Sequencing Lys-N Proteolytic Peptides by ESI and MALDI Tandem Mass Spectrometry

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.     

溴化氰裂解结合电喷雾串联质谱测定电泳分离蛋白的C端序列

C端测序是蛋白质及多肽一级结构确认的重要组成部分,也是重组蛋白药物质量控制的重要依据。建立了溴化氰裂解结合电喷雾串联质谱测定蛋白质C端序列的方法,并应用于重组人肿瘤坏死因子受体和纽兰格林的C端测序。首先根据待测蛋白序列进行溴化氰理论裂解,如果C-端肽段理论分子量在500~5000U之间,则将待测样品进行SDS-PAGE分离,考马斯亮兰染色,然后进行胶内溴化氰裂解,最后应用基质辅助激光解吸/电离飞行时间质谱(MALDI-TOF-MS)测定C-端肽段的分子量,电喷雾串联质谱对C端肽段进行测序。应用本方法分别测定了这两个蛋白质C端19个和11个氨基酸残基序列。研究结果表明:本方法灵敏、有效、实用性较强,可适用于部分重组蛋白药物的质量控制和蛋白质的结构确证,是对目前蛋白质C端测序方法的有效补充。     

多级质谱法测定Axinastatin 1环肽序列及其断裂机理研究

对具有抗癌活性的海洋环肽Axinastatin 1进行化学合成. 采用多级质谱法对合成环肽进行序列测定. 线性前体测序依据b_x－y_z断裂路径, 在同一张MS²谱中利用b和y离子所提供序列信息的互补来实现. 环肽测序依据b_x→b_x－1断裂路径, 每一级MS由b离子的C端碰撞掉一个氨基酸残基直到MS⁶, 得到2套b离子, 根据它们所提供序列信息的互补可准确测定环肽序列并推断其环结构, 同时观察到b离子重排现象. 讨论了上述断裂与重排的路径和机理, 并利用半经验量子化学PM3和AM1两种算法计算了碎片的生成焓, 验证了路径的合理性. 由离子b_5PN的生成焓偏高和其重排间的联系尝试提出过渡结构假设.     

刺五加中苷类化合物的电喷雾多级串联质谱

采用小柱层挤、电喷雾多级串联质谱方法，通过分子量及正负离子多级串联质谱的信息对刺五加提取物中的有效成分刺五加苷B、刺五加苷E进行了定性分析，并提出了其电喷雾质谱碎裂机理。该方法样品处理简单，分析速度快且灵敏度高，实验重复性好，适宜于作为刺五加有效成分鉴定的指纹图谱，对刺五加药品质量控制具有重要意义。     

利用(+)ESI-MS-MS方法分析研究脱氧四核苷酸的裂解特征

细胞中存在很多金属离子参与DNA及其组成部分的重要的生物过程.质谱作为1种重要的分析方法,能用以考察这些金属离子和DNA在分子水平的相互作用,确定金属离子和生物分子的结合位点,并检测金属离子对于生物分子的性质和反应性的影响.本研究利用(+)ESI-MS-MS考察了3个四碱基DNA分子d(TGAC)、d(GTAC)、d(ATAT)的[M+Na]+、[M+K]+的质谱行为,这些金属离子与同1分子的[M+H]+的裂解具有明显的差异;推测为不同的离子在DNA上的加合位置不同,导致质谱行为的差异.     

芍药苷的电喷雾串联质谱研究

采用电喷雾串联质谱(ESI-MSn)技术, 结合H/D交换方法, 在正、负离子检测模式下对白芍药材中主要成分芍药苷的质谱裂解规律进行了系统研究. 实验结果表明, 该化合物在正、负离子模式下均得到较好的质谱信息, 且在正离子模式下, 电喷雾质谱分析的灵敏度更高. 同时获得了其质谱裂解规律, 为白芍中其它化合物的分析鉴定提供了有效的质谱方法.     

Activation Energies of Fragmentations of Disaccharides by Tandem Mass Spectrometry

A simple multiple collision model for collision induced dissociation (CID) in quadrupole was applied for the estimation of the activation energy (E_o) of the fragmentation processes for lithiated and trifluoroacetated disaccharides, such as maltose, cellobiose, isomaltose, gentiobiose, and trehalose. The internal energy-dependent rate constants k(E_int) were calculated using the Rice-Ramsperger-Kassel-Marcus (RRKM) or the Rice-Ramsperger-Kassel (RRK) theory. The E_o values were estimated by fitting the calculated survival yield (SY) curves to the experimental ones. The calculated E_o values of the fragmentation processes for lithiated disaccharides were in the range of 1.4–1.7 eV, and were found to increase in the order trehalose < maltose < isomaltose < cellobiose < gentiobiose.

Structural Characterization of Chlorophyll-a by High Resolution Tandem Mass Spectrometry

A high resolution Fourier transform ion cyclotron resonance (FTICR) mass spectrometer is used for characterizing the fragmentation of chlorophyll-a. Three tandem mass spectrometry (MS/MS) techniques, including electron-induced dissociation (EID), collisionally activated dissociation (CAD), and infrared mutiphoton dissociation (IRMPD) are applied to the singly protonated chlorophyll-a. Some previously unpublished fragments are identified unambiguously by utilizing high resolution and accurate mass value provided by the FTICR mass spectrometer. According to this research, the two long aliphatic side chains are shown to be the most labile parts, and favorable cleavage sites are proposed. Even though similar fragmentation patterns are generated by all three methods, there are much more abundant peaks in EID and IRMPD spectra. The similarities and differences are discussed in detail. Comparatively, cleavage leading to odd electron species and H^? loss both seem more common in EID experiments. Extensive loss of small side groups (e.g., methyl and ethyl) next to the macrocyclic ring was observed. Coupling the high performance FTICR mass spectrometer with contemporary MS/MS techniques, especially IRMPD and EID, proved to be very promising for the structural characterization of chlorophyll, which is also suitable for the rapid and accurate structural investigation of other singly charged porphyrinic compounds.      

串联质谱法快速分析皮革中五氯酚残留

建立了皮革样品中五氯酚的串联质谱分析方法,以ｍ／ｚ２６６为母离子,碰撞电压为０．８Ｖ,以子离子ｎ／ｚ２３０为定量离子,外标法定量。线性范围为０．１￣１０．０ｍｇ／Ｌ,检出限为０．００５ｍｇ／ｋｇ,回收率为８５．４％￣１０２．５％,相对标准偏差为２．５５％￣３．５５％。方法具有快速、准确的特点。     

缩酚酸醚类化合物的电喷雾多级串联质谱研究

采用电喷雾多级质谱方法, 研究了三个从地衣中提取到的缩酚酸醚类化合物在电喷雾电离质谱中的裂解行为, 结果表明它们在电喷雾多级质谱中能得到较多碎片离子, 其裂解途径能体现它们的基本结构特征.     

脱水淫羊藿素的电喷雾多级串联质谱研究

研究了脱水淫羊藿素的电喷雾质谱行为，结果表明：该化合物在正、负离子模式下，均可得到较好的电喷雾质谱信息，且在负离子模式下电喷雾质谱分析的灵敏度较高；运用电喷雾碰撞诱导解离技术，发现正离子和负离子模式下的裂解方式虽有相似之处但并不完全相同；分别阐明了该化合物在正、负离子模式下的电喷雾质谱碎裂规律。     

SNP的电喷雾串联质谱研究

采用电喷雾串联质谱技术对两组单核苷酸多态性(SNP)样品进行了研究,初步研究结果显示应用串联质谱可快速鉴定SNP分型以及确定SNP位点。采用16.3 mmol/L三乙胺(TEA)-400 mmol/L六氟异丙醇(HFIP)缓存溶液,电喷雾负离子模式下,长度为20 bp的寡核苷酸的MS扫描谱图显示其母离子呈较宽的多电荷态分布。选择低电荷态[M-4H]4-母离子,碰撞能为54 eV时,MS/MS扫描得到丰富的碎片离子,通过鉴定特征离子碎片(w和a-B离子系列)可确定其序列。     

纳升电喷雾串联质谱法确证阿托西班的一级结构

利用电喷雾串联质谱对合成八肽阿托西班进行了二硫键还原前后的精确分子量测定和一级结构的确证.首先通过全扫描模式测定了其还原前后的精确分子量,然后选择母离子m/z 498.73(双电荷)通过串联质谱(MS/MS)得到碎片离子,采用Y离子的方法测定了阿托西班的序列并对其的修饰位点进行了确证.本方法具有灵敏度高、速度快、样品无需纯化等特点,在多肽类药物一级结构分析方面具有独特的优势.     

异烟肼及其代谢产物的串联质谱分析

报道了血样中异烟肼的串联质谱快速分析，利用这种分析方法，证实了一男性死者血样中异烟肼及其代谢产物的存在，从而为法庭诉讼提供了可靠的证据。这种方法具有快速性，高灵敏度和高选择性，适合于需要快速分析的场合。     

合成多肽的电喷雾质谱研究

利用电喷雾多极串联质谱对3种合成多肽进行了系统的鉴定和分析研究。首先通过全扫描模式测定了其分子量，然后选择[M H]^ 或[M 2H]^2 离子通过串联质谱(MS／MS)得到碎片离子，采用y离子和b离子互补的方法测定了多肽序列。利用文献数据对这种方法进行了验证，实验结果表明，该方法简便、快速、实用。     

Relative Stability of Peptide Sequence Ions Generated by Tandem Mass Spectrometry

We report the use of unimolecular dissociation by infrared radiation for gaseous multiphoton energy transfer to determine relative activation energy (E_a,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, k_d, is determined for each of a series of laser powers. Provided these conditions are met, a plot of the natural logarithm of k_d versus the natural logarithm of laser power yields a straight line, whose slope provides a measure of E_a,laser. This method reproduces the E_a 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 E_a,laser values that are systematic underestimates of theoretical barriers calculated with density functional theory (DFT). However, the relative E_a,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).     

组氨酸钴氧合配合物二聚体形态的电喷雾串联质谱验证研究

电喷雾质谱(ESI-MS)是一种软电离质谱技术,已在配合物的结构和机理的研究中显示了重要的作用。本文根据组氨酸钴(CoL2)(L=组氨酸)对分子氧活性很高,极易生成双核氧合配合物(CoL2-O2-CoL2)的特点,采用ESI-MS方法研究了组氨酸钴氧合物(CoL2-O2-CoL2)和组氨酸配合物ML2(M=Cu、Zn)。结果发现,质谱图中在相应于双核氧合配合物的高质荷比端(m/z>ML2),CoL2出现质谱峰,而组氨酸配合物ML2(M=Cu、Zn)在质谱图中高质荷比端没有出现质谱峰,只有配合物ML2的相关峰;这个结果证明了文献报道中的双核氧合配合物(CoL2-O2-CoL2)的存在,根据所得质谱结果,初步研究了组氨酸钴双核氧合配合物和ML2配合物的裂解规律。结果表明,可根据质谱数据中有无二聚体形态,做出Co配合物有无吸氧性能的初步判断,因此电喷雾质谱(ESI-MS)可做为研究Co配合物氧合反应和表征Co氧合配合物的有效分析手段。     

高效液相色谱-串联质谱法测定食品中硝磺草酮

1 引 言 硝磺草酮(Mesotrione),化学名2-(4-甲磺酰基2硝基苯甲酰基)环己烷1,3二酮,又名甲基磺草酮、硝磺酮,是瑞士先正达公司发明的玉米田芽前和苗后广谱选择性除草剂,因具有低毒性、高活性、对环境友好等特点[1,2],是近几年广泛使用的除草剂.但最近研究发现,硝磺草酮具有高效的起始活性和残留活性,长期食用含有硝磺草酮残留的食物会对人畜产生致癌作用,或引起胎儿畸形[3,4].欧盟和世界贸易组织对浆果、亚麻籽、越橘、栗草料等物质中硝磺草酮的限量为0.05 mg/kg.而美国、加拿大等国对芦笋、草杆、草料等物质中硝磺草酮的限量为0.01 mg/kg[10-13].     

Differentiation of Regioisomeric Esters of Sucrose by Ionspray Tandem Mass Spectrometry

Abstract

Structural characterization and differentiation of distinct regioisomenc esters of sucrose were obtained using lonspray ionization and low energy tandem mass spectrometry. Low energy CAD MS/MS analyses of the protonated molecules [M + H]⁺ provided characteristic fingerprint patterns, and permitted differentiation of the various regioisomers. MS/MS analyses of selected intermediate fragment ions formed during the ionization process provided additional structural data, and established the fragmentation routes of their [M + H]⁺ precursors.