MALDI-TOF MS和电子光谱技术研究海兔肝铁蛋白亚基电离和释放铁动力学特性

选用肽质量指纹谱(peptide mass fingerprint,PMF)技术鉴定质谱纯海兔肝铁蛋白(liver ferritin ofAplysia,ALF)。来源于基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)仪中的激光和基质芥子酸协同解吸海兔肝铁蛋白(ALF)为带双电荷、单电荷[M H] 和二聚体的亚基离子,并可供质谱分析。ALF亚基的质荷比m/z分别为9784.03[M 2H]2 、19678.42[M H] 和39387.80[2M H] ,其中亚基分子量[M H] 略小于鲨鱼肝铁蛋白(liver ferritin of shark,SLF)。在弱碱介质(pH8.0)条件下,电子光谱技术研究指出,抗坏血酸以1/2级反应方式参与ALF释放铁全过程,同时又使ALF以一级反应动力学方式释放铁,呈现两种不同的速率。推测这一异常现象可能与ALF含低铁量、亚基调节能力和海兔的进化地位有关。     

反相高效液相色谱-电喷雾质谱法分析食源血管紧张素转换酶抑制肽

利用反相高效液相色谱/电喷雾离子阱质谱法,直接分析从牦牛乳酪蛋白中酶水解得到的血管紧张素转换酶抑制肽粗产物。RP-HPLC显示具有活性的多肽粗产物含有3个主要成分,质谱同步测定各组分的分子量(m/z)分别为815.2,1680.1,962.2,然后选择[M H] 离子通过串联质谱(MS/MS)得到碎片离子,利用b离子和y离子互补的方法鉴定了多肽序列。三条肽分别为Leu-Pro-Tyr-Tyr,Pro-Leu-Pro-Leu-Leu-Gln,Phe-Leu-Pro-Pro-Tyr-Tyr。结果显示,所获得的多肽序列与牛乳酪蛋白一级结构中相应肽段的序列一致。     

质谱技术研究海兔大脑神经节超微量多肽内切酶和酸性多肽酶解产物

采用ESI-Q-TOF质谱分析了由27个氨基酸残基组成的酸性多肽(AP)的一级结构.选用RP-HPLC和MALDI-TOF质谱非在线技术分离与鉴定海兔大脑神经节(CG)多肽与蛋白质的组成与分布,发现CG中含有AP酶解的二聚体短肽.这些短肽序列分别为2(NKDEEQRELLKAISNLL)、2(NKDEEQRELLKAISNL)、2(SGVSLLTSNKDEEQREL)和2(LTSNKDEEQRE LL),均以L-R(氨基酸残基)方式断裂.以AP为探针,结合MALDI-TOF质谱分析技术,发现CG中含有超微量的L-R多肽内切酶,其分子量为78218.25Da.本研究中的分析方法也适合于研究其他生物体内超微量多肽及多肽酶分布与功能.     

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。     

脱氧雪腐镰刀菌烯醇的制备与鉴定

将禾谷镰刀菌(Fusarium graminearum)接种至大米培养基28℃培养4周,通过乙腈-水(84∶16)提取培养基。提取物先经自制净化小柱粗净化除去色素、鞣质等杂质后,以甲醇-水(30∶70)为流动相、Pursuit XRs C18(10 mm×250 mm,5μm)制备色谱柱进行分离,分离组分再以甲醇-水(5∶95)为流动相经Semipreparative SB-CN(9.4 mm×250 mm,5μm)制备色谱柱进一步纯化。所得脱氧雪腐镰刀菌烯醇(DON)经液相色谱-串联质谱(LC-MS/MS)定性,在正、负离子模式下,质谱图中母离子分别为m/z 319.1[M+Na]+,297.1[M+H]+,355.2[M+CH3COO]-,341.2[M+HCOO]-,295.1[M-H]-,子离子碎片分别为m/z175,231,235,249,179,233,237,265,证明该化合物为DON;经外标法测得DON纯度为(99.18±0.11)%。本研究建立的DON分离纯化方法简便易行,分离效果及稳定性好,成本低。     

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.     

质谱技术研究海兔大脑神经节超微量多肽内切酶和酸性多肽酶解产物

采用ESI-Q-TOF质谱分析了南27个氨基酸残基组成的酸性多肽（AP）的一级结构。选用RP-HPLC和MALDI-TOF质谱非在线技术分离与鉴定海兔大脑神经节（CG）多肽与蛋白质的组成与分布，发现CG中含有AP酶解的二聚体短肽。这些短肽序列分别为2（NKDEEQRELLKAISNLL）、2（NKDEEQRELLKAISNL）、2（SGVSLLTSNKDEEQREL）和2（LTSNKDEEQRE LL），均以L-R（氨基酸残基）方式断裂。以AP为探针，结合MALDI-TOF质谱分析技术，发现CG中含有超微量的L-R多肽内切酶，其分子量为78218.25Da。本研究中的分析方法也适合于研究其他生物体内超微量多肽及多肽酶分布与功能。     

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.     

丹参酮ⅡA和丹参酮Ⅰ的电子轰击与电喷雾电离质谱分析

采用电喷雾电离质谱(ESI-MS)和电子轰击质谱(EI-MS)两种质谱技术分别对传统中药丹参的主要脂溶性活性成分丹参酮ⅡA和丹参酮Ⅰ的化学结构和裂解途径进行系统研究。采用EI-MS从丹参酮ⅡA获得m/z 294[M] 、279、261、233、207等特征质谱峰,从丹参酮Ⅰ获得m/z 276[M] 、248、233、219、205等特征质谱峰;采用ESI-MS从丹参酮ⅡA获得m/z 295[M H] 、280、278、262、249等特征质谱峰,从丹参酮Ⅰ获得m/z 277[M H] 、259、249、231、221、193等特征质谱峰,并用Mass Frontier 3.0软件辅助解析了其中的主要特征碎片离子以及可能的裂解途径;比较了丹参酮ⅡA和丹参酮Ⅰ的电喷雾电离质谱和电子轰击质谱裂解规律,本研究为研究丹参二萜醌类主要特征活性成分的生物转化与结构修饰提供了依据。     

液相色谱-四极杆/静电场轨道阱高分辨质谱快速分析动物源性食品中多肽类药物残留

建立了液相色谱-四极杆/静电场轨道阱高分辨质谱(Q/Exactive)分析鸡肉、鸡肝、鸡肾、猪肉、猪肝、猪肾、牛奶和鸡蛋中粘杆菌素A、粘杆菌素B、多粘菌素B、杆菌肽A和维吉尼霉素M的方法。动物源性食品采用1%(体积分数)乙酸乙腈-水(体积比为8∶2)超声提取,提取液直接经C18液相色谱柱分离。在电喷雾正离子模式下,四极杆质谱在m/z 1.2隔离窗口下过滤一级带电离子,高分辨静电场轨道阱质谱在35 000分辨率下进行目标物子离子全扫描(targeted-MS2)。根据5种多肽类药物的高分辨分子离子峰、同位素分布、特征子离子信息建立数据库,采用Trace Finder 3.0软件实现定性检索。为解决多肽类药物残留测定结果重复性差的难题,系统地评价了5种多肽类药物的关键操作技术条件,发现导致该类药物降解的主要因素包括:提取时间、玻璃器皿、存放时间。该方法测定5种多肽类药物时,测定结果的质量精度均小于5×10-6,维吉尼霉素M的定量限(LOQ)为0.5μg/kg,另外4种多肽类药物的LOQ均为10μg/kg。在0.25~500μg/kg范围内,5种多肽类药物的峰面积与质量浓度呈良好的线性关系,相关系数R2≥0.993 1,方法回收率为67.4%~108.9%,相对标准偏差为4.5%~17.2%。该方法操作简单,测定结果准确,可用于动物源性食品中多肽类药物残留的高通量测定。     

Mass spectral studies on aryl-substituted N-carbamoyl/N-thiocarbamoyl narcotine and related compounds

Positive ion mass spectral fragmentation of new N-carbamoyl/N-thiocarbamoyl derivatives of narcotine and compounds closely related to it are reported and discussed. The techniques used include electron impact (EI), fast-atom bombardment (FAB), matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and electrospray ionization tandem mass spectrometry (ESI-MS/MS). Prominent peaks in the mass spectra of these compounds appear to involve C-C bond cleavage beta to the amine nitrogen with loss of the 4,5-dimethoxy(1H)isobenzofuranone moiety from their molecular ions, along with another prominent peak at m/z 382. No molecular ion peaks of these compounds were recorded in EI, whereas intense [M + H]+ ion peaks were observed in FAB and ESI spectra. MALDI also yielded [M + H]+ ion peaks in good agreement with FAB and ESI studies.     

Use of meso- tetrakis(pentafluorophenyl)porphyrin as a matrix for low molecular weight alkylphenol ethoxylates in laser desorption/ ionization time-of-flight mass spectrometry

The use of UV-absorbing molecules as matrices in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is well documented. The matrices that are currently used have low molecular weights (<300 Da) and thus, for a typical MALDI-TOF spectrum, the low-mass range (m/z 100-500) is dominated by matrix ions. Consequently, the applications of MALDI-TOFMS have been restricted mostly to the analysis of high molecular weight analytes. This report demonstrates the use of meso-tetrakis(pentafluorophenyl)porphyrin (F20TPP, MW 974.57) as a matrix in the MALDI-TOF mass spectrometric analysis of some commercial nonylphenol ethoxylates (4-(C(9)H(19))-C(6)H(4)-(OCH(2)CH(2))(n)-OH), in which the ethoxymer ion distribution ranges from 331-771 Da. When F20TPP was used without a sodium ion dopant, there were no MALDI signals for the ethoxylates. However, addition of sodium acetate to the sample produced MALDI spectra in which the ethoxymer molecules were sodiated to form [M + Na](+) ions. A comparison of the mass spectrometric data with those obtained when alpha-cyano-4-hydroxycinnamic acid (CHCA) was used as the matrix indicated that the F20TPP-induced spectra provided comparable data, with the advantage of having less matrix interference in the low-mass range (m/z 100-500). Thus, the use of F20TPP and similar porphyrins may provide the means to apply MALDI-TOF to the analysis of low molecular weight molecules with minimum interference from matrix signals. Copyright 1999 John Wiley & Sons, Ltd.     

Matrix-assisted laser desorption/ionization, fast atom bombardment and plasma desorption mass spectrometry of polyethylene glycol esters of (2-benzothiazolon-3-yl)acetic acid.

Fast atom bombardment (FAB), matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and plasma desorption (PD) mass spectra of newly synthesized polyethylene glycols (PEGs), (M(w) 600-4000 Da) chemically modified with biologically active (2-benzothiazolon-3-yl)acetyl end-groups are described (products 1-6). The spectra were also used for the determination of the molecular mass characteristics (number average (M(n)) and weight average (M(w)) molecular masses) of the initial and modified PEGs. As expected, M(n) and M(w) of the modified samples are higher than those of the non-modified samples. However, it is shown that molecular mass dispersity (determined by the comparison of the polydispersity indices (PDI = M(w)/M(n)) of both types of PEGs) essentially do not change during this modification. The FAB mass spectra, together with molecular species, show the presence of abundant [M + Na](+) ions of product 1 and [M + Na + H](+) species of 2 and 3, and [M + Na + 2H](+) of product 4. Two main series of fragment ions, derived from the cleavage of the ether bonds, are observed. The number fractions of the molecular adduct ions and fragment adduct ions, determined from the FAB and PD mass spectra of the modified PEGs, are compared. The MALDI-TOF mass spectra of compounds 1-6 show the presence of two series of polymers. The most abundant peaks are due to [M + Na](+) and [M + K](+) ions originating from the polymers, in which the two terminal hydroxyl groups of PEGs are esterified with (2-benzothiazolon-3-yl)acetic acid. The less abundant peaks are due to the monosubstituted polymers.     

Characterization of mesogen-jacketed liquid crystalline polymers by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

For synthetic polymers, a proper sample preparation method is essential for successful characterization by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. In this work, six synthetic mesogen-jacketed liquid crystalline polymers (MJLCPs) with different main-chain, spacer and mesogenic units were investigated by MALDI-TOF mass spectrometry. Several factors that affect the analysis of these polymers were examined. These factors include matrices used, cationization salts used, the concentration of polymers, and the ratio of sample to matrix. After testing different conditions, we found a suitable sample preparation method for these six polymers. The number average molecular weight (M(n)), weight average molecular weight (M(w)) and polydispersity (PD) were calculated using data obtained in the linear mode. The end groups of the polymers were proposed using data obtained in reflectron mode. Copyright 2000 John Wiley & Sons, Ltd.     

Determination of oligonucleotide composition from mass spectrometrically measured molecular weight

Extensive calculations for molecular mass versus subunit composition have been made for oligonucleotides from RNA and DNA to determine the extent to which base compositions might be derived from mass spectrometrically determined molecular weights. In the absence of compositional constraints (e.g., any numbers of A, U, G, C), measurement of molecular weight leads to only modest restrictions in allowable number of base compositions; however, if the compositional value for any one residue is known, such as from selective chemical modification or enzymatic cleavage, the number of allowable base compositions becomes unexpectedly low. For example, hydrolysis of RNA by ribonuclease T₁ produces oligonucleotides for which G=1, for which all base compositions can be uniquely specified up to the 14-mer level, solely by measurement of mass to within ±0,01%. The effects of methylation, phosphorylation state of nucleotide termini, and knowledge of chain length on the determination of subunit composition are discussed.     

Evaluation of mass discrimination effects in the quantitative analysis of polydisperse polymers by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using uniform oligostyrenes

Mass discrimination effects in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) were quantitatively investigated using equiweight and equimolar mixtures of uniform polystyrene (PS) oligomers. Uniform PS oligomers were separated by preparative super-critical fluid chromatography (SFC) from commercial standard PS samples. The separated PS oligomers, with degrees of polymerization n = 2–25, have absolutely no molecular weight distributions. Equiweight and equimolar mixtures of uniform PS oligomers were accurately prepared by weighing by microbalance, and their spectra were recorded using a MALDI-TOF mass spectrometer. In the lower molecular weight region (less than about 10³) the oligomers with lower molecular weights give lower mass spectral intensities, with no correlation with laser power. In contrast, higher laser powers yield a decrease of mass spectral intensities in the higher molecular weight region. These results clearly show that mass discrimination effects occur at lower and higher molecular weights depending on the laser power, and provide quantitative information about the discrimination. Using the data on equiweight and equimolar mixtures of PS oligomers, it was possible to calibrate the MALDI-TOF mass spectral data for an analysis of molecular weight distribution of a standard monodisperse PS sample with number-averaged molecular weight of 10³, and to compare it with the molecular weight distribution measured by analytical SFC. The result from the calibrated MALDI-TOF mass spectrum, however, does not agree perfectly with that from the SFC results, because undetectable peaks in MALDI-TOF mass spectra at lower and higher molecular weights could not be included in the calibration of peak intensities. Copyright © 2001 John Wiley & Sons, Ltd.     

基质辅助激光解吸电离飞行时间质谱技术研究人血清转铁蛋白稳定性及裂解产物

制备质谱纯人血清转铁蛋白(HTF),供分子结构分析。选用SDS-PAGE、胶外酶解、基质辅助激光解吸/电离质谱技术(MALDI-TOF)、数据库检索和比对技术鉴定铁饱和HTF、双铁HTF(HTF-2Fe3 )、单铁HTF(HTF-Fe3 )和脱铁HTF(apoHTF)的稳定性和裂解产物。以乙腈溶液作为洗脱相,发现铁饱和HTF在RP-HPLC分离纯化过程中产生裂解现象。铁饱和HTF和HTF-2Fe3 经乙腈处理后均能产生不同分子量的短肽裂解产物,指出HTF结构稳定性与络合铁离子数量有关。铁组分改善了HTF分子结构的稳定性。采用比对法,研究在乙腈作用下HTF裂解成为各种各样短肽的规律,初步阐明其裂解机理。在乙腈作用下,HTF可能通过蛋白质去折叠途径,形成不同多聚态HTF或多肽裂解产物。推测目前用于临床诊断先天性糖基化紊乱(CDG)和慢性酒精滥用(CAA)疾病低准确率的起因可能是受HTF裂解产物或多聚体的干扰。     

Synthesis and Investigation of Mass Spectra of 3-[5′-(2′-Substituent)thienyl]benzo[5,6]coumarins

3-[5'-(2'-Hydroxycarbonyl)thienyl]benzo[5,6]coumarin ( 3 ) was prepared via condensation of 2 with thioglycolic acid in the presence of AcONa and Ac 2 O. Esterification of 3 with alcohols gave 3-[5'-(2'-alkoxycarbonyl)thienyl]benzo[5,6]-coumarins ( 4a , b ). The chemical behavior of 4 toward nucleophilic reagents (such as ammonia, hydroxyl amine, and hydrazine derivatives) is described. The electron impact ionization mass spectra of compounds 4b , 5 , and 8a , b show a weak molecular ion peak and a base peak of m / z 278 resulting from a cleavage fragmentation. In contrest compounds 3 and 4a show a base peak of m / z 250 and m / z 74 resulting from fragmentation. Compounds 9 and 10 give a characteristic fragmentation pattern with a very stable fragment of m / z 305.     

高效液相色谱–质谱法分析僵蚕蛋白酶解多肽

分析僵蚕蛋白酶解多肽类成分的相对分子质量和氨基酸组成。采用高效液相色谱–质谱联用正离子模式进行分析,以Hola C_(18)色谱柱(100 mm×2.1 mm,2.7μm)为分离色谱柱,以0.05%甲酸水溶液和0.05%甲酸–乙腈溶液为流动相,根据质谱一级、二级碎片离子信息,确定酶解多肽类相对分子质量信息和氨基酸组成。僵蚕样品经酶解后得到相对分子质量在500~1 000之间的多肽,经LC–MS分析,多肽由低于10个的氨基酸组成。高效液相色谱–质谱法分析平台可用于分析多肽化合物的相对分子质量和氨基酸组成,这有利于酶解多肽的生物活性分析。