天然产物的质谱智能解析研究

串联质谱在鉴定天然产物的分子结构方面起着重要的作用.每一类天然产物,在串联质谱中除有常规的质谱裂解方式外,通常还具有因特定骨架类型而产生的特征裂解.因此可以通过天然产物各类成分的特征质谱裂解所产生的特征碎片离子(diagnostic ions)来确认该类天然产物的结构类型,然后再由常规的裂解方式来推测其周边可能的取代基团.基于以上思路,我们设计并构建了一种天然产物质谱智能解析软件SmartMass.     

质谱中若干金属配合物及簇合物的碎片重组反应

在质谱实验中,我们发现了与一般单分子分解反应相悖的四种类型碎片产物的重组反应物即(Ⅰ)裂解碎片的自组合产物如S_8~+及不同聚合度的聚硫离子;(Ⅱ)裂解碎片之间的相互组合物如[Fe(S_2CNC_4H_8)_3]~+和[Fe(S_2CNEt_2)_3]~+;(Ⅲ)裂解碎片的阴离子和阳离子部分碎片间的组合物如[Fe_2S_2(NO)_4(CH_3)_2]~+;(Ⅳ)歧化反应产物Cp_3Yb~+和YbL_3~+(Cp=C_5H_5),L=β-二酮)。本文阐述了这种重组反应或离子—分子反应在质谱条件下产生的可能性及其理论根据。     

基于密度泛函理论的橘霉素衍生物质谱裂解行为研究

采用电喷雾离子阱质谱(ESI-IT-MSn),研究了橘霉素衍生物正离子模式下的裂解行为,并结合密度泛函理论(DFT)证明裂解途径的可靠性,为同类化合物的准确鉴定提供实验和理论基础。核磁共振仪对海莲内生真菌代谢产物分离得到的橘霉素衍生物结构进行确认,电喷雾离子阱质谱正离子模式扫描,测定化合物氢氘交换产物多级裂解质谱图,并结合键断裂能和Mulliken电荷分布,进一步验证裂解规律。实验结果表明,分子结构中-COOH和CH3COO-在一级裂解过程中易脱除CO2和COCH2,CID-MS2观测到丢失H2O/HDO、H2O和-CH3或CHCO/CDCO的碎片离子峰,且丰度依次降低,理论计算也证明,这些碎片离子的总能量依次增大,稳定性降低,键断裂能逐渐增大。该结果丰富了橘霉素衍生物的电喷雾质谱裂解规律,有助于橘霉素衍生物结构的准确鉴定,为该类化合物的检测和痕量分析提供了更多支持。     

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.     

有机膦酸配合物热分解法制备三维网状形貌的偏磷酸铁

用三氯化铁和乙二胺四甲叉膦酸(edtmpH8)为原料合成膦酸铁(FeIII-edtmpH5)配合物前驱体,再将该前驱体在空气氛下焙烧即制得偏磷酸铁Fe(PO3)3.利用电喷雾质谱(ESI/MS)、红外光谱(FT-IR)、热重-差热分析(TG-DTA)测试确定了FeIII-edtmpH5配合物的组成和可能结构.对焙烧后产物进行X射线衍射(XRD)、扫描电镜(SEM)及透射电镜(TEM)测试,结果表明焙烧产物为具有三维网状形貌的高纯Fe(PO3)3.     

可溶性聚酰亚胺的热分解

用高分辨裂解气相色谱 质谱 (HRPyGC MS)研究了两种可溶性聚酰亚胺的热分解行为 ,考察了相应裂解产物的组成、分布及其与高分子结构的关系 ,并用热重法 (TG)测定了它们的热分解反应动力学参数 ,提出了其热分解反应机理     

5-溴粉防己碱合成产物中微量杂质的ESI-MSn快速鉴定

采用电喷雾串联质谱分析了5-溴粉防己碱合成产物中的微量杂质成分。通过多级质谱的解析推测出2个微量杂质可能的分子结构,并从中总结出该类化合物的ESI—MS^n裂解规律。     

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。     

纳米-微米铁修饰的维氏体氨合成催化剂

我们以商业预还原的维氏体(Fe1-xO)氨合成催化剂为载体,采用Fe(NO)3 ·9H2O和H2C2O4·2H2O进行原位室温固相反应制备纳米铁或微米铁修饰的铁基氨合成催化剂,并通过XRD、SEM、TG-DTG、H2-TPR等进行了表征.结果表明:Fe(NO)3·9H2O和H2C2O4·2H2O室温固相反应完全生成产物Fe2(C2O4)3·5H2O,且产物分散于载体维氏体催化剂表面.通过纳米铁-微米铁的修饰,催化剂的氨合成活性有很大提高且稳定性好.催化剂活性随着Fe负载量的增加先增加后降低,负载量5％时催化活性最好,反应器出口氨浓由450℃(12.4％)、425℃(11.0％)、400℃(9.4％)分别提升至450℃(15.6％)、425℃(14.8％)、400℃(13％).通过一步简单的修饰,维氏体催化剂的氨合成活性提高约25％ ～38％.由于焙烧和还原,生成的Fe1xO或铁粒子与铁催化剂表面发生强相互作用,因此,反应过程中纳米铁或微米铁粒子能稳定存在,催化剂有较高的稳定性.     

质谱在生命有机磷化学中的一些应用

讨论了质谱在生命有机磷化学中的应用,包括N-磷酰氨基酸、肽、五配位磷化合物、核苷-氨基酸磷酰胺的质谱裂解途径,有机磷试剂辅助下氨基酸的自组装成肽产物和机理。     

Mapping low-resolution three-dimensional protein structures using chemical cross-linking and Fourier transform ion-cyclotron resonance mass spectrometry

Techniques in mass spectrometry (MS) combined with chemical cross-linking have proven to be efficient tools for the rapid determination of low-resolution three-dimensional (3-D) structures of proteins. The general procedure involves chemical cross-linking of a protein followed by enzymatic digestion and MS analysis of the resulting peptide mixture. These experiments are generally fast and do not require large quantities of protein. However, the large number of peptide species created from the digestion of cross-linked proteins makes it difficult to identify relevant intermolecular cross-linked peptides from MS data. We present a method for mapping low-resolution 3-D protein structures by combining chemical cross-linking with high-resolution FTICR (Fourier transform ion-cyclotron resonance) mass spectrometry using cytochrome c and hen egg lysozyme as model proteins. We applied several homo-bifunctional, amine-reactive cross-linking reagents that bridge distances from 6 to 16 A. The non-digested cross-linking reaction mixtures were monitored by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) to determine the extent of cross-linking. Enzymatically digested reaction mixtures were separated by nano-high-performance liquid chromatography (nano-HPLC) on reverse-phase columns applying water/acetonitrile gradients with flow rates of 200 nL/min. The nano-HPLC system was directly coupled to an FTICR mass spectrometer equipped with a nano-ESI (electrospray ionization) source. Cross-linking products were identified using a combination of the GPMAW software and ExPASy Proteomics tools. For correct assignment of the cross-linking products the key factor is to rely on a mass spectrometric method providing both high resolution and high mass accuracy, such as FTICRMS. By combining chemical cross-linking with FTICRMS we were able to rapidly define several intramolecular constraints for cytochrome c and lysozyme.     

基于M13噬菌体展示单链抗体文库的新型蛋白质均衡器的研制及其在肾病患者尿蛋白分析中的应用

通过将展示单链抗体(scFv)文库的M13噬菌体固定于冷凝胶整体柱表面制备了一种新型蛋白质均衡器。由于展示的scFv片段具有种类多、数目大以及与蛋白质结合的特异性强等优点,因此其非常适合用于复杂蛋白质样品的预处理。将肾病患者尿蛋白在平衡器上反复上样5次后,依次使用2 mol/L NaCl、50 mmol/L Gly-HCl(pH 2.5)和凝血酶溶液洗脱与均衡器结合的蛋白质,收集各馏分,并采用串联微柱反相液相色谱-电喷雾质谱进行蛋白质的分离鉴定。与未经均衡器处理的样品相比,鉴定的蛋白质数目由142个提高到396个。此外,凝胶电泳的分析结果显示,在上样流出液中蛋白质的浓度差异明显变小,大量的高丰度蛋白质存在于NaCl洗脱液中。上述结果表明,基于M13噬菌体展示scFv文库的新型蛋白质均衡器能够有效减小样品中蛋白质的浓度差异,有利于发现更多的低丰度蛋白质。     

高效液相色谱-串联质谱法测定肉制品和调味料中7种水产品过敏原

基于高效液相色谱-串联质谱系统建立了食品中水产品过敏原的快速筛查和定量检测方法。样品经蛋白质提取、纯化、胰蛋白酶解后，利用超高效液相色谱-四极杆/静电场轨道阱高分辨质谱（UPLC-Q/Exactive-HRMS）结合ProteinPilot软件，基于母离子和碎片离子谱分析，实现蛋白质和多肽的鉴定。再通过基本局部比对搜索工具（BLAST）与Uniprot数据库对比分析，筛选出南美白虾、大闸蟹、青蟹、金枪鱼、大西洋鲑鱼的7种过敏原蛋白的30个特征肽。利用高效液相色谱-三重四极杆质谱（UPLC-QqQ-MS）系统对特征肽进行验证和多反应监测（MRM）定量研究。结果表明，该方法在5~250 mg/kg范围内线性关系良好，检出限为2~3.5 mg/kg，平均回收率为88.7%~110.2%。该方法重现性好，通量高，可应用于肉制品和调味料中7种过敏原的快速筛查和定量分析。     

Enrichment and analysis of phosphopeptides under different experimental conditions using titanium dioxide affinity chromatography and mass spectrometry

Titanium dioxide metal oxide affinity chromatography (TiO₂‐MOAC) is widely regarded as being more selective than immobilized metal‐ion affinity chromatography (IMAC) for phosphopeptide enrichment. However, the widespread application of TiO₂‐MOAC to biological samples is hampered by conflicting reports as to which experimental conditions are optimal. We have evaluated the performance of TiO₂‐MOAC under a wide range of loading and elution conditions. Loading and stringent washing of peptides with strongly acidic solutions ensured highly selective enrichment for phosphopeptides, with minimal carryover of non‐phosphorylated peptides. Contrary to previous reports, the addition of glycolic acid to the loading solution was found to reduce specificity towards phosphopeptides. Base elution in ammonium hydroxide or ammonium phosphate provided optimal specificity and recovery of phosphorylated peptides. In contrast, elution with phosphoric acid gave incomplete recovery of phosphopeptides, whereas inclusion of 2,5‐dihydroxybenzoic acid in the eluant introduced a bias against the recovery of multiply phosphorylated peptides. TiO₂‐MOAC was also found to be intolerant of many reagents commonly used as phosphatase inhibitors during protein purification. However, TiO₂‐MOAC showed higher specificity than immobilized gallium (Ga³⁺), immobilized iron (Fe³⁺), or zirconium dioxide (ZrO₂) affinity chromatography for phosphopeptide enrichment. Matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) was more effective in detecting larger, multiply phosphorylated peptides than liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS), which was more efficient for smaller, singly phosphorylated peptides. Copyright © 2009 Crown in the right of Canada. Published by John Wiley & Sons, Ltd.     

Nitrite anions induce nitrosative deamination of peptides and proteins

In the present study, reactions of sodium nitrite with proteins/peptides were characterized with mass spectrometry. The reaction generates two major products: replacement of the amino group by a hydroxyl group and formation of an alkene derivative by loss of a NH3 group at the N-terminus and the side chain of lysine residues of proteins/peptides. The reaction proceeds rapidly in weak acidic solution and at 37 degrees C in the presence of a millimolar concentration of nitrite, demonstrating that nitrite induces nitrosative deamination in proteins and peptides. The facile nitrite-induced modification of amino groups of protein/peptides changes the chemical nature of proteins and may have various applications in peptide synthesis, analytical chemistry, and protein engineering. It also provides information to enhance our understanding of functions of nitrite ions in biology and food preservation.     

Evaluation of enzymatic digestion and liquid chromatography-mass spectrometry peptide mapping of the integral membrane protein bacteriorhodopsin

A method for the complete peptide mapping of the model integral membrane protein bacteri-orhodopsin is demonstrated. Utilizing more effective enzymatic digestion, procedures with capillary liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) and tandem mass spectrometry (MS/MS), all predicted tryptic digestion products were detected, as well as peptides from all previously reported post-translational modifications of bacteriorhodopsin. A significant contribution of chymotryptic-like digestion products was also observed. A characterization of the behavior of hydrophobic integral membrane peptides in a reversed-phase liquid chromatographic separation is also provided. The method reported here offers improved compatibility of the solubilizing reagents with both the chromatography and mass spectrometry, rendering it suitable for high-throughput proteomic applications.     

Selective enrichment and identification of azide-tagged cross-linked peptides using chemical ligation and mass spectrometry

Protein-protein interaction is one of the key regulatory mechanisms for controlling protein function in various cellular processes. Chemical cross-linking coupled with mass spectrometry has proven to be a powerful method not only for mapping protein-protein interactions of all natures, including weak and transient ones, but also for determining their interaction interfaces. One critical challenge remaining in this approach is how to effectively isolate and identify cross-linked products from a complex peptide mixture. In this work, we have developed a novel strategy using conjugation chemistry for selective enrichment of cross-linked products. An azide-tagged cross-linker along with two biotinylated conjugation reagents were designed and synthesized. Cross-linking of model peptides and cytochrome c as well as enrichment of the resulting cross-linked peptides has been assessed. Selective conjugation of azide-tagged cross-linked peptides has been demonstrated using two strategies: copper catalyzed cycloaddition and Staudinger ligation. While both methods are effective, Staudinger ligation is better suited for enriching the cross-linked peptides since there are fewer issues with sample handling. LC MSⁿ analysis coupled with database searching using the Protein Prospector software package allowed identification of 58 cytochrome c cross-linked peptides after enrichment and affinity purification. The new enrichment strategy developed in this work provides useful tools for facilitating identification of cross-linked peptides in a peptide mixture by MS, thus presenting a step forward in future studies of protein-protein interactions of protein complexes by cross-linking and mass spectrometry.     

Further investigation of a peptide extraction method with mesoporous silica using high‐performance liquid chromatography coupled with tandem mass spectrometry

Mobil Composition of Matter No. 41 (MCM‐41) was the most frequently used mesoporous silica material to extract peptides from complex biological samples. However, there were confusing extraction conditions and large extraction efficiency variance among related reports, which resulted from unclear understanding about the interaction between the material and peptides. In this study, the extraction mechanism was investigated with one set of tryptic peptides by using high‐performance liquid chromatography coupled with triple quadrupole mass spectrometry. Generally, hydrophobic interaction and electrostatic attraction were two major driving forces for extraction of peptides, while electrostatic repulsion greatly weakened the interaction between the material and peptides with isoelectric points below the pH. With most peptides positively charged and MCM‐41 slightly negatively charged, most efficient extraction was obtained at pH 3, and it was proved that electrostatic and hydrophobic interaction acted in synergy for extraction of all the peptides. A mixed solution of acetonitrile with buffers of high pH or ion strength was demonstrated to be favorable for elution, which performed much better than the commonly used eluate (mixture of acetonitrile with 0.1% trifluoroacetic acid). Finally, under optimum conditions, it was found that extraction efficiency of MCM‐41 for protein digest and human serum was greatly improved.     

9,10-2H-9-氧杂-10-磷杂菲-10-氧化物基1,3-苯并噁嗪树脂的合成与表征

以水杨醛、对甲基苯胺(或对氟苯胺,对甲氧基苯胺)、9,10-2H-9-氧-10-磷杂菲-10-氧化物(DOPO)及甲醛为原料,通过三步反应合成了三个含DOPO基的1,3-苯并噁嗪化合物3a-3c.第一步,水杨醛与对位取代苯胺进行缩合反应生成亚胺(1a-1c);第二步,DOPO对亚胺进行加成反应得到仲胺(2a-2c);第三步,仲胺与甲醛在强酸性离子交换树脂催化下进行关环反应形成含DOPO的1,3-苯并噁嗪树脂.采用红外光谱、核磁共振谱和质谱等表征了化合物3a-3c,同时采用热失重分析技术测定了其热稳定性.结果表明,3a-3c由两对对映体组成,质谱条件下2a-2c和3a-3c均经裂解失去稳定的DOPO基团;3a-3c热降解反应包含两个热失重过程,分别在250和400℃下出现最大热释放速率.     

Operational variables in high-performance liquid chromatography-electrospray ionization mass spectrometry of peptides and proteins using poly(styrene-divinylbenzene) monoliths

Capillary reversed-phase high-performance liquid chromatography (RP-HPLC) utilizing monolithic poly(styrene-divinylbenzene) columns was optimized for the coupling to electrospray ionization mass spectrometry (ESI-MS) by the application of various temperatures and mobile phase additives during peptide and protein analysis. Peak widths at half height improved significantly upon increasing the temperature and ranged from 2.0 to 5.4 s for peptide and protein separations at 70 degrees. Selectivity of peptide elution was significantly modulated by temperature, whereas the effect on proteins was only minor. A comparison of 0.10% formic acid (FA), 0.050% trifluoroacetic acid (TFA), and 0.050% heptafluorobutyric acid (HFBA) as mobile phase additives revealed that highest chromatographic efficiency but poorest mass spectrometric detectabilities were achieved with HFBA. Clusters of HFBA, water, and acetonitrile were observed in the mass spectra at m/z values >500. Although the signal-to-noise ratios for the individual peptides diverged considerably both in the selected ion chromatograms and extracted mass spectra, the average mass spectrometric detectabilities varied only by a factor of less than 1.7 measured with the different additives. Limits of detection for peptides with 500 nl sample volumes injected onto a 60 mm x 0.20 mm monolithic column were in the 0.2-13 fmol range. In the analysis of hydrophobic membrane proteins, HFBA enabled highest separation selectivity at the cost of lower mass spectral quality. The use of 0.050% TFA as mobile phase additive turned out to be the best compromise between chromatographic and mass spectrometric performance in the analysis of peptides and proteins by RP-HPLC-ESI-MS using monolithic separation columns.