解吸电喷雾电离技术

本文介绍了一种新近发展起来的质谱离子化方法——解吸电喷雾电离（desorption electrospray ionization, DESI）及其最新研究进展。该方法首次于2004年提出后,由于其具有样品无需前处理就可以在常压条件下从各种载物表面直接分析固相,或凝固相样品等优势而得到了迅速的发展。本文描述了DESI的基本原理、离子源的结构和相关优化的参数,并对该离子化方法中所用的载物表面进行了总结。在实际应用方面,本文综述了DESI技术在常压气相化学反应产物监测,合成高聚物表征,爆炸物和化学战毒剂检测,以及在药品,生物代谢产物和生物组织表面分析方面的应用成果,同时对DESI的基础应用研究方向进行了分析和展望。     

Deconstructing Desorption Electrospray Ionization: Independent Optimization of Desorption and Ionization by Spray Desorption Collection

Spray desorption collection (SDC) and reflective electrospray ionization (RESI) were used to independently study the desorption and ionization processes that together comprise desorption electrospray ionization (DESI). Both processes depend on several instrumental parameters, including the nebulizing gas flow rate, applied potential, and source geometries. Each of these parameters was optimized for desorption, as represented by the results obtained by SDC, and ionization, as represented by the results obtained by RESI. The optimized conditions were then compared to the optimization results for DESI. Our results confirm that optimal conditions for desorption and ionization are different and that in some cases the optimized DESI conditions are a compromise between both sets. The respective results for DESI, RESI, and SDC for each parameter were compared across the methods to draw conclusions about the contribution of each parameter to desorption and ionization separately and then combined within DESI. Our results indicate that desorption efficiency is (1) independent of the applied potential and (2) the impact zone to inlet distance, and that (3) gas pressure settings and (4) sprayer to impact zone distances above optimal for DESI are detrimental to desorption but beneficial for ionization. In addition, possible interpretations for the observed trends are presented.     

Kinetic Control of Protonation in Electrospray Ionization

The site of protonation in a molecule can greatly affect the fragments observed in product ion MS/MS spectra. In electrospray positive ionization mass spectra, protonation usually occurs predominantly on the most basic site on the molecule to produce the thermodynamically favored protonated species. However, the literature is unclear whether liquid phase or gas phase thermodynamics has the greater influence. This paper describes the protonation and fragmentation behavior of crizotinib and two of its impurities. Crizotinib has two possible protonation sites, a pyridine nitrogen and a secondary amine, piperidine nitrogen; the former is the favored site in the gas phase and the latter the more favored site in the liquid phase. The impurities contain alkyl substitution on the piperidine nitrogen, producing tertiary amine species. Literature precedence suggests that in the liquid phase, the piperidine nitrogen is still the most basic site but, in the gas phase, the pyridine nitrogen and the piperidine nitrogen have very similar basicities. Fragmentation data for the three molecules suggest that the secondary and tertiary amines protonate preferentially and almost exclusively on different sites. We propose that the secondary amine protonates on the piperidine nitrogen (influenced by solution thermodynamics) and the two tertiary amine structures protonate on the pyridine nitrogen because of steric hindrance at the most basic site of the molecule, allowing kinetic control of the protonation process.     

Laser Desorption Ionization Versus Electrospray Ionization Mass Spectrometry: Applications in the Analysis of Cluster Anions

Mass spectra of transition metal carbonyl cluster anions were recorded using laser desorption ionization time-of-flight (LDI-TOF) and electrospray ionization (ESI) techniques. The LDI spectra generally contain peaks corresponding the intact cluster together with extensive CO loss fragments ions whereas the ESI spectra exhibit peaks corresponding the intact cluster together with few (if any) CO loss fragment ions. The parameters of both techniques can be modified to vary the extent of fragmentation. In all cases no fragmentation of the metal core is observed. Overall, ESI is a more informative method for the analysis of these types of cluster anions.     

自建电喷雾解吸电离源（DESI）在大分子分析中的应用及离子化机理探索

自建了简易的电喷雾解吸电离源(DESI),优化了DESI源喷口的位置和角度,并将其用于常见多肽和蛋白质的分析。多肽和小质量蛋白质(<20 kDa)可以容易地从表面解吸电离,生成清晰的质谱。而牛血清白蛋白(66.4 kDa)不能产生清晰的多电荷分布的质谱,说明当前DESI源的设计可能存在一个电离的分子量上限。通过比较不同的实验条件并对比ESI-MS,发现溶剂分子的挥发过程对电荷分布以及峰宽均有显著影响,可能是由于ESI更软引起。载样表面的性质对DESI-MS的信号强度有较大影响。金表面的自组装单分子膜(SAM)相对于纯金表面有较好的绝缘性,并有助于产生较强信号,说明来自表面的电子转移(电中和)是电喷雾解吸电离过程中的一个重要因素。该文的研究有助于对DESI-MS的实验条件和载样表面的选择,同时增进了对电喷雾解吸电离机理的了解。     

Characterization of Rhodamine Self-Assembled Films Using Desorption Electrospray Ionization Mass Spectrometry

Growth process information and molecular structure identification are very important for characterization of self-assembled films. Here, we explore the possible application of desorption electrospray ionization mass spectrometry (DESI-MS) that provides the assembled information of rhodamine B (Rh B) and rhodamine 123 (Rh 123) films. With the help of lab-made DESI source, two characteristic ions [Rh B]⁺ and [Rh 123]⁺ are observed directly in the open environment. To evaluate the reliability of this technique, a comparative study of ultraviolet-visible (UV-vis) spectroscopy and our method is carried out, and the result shows good correlation. According to the signal intensity of characteristic ions, the layer-by-layer adsorption process of dyes can be monitored, and the thicknesses of multilayer films can also be comparatively determined. Combining the high sensitivity, selectivity, and speed of mass spectrometry, the selective adsorption of similar structure molecules under different pH is recognized easily from extracted ion chronograms. The variation trend of dyes signalling intensity with concentration of polyelectrolyte is studied as well, which reflects the effect of surface charge on dyes deposition. Additionally, the desorption area, surface morphology, and thicknesses of multilayer films are investigated using fluorescence microscope, scanning electron microscope (SEM), and atomic force microscopy (AFM), respectively. Because the desorption area was approximately as small as 2 mm², the distribution situation of organic dyes in an arbitrary position could be gained rapidly, which means DESI-MS has advantages on in situ analysis.

A Combined Desorption Ionization by Charge Exchange (DICE) and Desorption Electrospray Ionization (DESI) Source for Mass Spectrometry

A source that couples the desorption ionization by charge exchange (DICE) and desorption electrospray ionization (DESI) techniques together was demonstrated to broaden the range of compounds that can be analyzed in a single mass spectrometric experiment under ambient conditions. A tee union was used to mix the spray reagents into a partially immiscible blend before this mixture was passed through a conventional electrospray (ES) probe capillary. Using this technique, compounds that are ionized more efficiently by the DICE method and those that are ionized better with the DESI procedure could be analyzed simultaneously. For example, hydroquinone, which is not detected when subjected to DESI-MS in the positive-ion generation mode, or the sodium adduct of guaifenesin, which is not detected when examined by DICE-MS, could both be detected in one experiment when the two techniques were combined. The combined technique was able to generate the molecular ion, proton and metal adduct from the same compound. When coupled to a tandem mass spectrometer, the combined source enabled the generation of product ion spectra from the molecular ion and the [M + H]⁺ or [M + metal]⁺ ions of the same compound without the need to physically change the source from DICE to DESI. The ability to record CID spectra of both the molecular ion and adduct ions in a single mass spectrometric experiment adds a new dimension to the array of mass spectrometric methods available for structural studies.     

电喷雾解吸电离质谱快速测定吴茱萸中生物碱

生物碱是许多中草药的活性有效成分,其含量的多少和种类的差异是导致中草药品质差异的重要因素.本文利用电喷雾解吸电离质谱(DESI-MS)能够在不需要样品预处理的前提下进行复杂基体样品分析的特点,采用酸性甲醇-水混合溶液作为喷雾试剂,在优化了的实验条件下快速获得了吴茱萸的DESI-MS指纹谱图,然后利用串联质谱对其中有重要活性的5种生物碱进行了结构鉴定.实验表明,基于固体表面解吸电离质谱分析的方法不需要萃取-分离手续,单个样品测定时间不超过1.5 min,大幅度提高了分析速度,有望在药品品质的在线监测和工艺过程控制中发挥重要作用.     

解析电喷雾质谱法对感冒药中对乙酰氨基酚的快速检测

基于常压质谱的直接、快速、无需样品预处理检测的优势,该文拓展了其在药物质检领域的应用。采用自行搭建的解析电喷雾(DESI)装置,对常用感冒药对乙酰氨基酚片进行快速质谱检测,无需样品预处理,直接获得药片中有效成分的分子结构信息。为克服基质差异对定量分析的影响,以淀粉为基质构建对乙酰氨基酚模拟药片,在优化的基础上进行定量实验。针对实际药片中高浓度对乙酰氨基酚检测的需要,研究了其在较高浓度范围的定量关系,结果显示,方法对0.35～4.52 mg/mm²范围内的对乙酰氨基酚具有较好的线性关系(r²=0.998 2),定量下限为1.903 ng/mm²,检出限为0.237 ng/mm²,加标回收率为102%～114%。对3种市售的对乙酰氨基酚片进行直接检测,与厂家提供的标准数据相比,相对标准偏差(RSD)为7.2%～12%,表明方法具有较好准确度。该工作很好地证明了DESI-MS在药品快检中的优势,从而为药品质检提供了潜在的高效、可靠的检测手段。     

电喷雾解吸电离质谱法用于临床尿样的直接分析

将电喷雾解吸电离质谱(DESI-MS)用于临床尿样的分析, 优化了电喷雾溶剂流速、电喷雾电压和喷雾锥距离等重要参数. 采用普通滤纸作为样品载体, 在不需要样品预处理的前提下同时快速测定了临床尿样中的钾、钠、尿素、尿酸、丙酮酸和肌苷等多种成分, 并对各种成分的主要离子进行了串联质谱鉴定. DESI-MS在进行多组分同时测定时不需要进行样品预处理, 缩短了测定时间, 单个样品的分析时间不到1 min. 同时, 采用内标法对所测定组分进行了半定量分析.     

Native Desorption Electrospray Ionization Liberates Soluble and Membrane Protein Complexes from Surfaces

Mass spectrometry (MS) applications for intact protein complexes typically require electrospray (ES) ionization and have not been achieved via direct desorption from surfaces. Desorption ES ionization (DESI) MS has however transformed the study of tissue surfaces through release and characterisation of small molecules. Motivated by the desire to screen for ligand binding to intact protein complexes we report the development of a native DESI platform. By establishing conditions that preserve non‐covalent interactions we exploit the surface to capture a rapid turnover enzyme–substrate complex and to optimise detergents for membrane protein study. We demonstrate binding of lipids and drugs to membrane proteins deposited on surfaces and selectivity from a mix of related agonists for specific binding to a GPCR. Overall therefore we introduce this native DESI platform with the potential for high‐throughput ligand screening of some of the most challenging drug targets including GPCRs.     

Gas-Phase Stability of Negatively Charged Organophosphate Metabolites Produced by Electrospray Ionization and Matrix-Assisted Laser Desorption/Ionization

The formation mechanisms of singly and multiply charged organophosphate metabolites by electrospray ionization (ESI) and their gas phase stabilities were investigated. Metabolites containing multiple phosphate groups, such as adenosine 5'-diphosphate (ADP), adenosine 5'-triphosphate (ATP), and D-myo-inositol-1,4,5-triphosphate (IP₃) were observed as doubly deprotonated ions by negative-ion ESI mass spectrometry. Organophosphates with multiple negative charges were found to be unstable and often underwent loss of PO₃ ^–, although singly deprotonated analytes were stable. The presence of fragments due to the loss of PO₃ ^– in the negative-ion ESI mass spectra could result in the misinterpretation of analytical results. In contrast to ESI, matrix-assisted laser desorption ionization (MALDI) produced singly charged organophosphate metabolites with no associated fragmentation, since the singly charged anions are stable. The stability of an organophosphate metabolite in the gas phase strongly depends on its charge state. The fragmentations of multiply charged organophosphates were also investigated in detail through density functional theory calculations.

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Toward Depth-Resolved Analysis of Plant Metabolites by Nanospray Desorption Electrospray Ionization Mass Spectrometry

Nanospray desorption electrospray ionization (nano-DESI) is one of the ambient desorption ionization methods for mass spectrometry (MS), and it utilizes a steady-state liquid junction formed between two microcapillaries to directly extract analytes from sample surfaces with minimal sample damage. In this study, we employed nano-DESI MS to perform a metabolite fingerprinting analysis directly from a Hypericum leaf surface. Moreover, we investigated whether changes in metabolite fingerprints with time can be related to metabolite distribution according to depth. From a raw Hypericum leaf, the mass spectral fingerprints of key metabolites, including flavonoids and prenylated phloroglucinols, were successfully obtained using ethanol as a nano-DESI solvent, and the changes in their intensities were observed with time via full mass scan experiments. In addition, the differential extraction patterns of the obtained mass spectral fingerprints were clearly visualized over time through selected ion monitoring and pseudo-selected reaction monitoring experiments. To examine the correlation between the time-dependent changes in the metabolite fingerprints and depth-wise metabolite distribution, we performed a nano-DESI MS analysis against leaves whose surface layers were removed multiple times by forming polymeric gum Arabic films on their surfaces, followed by detaching. The preliminary results showed that the changes in the metabolite fingerprints according to the number of peelings showed a similar pattern with those obtained from the raw leaves over time.     

基质辅助激光解析电离质谱和电喷雾质谱共同快速确证达托霉素结构

运用基质辅助激光解析电离-飞行时间串联质谱(MALDI-TOF/TOF MS)和电喷雾-四级杆-飞行时间质谱(ESI-Q-TOF MS)快速确证环脂肽达托霉素的结构。首先,ESI检测达托霉素相对分子量为1619.7107,与理论值偏差0.0007。选择其双电荷峰m/z 809.848作为母离子进行ESI串联质谱(MS/MS)测定,成功匹配了达托霉素环外氨基酸序列C9H19CO-Trp-Asn-Asp。其次,优化Li OH裂解达托霉素的实验条件,以MALDITOF/TOF MS监测开环效果,获得95%以上的开环样本后,分别运用MALDI和ESI进行MS/MS测定,达托霉素开环产物的b+和y+全部被匹配,达托霉素全部氨基酸序列得到确证。最后,对开环产物的ESI-MS/MS条件进一步优化,获得了丰富的低端碎片离子,解析了脂肪酸链结构,并绘制了脂肪酸链的裂解图。本方法快速、简便、准确,是确证环脂肽类化合物结构的可靠方法。     

OmniSpect: An Open MATLAB-Based Tool for Visualization and Analysis of Matrix-Assisted Laser Desorption/Ionization and Desorption Electrospray Ionization Mass Spectrometry Images

We present omniSpect, an open source web- and MATLAB-based software tool for both desorption electrospray ionization (DESI) and matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI) that performs computationally intensive functions on a remote server. These functions include converting data from a variety of file formats into a common format easily manipulated in MATLAB, transforming time-series mass spectra into mass spectrometry images based on a probe spatial raster path, and multivariate analysis. OmniSpect provides an extensible suite of tools to meet the computational requirements needed for visualizing open and proprietary format MSI data.      

三氟乙酸增强的解吸电喷雾电离质谱成像用于脂质空间分布研究

将三氟乙酸添加到解吸电喷雾电离质谱的喷雾溶剂中,提高了对脂质的成像检测能力,使小鼠脑组织切片中能检测到的脂质种类从59种提高到71种.此外,该方法还可以促进脂质的质子化峰的形成并抑制钠和钾加合物的形成,有利于简化谱图解析过程并实现更好的定量分析.利用该方法实现了小鼠脑组织切片上多种脂质的空间相关性研究,为探索脂质间潜在的代谢途径提供了新手段,在神经学、临床等领域具有巨大的应用前景.     

电喷雾解吸电离质谱法直接测定蔬菜表面的莠去津残留

莠去津为一种选择性的高效除草剂,长期以来广泛用于我国东北和华北玉米、高粱、果园地除草.     

Comparing the Effects of Additives on Protein Analysis Between Desorption Electrospray (DESI) and Electrospray Ionization (ESI)

It is frequently said that DESI-MS follows a similar ionization mechanism as ESI because of similarities usually observed in their respective mass spectra. However, practical use of DESI-MS for protein analysis is limited to proteins with lower molecular weights (<?25 kDa) due to a mass-dependent loss in signal intensity. Here we investigated commonly used volatile acids and their ammonium salt buffers for DESI-MS analysis of protein. We noticed that, surprisingly, some additives influence the analysis differently in DESI compared to ESI. Improved signal intensities with both DESI and ESI were obtained when acetic and formic acid were added into aqueous methanol spray solvents with both DESI and ESI. On the other hand, while with ESI the addition of ammonium salts into spray solutions strongly reduced both signal and S/N, with DESI signal intensities and S/N were improved dramatically. Ammonium bicarbonate when used with DESI reduced the total amount of adduction and delivered excellent signal-to-noise ratios with high intensity; however, it also denatures protein. When native state protein mass spectra are preferred, ammonium acetate would also deliver reasonable adduct removal and improved S/N. The amount of total adduction of individual adducting species and of all species could not be correlated with differences in either solutions pH values or with proton affinities of the anions. An obvious difference between DESI and ESI mass spectrometry is the effects of protein solubility during droplet pickup (desorption), but differences in the sizes, velocities, and composition of ionizing droplets were also discussed as important factors.

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The Effect of Organic Modifiers on Electrospray Ionization Charge-State Distribution and Desorption Efficiency for Oligonucleotides

The chemical composition of the solution has a critical impact on the electrospray desorption efficiency of oligonucleotides. Several physiochemical properties of various organic modifiers were investigated with respect to their role in the desorption process of oligonucleotides. The Henry’s Law Constant, which reflects the volatility of alkylamines, was found to have a prominent effect on both the electrospray charge state distribution and desorption efficiency of oligonucleotides. Alkylamines with higher $ \mathrm{k}_{\mathrm{H,cc}}\left( {\mathrm{aq}/\mathrm{gas}} \right) $ values such as hexylamine, piperidine, and imidazole reduced the charge state distribution by forming complexes with the oligonucleotide and dissociating from it in the gas phase, while alkylamines with extremely low $ \mathrm{k}_{\mathrm{H,cc}}\left( {\mathrm{aq}/\mathrm{gas}} \right) $ values reduced the electrospray charge state distribution by facilitating ion emission at an earlier stage of the electrospray desorption process. Ion-pairing agents with moderate $ \mathrm{k}_{\mathrm{H,cc}}\left( {\mathrm{aq}/\mathrm{gas}} \right) $ values do not alter the electrospray charge state distribution of oligonucleotides and their ability to enhance oligonucleotide ionization followed the order of decreasing $ \mathrm{k}_{\mathrm{H,cc}}\left( {\mathrm{aq}/\mathrm{gas}} \right) $ values. The Henry’s Law Constant also correlated to the impact of the acidic modifiers on oligonucleotide ionization efficiency. Ionization enhancement effects were observed with hexafluoroisopropanol, and this effect was attributed to its low $ \mathrm{k}_{\mathrm{H,cc}}\left( {\mathrm{aq}/\mathrm{gas}} \right) $ and moderate acidity. The comprehensive effects of both alkylamine and hexafluoroisoproapnol on the electrospray ionization desorption of oligonucleotides were also evaluated, and acid-base equilibrium was found to play a critical role in determining these effects.      

Ambient Mass Spectrometry Imaging: A Comparison of Desorption Ionization by Sonic Spray and Electrospray

Easy ambient sonic spray ionization (EASI) and desorption electrospray ionization (DESI) were used for imaging of a number of samples, including sections of rat brain and imprints of plant material on porous Teflon. A novel approach termed Displaced Dual-mode Imaging was utilized for the direct comparison of the two methods: Images were recorded with the individual rows alternating between EASI and DESI, yielding a separate image for each technique recorded under perfectly similar conditions on the same sample. EASI works reliably for imaging of all samples, but the choice of spray solvent and flow rate is more critical in tissue imaging with EASI than with DESI. The overall sensitivity of EASI is, in general, slightly lower than that of DESI, and the representation of the dynamic range is different in images of the two techniques for some samples. However, for abundant compounds, EASI works well, resulting in images of similar quality as DESI. EASI can thus be used in imaging experiments where the application of high voltage is impractical or undesirable. The present study is in its nature also a comparison of the characteristics of the two techniques, showing results also applicable for non-imaging work, with regards to sensitivity and experimental conditions.