生物质谱

质谱已成为生物和生物化学研究的一个重要的分析工具，特别是在蛋白质组学研究的作用更显突出，它的分析速度、准确性和灵敏度都是传统分析技术所不可比拟的。主要介绍了两种近年来发展最为迅速、应用最为广泛的软离子化质谱技术：即基体辅助激光解吸离子化质谱（MALDI－MS）和电喷了子化质谱（ESI－MS）的原理、技术的最新进展，并简单介绍了它们在蛋白质和多肽分析中的应用。     

复杂样品质谱分析技术的原理与应用

原位、实时、在线、非破坏、高通量、低耗损的质谱学方法是质谱分析技术发展的重要趋势.在无需样品预处理的条件下对复杂基体样品中痕量待测物直接离子化技术的出现,极大地提高了质谱分析的效率,使实际样品的快速质谱分析成为可能.本文着重综述了能够在无需样品预处理情况下对复杂基体样品离子化的新兴质谱技术及其应用研究,系统阐述了直接离子化技术的基本原理和方法,介绍了几种典型的常压直接离子化技术和装置,对直接离子化质谱分析技术在食品、药品、环境、活体分析、代谢组学、蛋白质组学以及生物组织质谱成像等领域的典型应用进行了述评,讨论了提高复杂样品快速质谱分析选择性的可能方法,并展望了常压直接离子化技术未来发展的可能趋势.     

质谱与生命科学

本文以实验表明了谱学，特别是快原子轰击质谱（ＦＡＢ－ＭＳ）、电喷雾电离质谱（ＥＳＩ－ＭＳ）、串联质谱（ＭＳ－ＭＳ）以及有底物的激光解吸电离飞行时间质谱（ＭＡＬＤＩ－ＴＯＦ－ＭＳ）等技术在解决生物物质－肽－蛋白质等的一级结构难题中的特定作用，而这些结构难题往往是常规的生物不方法－Ｅｄｍａｎ降解法、ＤＮＡ转泽法等所无能为力的。虽然文中讨论的例子仅涉及到肽、蛋白质的结构修饰，同理，它也可以广泛应用于其他     

毛细管电泳-质谱联用技术及其在蛋白质组学中的应用

蛋白质组学研究在生物学、精准医学等方面发挥着重要的作用。然而研究面临的巨大挑战来自生物样品的复杂性,因此在质谱（MS）鉴定技术不断革新的同时,发展分离技术以降低样品复杂度尤为重要。毛细管电泳（CE）技术具有上样体积小、分离效率高、分离速度快等优势,其与质谱的联用在蛋白质组学研究中越来越受到关注。低流速鞘流液和无鞘流液接口的发展及商品化推动了CE-MS技术的发展。目前毛细管区带电泳（CZE）、毛细管等电聚焦（CIEF）、毛细管电色谱（CEC）等分离模式已与质谱联用,其中CZE-MS应用最广泛。目前被广泛采用的蛋白质组学研究策略主要是基于酶解肽段分离鉴定的"自下而上（bottom-up）"策略。首先,CE-MS技术对酶解肽段的检测灵敏度高达1 zmol,已成功应用于单细胞蛋白质组学;其次,毛细管电泳技术与反相液相色谱互补,为疏水性质相近的肽段（尤其是翻译后修饰肽段）的分离鉴定提供了新的途径。基于整体蛋白质分离鉴定的自上而下"top-down"策略可以直接获得更精准、更完整的蛋白质信息。CE技术在蛋白质大分子的分离方面具有分离效率高、回收率高的优势,其与质谱的联用提高了整体蛋白质的鉴定灵敏度和覆盖度。非变性质谱（native MS）是一种在近生理条件下从完整蛋白质复合物水平上进行分析的质谱技术。CE与非变性质谱联用已被尝试用于蛋白质复合体的分离鉴定。该文引用了与CE-MS和蛋白质组学应用相关的93篇文献,综述了以上介绍的CE-MS的研究进展以及在蛋白质组学分析中的应用优势,并总结和展望了其应用前景。     

复杂生物体系中蛋白质高效分离分析技术的新进展

 继人类基因组计划完成之后,作为一种新的研究策略,蛋白质组学在生命科学研究中发挥着愈来愈重要的作用。由于生物体系的复杂性和多样性,使得分离效率高、灵敏度高、通量高和动态范围宽的分离分析技术平台的研究和应用已成为蛋白质组学研究的重点和热点之一。着重介绍了近年来应用日益广泛的多维色谱预分离、毛细管液相色谱-质谱联用、毛细管电泳及其与质谱联用等高效分离分析技术在复杂生物体系的蛋白质分析中的最新进展。引用相关文献40篇。     

串联质谱系统在毒鼠强测定中的应用

采用串联质谱技术（ＭＳ－ＭＳ）,对以生物为基体的毒鼠强中毒检品分析方法进行了研究,被检测物含量在５＊１０＾－１１ｇ时,信噪比Ｓ／Ｎ达２６,在（４０－４００）＊１０＾－１２ｇ范围内获得良好的线性关系。该方法同样适合于其它复杂基体的超微量目标化合物的分析。     

生命科学中的分析化学--蛋白质组分析

概述了分析化学在生命科学中的重要作用和蛋白质组分析的现状与展望。介绍了生物试样的制备、双向凝胶电泳分离蛋白质以及生物质谱鉴定蛋白质等蛋白质组分析的主要技术平台。     

蛋白质非共价化合物的电喷雾质谱研究

介绍了近几年来利用电喷雾离子化质谱技术研究蛋白质的基本构象及蛋白质非共价化合物的基本化学信息的进展。许多研究表明,电喷雾质谱在研究蛋白质的复杂结构和功能方面有十分广阔的前景。文中列举了若干实例说明了电喷雾离子化质谱技术在分析蛋白质非共价化合物的应用和一般方法。     

气相色谱-质谱联用法在生物样品分析中的应用

概述了气相色谱-质谱技术在生物样品分析及药物测定中的应用.     

加速溶剂萃取-气相色谱串联质谱测定菊花中的3种菊酯类农药残留量

建立了加速溶剂萃取菊花中氯氰菊酯、氰戊菊酯、溴氰菊酯的气相色谱串联质谱农残分析检测方法。探讨了选择性加速溶剂萃取技术在农残前处理上的应用,开发了集萃取、净化于一体的快速新型样品前处理技术。同时还对气相色谱串联质谱的质谱条件进行了优化,得到了一种快速准确高灵敏的农残检测方法。方法的回收率在80%-106%之间,精密度在5.6%-13%,并在1.0-6000μg/L范围内有良好的线性关系（R≥0.9967）,检出限（S/N=3）〈0.5μg/L;定量限（S/N=10）〈1.67μg/L。     

Sheathless capillary electrophoresis-mass spectrometry using a pulsed electrospray ionization source

A sheathless interface has been developed for coupling CE with electrospray IT mass spectrometer. This interface utilized a pulsed ESI source. The use of a pulsed electrospray source allows the use of a sprayer with larger orifice, and thus alleviates the problem of column clogging during conductive coating and CE analysis. A pulsed ESI source operated at 20 Hz and 20% duty cycle was found to produce the optimal signals. For better signals, the maximum ion injection time in the IT mass spectrometer has to be set to a value close to the actual spraying time (10 ms). Using a sprayer with 50 microm od, more stable and enhanced signals were obtained in comparison with continuous CE-ESI-MS under the same flow rate (150 nL/min). The utility of this design is demonstrated with the analysis of synthetic drugs by CE-MS.     

Pulsed dual electrospray ionization for In/In reactions

A pulsed dual electrospray ionization source has been developed to generate positive and negative ions for subsequent ion/ion reaction experiments. The two sprayers, typically a nano-electrospray emitter for analytes and an electrospray emitter for reagents, are positioned in a parallel fashion close to the sampling orifice of a triple quadrupole/linear ion trap tandem mass spectrometer (Sciex Q TRAP). The potentials applied to each sprayer are alternately pulsed so that ions of opposite polarity are generated separately in time. Ion/ion reactions take place after ions of each polarity are sequentially injected into a high-pressure linear ion trap, where axial trapping is effected by applying an auxiliary radio frequency voltage to the end lenses. The pulsed dual electrospray source allows optimization of each sprayer and can be readily coupled to any spray interface with no need for instrument modifications, provided the potentials required to transmit the ion polarity of interest can be alternated in synchrony with the emitter potentials. Ion/ion reaction examples such as charge reduction of multiply charged protein ions, charge inversion of peptides ions, and protein-protein complex formation are given to illustrate capabilities of the pulsed dual electrospray source in the study of gas-phase ion/ion chemistry.     

Polarization induced electrospray ionization mass spectrometry for the analysis of liquid,viscous and solid samples

In this study, a polarization‐induced electrospray ionization mass spectrometry (ESI‐MS) was developed. A micro‐sized sample droplet was deposited on a naturally available dielectric substrate such as a fruit or a stone, and then placed close to (~2 mm) the orifice of a mass spectrometer applied with a high voltage. Taylor cone was observed from the sample droplet, and a spray emitted from the cone apex was generated. The analyte ion signals derived from the droplet were obtained by the mass spectrometer. The ionization process is similar to that in ESI although no direct electric contact was applied on the sample site. The sample droplet polarized by the high electric field provided by the mass spectrometer initiated the ionization process. The dielectric sample loading substrate facilitated further the polarization process, resulting in the formation of Taylor cone. The mass spectral profiles obtained via this approach resembled those obtained using ESI‐MS. Multiply charged ions dominated the mass spectra of peptides and proteins, whereas singly charged ions dominated the mass spectra of small molecules such as amino acids and small organic molecules. In addition to liquid samples, this approach can be used for the analysis of solid and viscous samples. A small droplet containing suitable solvent (5–10 µl) was directly deposited on the surface of the solid (or viscous) sample, placed close the orifice of mass spectrometer applied with a high voltage. Taylor cone derived from the droplet was immediately formed followed by electrospray processes to generate gas‐phase ions for MS analysis. Analyte ions derived from the main ingredients of pharmaceutical tablets and viscous ointment can be extracted into the solvent droplet in situ and observed using a mass spectrometer. Copyright © 2015 John Wiley & Sons, Ltd.     

Applying a dynamic method to the measurement of ion mobility

A dynamic method is applied to measure the mobility of gas-phase ions in the dual ion funnel interface of the electrospray source of a quadrupole orthogonal time-of-flight mass spectrometer. In a new operational mode, a potential barrier was formed in the second ion funnel of the mass spectrometer and then progressively increased. In this region, a flow of gas drags the ions into the mass spectrometer while the electric force applied by the potential barrier decelerates them. Ions with lower mobility can be carried by the gas flow more easily than those with high mobility. Thus, electrical forces can block the more mobile ions more easily. Hence, the electric barrier formed in the ion funnel permits only ions below a certain mobility threshold to enter the mass spectrometer. When the barrier voltage is increased, this threshold moves from high to low mobilities. Ions with mobilities above the threshold cannot enter the mass spectrometer, and their signal decreases to zero. Thus, in a barrier voltage scan, mass spectrometric signals of ions sequentially disappear. Differentiation of these decreasing ion signal curves produces peaks from which an ion mobility spectrum can be reconstructed. Blocking voltages, i.e., the positions of the peaks on the barrier voltage scale are directly related to the mobility of these ions. An internal calibration using ions with known mobility values helps determine the unknown ion mobilities and allows calculation of ionic cross sections.     

Multitrack electrospray chips

Multitrack electrospray chips (MTEC) were fabricated by UV-photoablation of polyethylene terephthalate (PET) substrates. They are composed of an array of up to six microchannels that are successively used as electrospray ionization (ESI) emitters for mass spectrometry (MS). There is no requirement for alignment of the different spraying microchannels with the mass spectrometer orifice. The MTEC is thus fixed in front of the mass spectrometer and the successive MS analyses are performed without moving the chip. The sequential electrospraying by successive application of an identical high voltage in each off-axis microchannel was evaluated for the fast screening of peptides and proteins. The counting of cysteines in peptides through chemical modification and the relative quantification of a peptide in two samples are presented herein as two original strategies based on this new analytical tool.     

Novel microfabricated device for electrokinetically induced pressure flow and electrospray ionization mass spectrometry

A novel microchip device for electrospray ionization has been fabricated and interfaced to a time-of-flight mass spectrometer. Fluid is electrokinetically transported through the chip to a fine fused-silica capillary inserted directly into a channel at the edge of the device. Electrospray is established at the tip of the capillary, which assures a stable, efficient spray. The electric potential necessary for electrospray generation and the voltage drop for electroosmotic pumping are supplied through an electrically permeable glass membrane contacting the fluidic channel holding the capillary. The membrane is fabricated on the microchip using standard photolithographic and wet chemical etching techniques. Performance relative to other microchip electrospray sources has been evaluated and the device tested for potential use as a platform for on-line electrophoretic detection. Sensitivity was found to be approximately three orders of magnitude better than spraying from the flat edge of the chip. The effect of the capillary on electroosmotic flow was examined both experimentally and theoretically.     

Aerodynamic mass spectrometry interfacing of microdevices without electrospray tips

A new concept for electrospray coupling of microfluidic devices with mass spectrometry was developed. The sampling orifice of the time-of-flight mass spectrometer was modified with an external adapter assisting in formation and transport of the electrosprayed plume from the multichannel polycarbonate microdevice. The compact disk sized microdevice was designed with radial channels extending to the circumference of the disk. The electrospray exit ports were formed by the channel openings on the surface of the disk rim. No additional tips at the channel exits were used. Electrospray was initiated directly from the channel openings by applying high voltage between sample wells and the entrance of the external adapter. The formation of the spatially unstable droplet at the electrospray openings was eliminated by air suction provided by a pump connected to the external adapter. Compared with the air intake through the original mass spectrometer sampling orifice, more than an order of magnitude higher flow rate was achieved for efficient transport of the electrospray plume into the mass spectrometer. Additional experiments with electric potentials applied between the entrance sections of the external adapter and the mass spectrometer indicated that the air flow was the dominant transport mechanism. Basic properties of the system were tested using mathematical modeling and characterized using ESI/TOF-MS measurements of peptide and protein samples.     

Online monitoring of chemical reactions by polarization-induced electrospray ionization

Polarization-induced electrospray ionization (PI-ESI) is a simple technique for instant generation of gas-phase ions directly from a microliter-sized droplet for mass spectrometric analysis. A sample droplet was placed over a dielectric substrate and in proximity (2–3 mm) to the inlet of a mass spectrometer. Owing to the polarization effect induced by the high electric field provided by the mass spectrometer, the droplet was polarized and the electrospray was generated from the apex of the droplet. The polarization-induced electrospray could last for tens of seconds, which was sufficiently long to monitor fast reactions occurring within few seconds. Thus, we demonstrated the feasibility of using the droplet-based PI-ESI MS for the online monitoring of fast reactions by simply mixing two droplets (5–10 μL) containing reactants on a dielectric substrate placed in front of a mass spectrometer applied with a high voltage (−4500 V). Schiff base reactions and oxidation reactions that can generate intermediates/products within a few seconds were selected as the model reactions. The ionic reaction species generated from intermediates and products can be simultaneously monitored by PI-ESI MS in real time. We also used this approach to selectively detect acetone from a urine sample, in which acetone was derivatized in situ. In addition, the possibility of using this approach for quantitative analysis of acetone from urine samples was examined.     

A pulsed triple ionization source for sequential ion/ion reactions in an electrodynamic ion trap

A pulsed triple ionization source, using a common atmosphere/vacuum interface and ion path, has been developed to generate different types of ions for sequential ion/ion reaction experiments in a linear ion trap-based tandem mass spectrometer. The triple ionization source typically consists of a nano-electrospray emitter for analyte formation and two other emitters, an electrospray emitter and an atmospheric pressure chemical ionization emitter or a second nano-electrospray emitter for formation of the two different reagent ions. The three emitters are positioned in a parallel fashion close to the sampling orifice of the tandem mass spectrometer. The potentials applied to each emitter are sequentially pulsed so that desired ions are generated separately in time and space. Sequential ion/ion reactions take place after analyte ions of interest and different set of reagent ions are sequentially injected into a linear ion trap, where axial trapping is effected by applying an auxiliary radio frequency voltage to the end lenses. The pulsed triple ionization source allows independent optimization of each emitter and can be readily coupled to any atmospheric pressure ionization interface with no need for instrument modifications, provided the potentials required to transmit the ion polarity of interest can be synchronized with the emitter potentials. Several sequential ion/ion reactions examples are demonstrated to illustrate the analytical usefulness of the triple ionization source in the study of gas-phase ion/ion chemistry.     

基于敞开式直接电离质谱技术的尿液中毒品检测北大核心CSCD

尿液作为一种易于获取的体内毒品检材,在吸毒人员快速筛查中被广泛应用。针对传统快速筛查技术存在假阳性率高、定量能力不足以及实验室质谱技术在快速检测中存在前处理复杂、检测耗时长、使用环境苛刻等问题,该文提出了一种基于敞开式直接电离质谱技术的生物样本快速检测方法。该研究采用探针式电喷雾离子源与便携式质谱仪联用快速检测平台,优化了喷雾电压和质谱入口毛细管温度,开发了高效快速的前处理技术。基于该平台和前处理技术,5种常规毒品(甲基苯丙胺、氯胺酮、可卡因、O^(6)-单乙酰吗啡和3,4-亚甲双氧甲基苯丙胺)的尿液加标溶液的检出限为0.5~30 ng/mL,且其中4种毒品定量检测的线性相关系数大于0.99。除此之外,5种常规毒品在3个不同水平下的加标回收率为56.1%~103.7%,多次检测结果的相对标准偏差为9.0%~27.8%,说明联用检测平台与前处理方法结合可以达到良好的准确度。为了进一步检验该联用仪器的实战能力,测试了某社区戒毒康复中心40份阳性和110份阴性实际尿液样本,总体检测的准确率接近99%,且通过一次进样在20 s内可同时检测多种毒品。该研究成果有利于推动快速检测技术的发展,促进敞开式直接电离质谱仪技术的推广应用,提升一线执法服务水平。