitin D and [M + H]⁺ m/z 629.3 for the IS. The standard curve was linear (R² ≥0.995) over the concentration range 9.98–2080 ng/mL and had good back‐calculated accuracy and precision. The intra‐ and interday precision and accuracy determined on three quality control samples (29.94, 166.4 and 1872 ng/mL) were ≤12.8 and –8.9–3.6%, respectively. The extraction recovery was ≥88.2% and the lower limit of quantification was 9.98 ng/mL. The method was successfully applied to evaluate pharmacokinetics of heteroclitin D in Sprague–Dawley rats following a single intravenous bolus injection of 2.0 mg/kg heteroclitin. Copyright © 2014 John Wiley & Sons, Ltd.     

Structural determination by atmospheric pressure photoionization tandem mass spectrometry of some compounds isolated from the SARA fractions obtained from bitumen

We have identified compounds obtained from the SARA fractions of bitumen by using atmospheric pressure photoionization mass spectrometry and low‐energy collision tandem mass spectrometric analyses with a QqToF‐MS/MS hybrid instrument. The identified compounds were isolated from the maltene saturated oil and the aromatic fractions of the SARA components of a bitumen. The QqToF instrument had sufficient mass resolution to provide accurate molecular weight information and to enhance the tandem mass spectrometry results. The APPI‐QqToF‐MS analysis of the separated compounds showed a series of protonated molecules [M + H]⁺ and molecular ions [M]^+? of the same mass but having different chemical structures, in the maltene saturated oil and the aromatic SARA fractions. These isobaric ions were a molecular ion [M₂]^+? at m/z 418.2787 and a protonated molecule [M₅ + H]⁺ at m/z 287.1625 in the saturated oil fraction, and molecular ions [M₆]^+? at m/z 418.1584 and [M₇]^+? at m/z 287.1285 in the aromatic fraction. The identification of this series of chemical compounds was achieved by performing CID‐MS/MS analyses of the molecular ions [M]^+? ([M₁]^+? at m/z 446. 2980, [M₂]^+? at m/z 418.2787, [M₃]^+? at m/z 360.3350 and [M₄]^+? at m/z 346.2095) in the saturated oil fraction and of the [M₅ + H]⁺ ion at m/z 287.1625 also in the saturated oil fraction. The observed CID‐MS/MS fragmentation differences were explained by proposed different breakdown processes of the precursor ions. The presented tandem mass spectrometric study shows the capability of MS/MS experiments to differentiate between different classes of chemical compounds of the SARA components of bitumen and to explain the reasons for the observed mass spectrometric differences. However, greater mass resolution than that provided by the QqToF‐MS/MS instrument would be required for the analysis of the asphaltene fraction of bitumen. Copyright © 2011 John Wiley & Sons, Ltd.