适用小分子化合物MALDI分析的基质研究

基质辅助激光解吸电离技术（matrix assisted laser desorption/ionization,MALDI）是20世纪80年代发展起来的一种应用于质谱分析的电离化技术。MALDI技术在生物大分子的分析和检测方面获得了良好的应用。由于受有机基质分子的干扰,MALDI在小分子化合物分析方面的应用受到很大的限制。近年来为解决这一问题,一些用于MALDI分析的新型材料被设计和开发出来。这些新型材料主要包括：碳、硅、纳米金属等无机材料和新型有机分子等。除此之外,在传统基质中添加表面活性剂和对分析物衍生化等方法也被成功应用于小分子化合物的MALDI质谱分析中。本文对这些可应用于小分子化合物分析的新型MALDI基质进行了综述和展望。     

质谱分子成像技术与应用进展

该文总结了二次离子质谱、基质辅助激光解吸电离质谱和常压敞开式离子化质谱三大类型质谱分子成像（MSI）技术的概况、技术与方法及其应用新进展。MSI技术作为免标记、高覆盖、高灵敏、检测范围广的可视化分析手段,不局限于生物组织或细胞中某种特定分子的检测,可对已知和未知多种分子进行同时成像分析,获得不同分子的空间分布、相对含量及结构信息,实现其分子的定性、定量与定位分析;还可提供不同生理及病理过程中功能分子的动态时空变化信息等。因此,MSI技术成为质谱领域以及分析化学等领域的研究前沿与热点方向之一,并在化学、医学、生命科学、药学和环境科学等领域显示出重大应用前景。此外,MSI技术是单细胞可视化分析和空间分辨代谢组学的强有力分析手段,可从动物或器官组织的整体、微区、单细胞等不同空间尺度,获取具有空间分布特征、时空动态变化的功能分子全景轮廓信息等而备受关注。     

蛋白质组学中基质辅助激光解吸电离的基质研究进展

基质辅助激光解吸电离(MALDI)技术是近年来发展起来的新的质谱离子化技术。本文较为系统地综述了应用于蛋白质组学中的MALDI基质的最新进展以及不同的基质的优缺点及应用范围,并且归纳了其发展趋势。     

基质辅助激光解吸电离质谱分析糖类物质

基质辅助激光解吸电离质谱（MALDI-MS）是一种样品无需衍生、图谱解析简单、灵敏度高、快速便捷的分析生物样品结构的方法,已被广泛用于糖类物质的结构分析。此技术与HPLC、糖苷酶外切技术以及各种串联质谱等技术结合使用,可给出糖类物质详细的结构信息。本文介绍了基质辅助激光解吸（MALDI）离子化技术的原理、特点、与飞行时间质量分析器（TOF）联用时的相关技术和裂解方式,以及MALDI-MS在分析糖类物质时选用的基质、样品的制备、糖链碎片分析的方法和在不同糖型分析中的应用,展示了它的发展前景。随着MALDI对糖类物质分析时基质的改进、质谱分辨率的提高、质量检测范围的扩大,MALDI-MS技术必将成为糖类物质分析中强有力的工具。     

脂质组学分析中样品前处理技术的研究进展

脂质不仅是细胞膜的主要组成部分,还参与一些生命活动如能量存储、信号传导等,在生命体中发挥着重要作用。近年来,越来越多的研究表明脂质的变化与一些重大疾病的发生发展密切相关,脂质组学研究对理解疾病的发生机制及过程具有重要意义。在脂质分析过程中,由于样品基质的干扰或被分析物浓度的限制,通常需要对样品进行前处理,以得到最佳的分析性能。该文综述了脂质组学分析中的样品前处理技术,包括脂质的提取方法（如液液萃取、固相萃取等）和针对不同类脂质的化学衍生化技术在各领域,尤其是生命分析和代谢组学中的应用,并对脂质组学分析中的样品前处理技术的发展进行了展望。     

用MALDI-TOF-MS法表征系列环状预聚体

应用基质辅助激光解吸电离飞行时间质谱（MALDI－TOF－MS）技术对系列环状预聚体进行了表征,分别确定了系列环状预聚体各自不同的聚合度,同时对它们的结构进行了确认,获得了满意的结果。实验结果表明MALDI－TOF－MS是分析环状预聚体准确、快速工具之一。     

基质辅助激光解吸离子化质谱及其在生物大分子分析中的应用

近年来质谱离子化技术方面有两项重要成果:基质辅助激光解吸离子化(matrix-assisted laser desorptionionization,MALDI)和电喷雾离子化(electrospray ionization,ESI)。MALDI和ESI的应用使质谱在生物大分子研究方面取得重大突破。本文仅就MALDI的原理、特点、样品准备方法、基质的选择、仪器条件及其在生物大分子应用方面的最新进展进行简要的综述。     

基于衍生化技术的甘油磷脂分析方法研究进展

磷脂是所有生物细胞膜的主要成分,在许多生命活动过程中具有重要的功能。但由于生物样本中的磷脂种类繁多,含量极低,存在基质抑制效应,且结构中缺少易电离的官能团,从而导致对磷脂的定性和定量分析较困难。利用化学衍生化技术对其进行结构修饰可以提高离子化效率、改善色谱分离度且提高质谱(MS)检测的灵敏度和选择性。MS与衍生化方法结合已被广泛用于蛋白组学、糖组学、代谢物等的分析。近年来,这一策略逐渐被应用于脂质组学的分析研究。该文综述了国内外近10年基于衍生化技术的甘油磷脂分析方法及其应用研究进展,以激发衍生化技术在脂质组学分析中的应用潜能。     

脂质组学及其分析方法

脂质组学的研究属于生命科学的范畴,与人类的健康密切相关。目前,脂质组学已成为代谢组学最重要的分支之一,且是一个非常活跃的研究领域,尤其在研究疾病方面的重要性已经引起了科学界的广泛关注。该文简要介绍了脂质组学的研究内容,重点评述了脂质组学分析方法,包括样品处理、轮廓分析、目标分析、成像分析以及数据处理。最后提出了脂质组学分析技术和方法的展望。     

MALDI质谱技术在酶活性分析中的应用

酶作为生物催化剂参与很多重要的生理过程,同时也是一类重要的生物分子。酶的活性分析对于疾病诊断和治疗具有重要意义。基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)具有操作简单、分析速度快、灵敏度高和易于实现高通量分析的特点,已被广泛用于各种组学研究和生物分子的检测,在酶的检测和活性分析中亦发挥了重要作用。该文综述了国内外利用MALDI-TOF MS分析酶活性和进行药物筛选的策略,总结了各种方法的优缺点,提出了MALDI质谱技术在酶活性分析领域存在的问题和挑战,并对其发展前景进行了展望。     

Matrix-free mass spectrometric imaging using laser desorption ionisation Fourier transform ion cyclotron resonance mass spectrometry

Mass spectrometry imaging (MSI) is a powerful tool in metabolomics and proteomics for the spatial localization and identification of pharmaceuticals, metabolites, lipids, peptides and proteins in biological tissues. However, sample preparation remains a crucial variable in obtaining the most accurate distributions. Common washing steps used to remove salts, and solvent-based matrix application, allow analyte spreading to occur. Solvent-free matrix applications can reduce this risk, but increase the possibility of ionisation bias due to matrix adhesion to tissue sections. We report here the use of matrix-free MSI using laser desorption ionisation performed on a 12 T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. We used unprocessed tissue with no post-processing following thaw-mounting on matrix-assisted laser desorption ionisation (MALDI) indium-tin oxide (ITO) target plates. The identification and distribution of a range of phospholipids in mouse brain and kidney sections are presented and compared with previously published MALDI time-of-flight (TOF) MSI distributions.     

质谱成像技术及其在乳腺癌研究中的应用

乳腺癌是女性最常见的恶性肿瘤,其发病率在世界范围内呈现上升趋势,是威胁女性健康的重要疾病之一。随着现代医学技术的快速发展,早期有效的诊断和筛查方法能够改善乳腺癌患者生存率和提高其生活质量。由于乳腺癌肿瘤具有非常显著的异质性,这对于诊断和筛查带来了较大困难,亟须在肿瘤演进时间信息中,继续引入生物分子的空间信息,从而对其异质性、肿瘤微环境等进行准确的追踪。质谱成像技术,可在免标记的前提下利用离子质荷比的特性发现生物组织中的各种分子,并研究这些分子的时间和空间信息,对其进行准确的定性、定量和空间定位。目前,通过质谱成像技术可直接获取药物及其代谢物、内源性代谢物、脂质、多肽和蛋白质等在组织中的空间分布信息,为肿瘤分子分型诊断和确认以及相关抗肿瘤药物的筛选提供了新的思路和研究方向。该综述以乳腺癌相关的生物样品制备和研究进展为主要内容,从小分子样本、大分子样本、石蜡包埋样本、基质喷涂方式、常用离子源等方面阐述质谱成像中样本制备的重要性以及样品制备过程中存在的难点问题。同时,以细胞模型、动物模型和临床肿瘤标本为研究对象,汇总了质谱成像技术在乳腺癌方面的应用进展,并进行了展望,为开展癌症精准分型研究和药物药效的快速筛查提供了重要依据。     

In situ isobaric lipid mapping by MALDI–ion mobility separation–mass spectrometry imaging

The highly diverse chemical structures of lipids make their analysis directly from biological tissue sections extremely challenging. Here, we report the in situ mapping and identification of lipids in a freshwater crustacean Gammarus fossarum using matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) in combination with an additional separation dimension using ion mobility spectrometry (IMS). The high‐resolution trapped ion mobility spectrometry (TIMS) allowed efficient separation of isobaric/isomeric lipids showing distinct spatial distributions. The structures of the lipids were further characterized by MS/MS analysis. It is demonstrated that MALDI MSI with mobility separation is a powerful tool for distinguishing and localizing isobaric/isomeric lipids.     

Novel approach to enhance sensitivity in surface‐assisted laser desorption/ionization mass spectrometry imaging using deposited organic‐inorganic hybrid matrices

Sample pretreatment is key to obtaining good data in matrix‐assisted laser desorption/ionization mass spectrometry imaging (MALDI‐MSI). Although sublimation is one of the best methods for obtaining homogenously fine organic matrix crystals, its sensitivity can be low due to the lack of a solvent extraction effect. We investigated the effect of incorporating a thin film of metal formed by zirconium (Zr) sputtering into the sublimation process for MALDI matrix deposition for improving the detection sensitivity in mouse liver tissue sections treated with olanzapine. The matrix‐enhanced surface‐assisted laser desorption/ionization (ME‐SALDI) method, where a matrix was formed by sputtering Zr to form a thin nanoparticle layer before depositing MALDI organic matrix comprising α‐cyano‐4‐hydroxycinnamic acid (CHCA) by sublimation, resulted in a significant improvement in sensitivity, with the ion intensity of olanzapine being about 1800 times that observed using the MALDI method, comprising CHCA sublimation alone. When Zr sputtering was performed after CHCA deposition, however, no such enhancement in sensitivity was observed. The enhanced sensitivity due to Zr sputtering was also observed when the CHCA solution was applied by spraying, being about twice as high as that observed by CHCA spraying alone. In addition, the detection sensitivity of these various pretreatment methods was similar for endogenous glutathione. Given that sample preparation using the ME‐SALDI‐MSI method, which combines Zr sputtering with the sublimation method for depositing an organic matrix, does not involve a solvent, delocalization problems such as migration of analytes observed after matrix spraying and washing with aqueous solutions as sample pretreatment are not expected. Therefore, ME‐Zr‐SALDI‐MSI is a novel sample pretreatment method that can improve the sensitivity of analytes while maintaining high spatial resolution in MALDI‐MSI.     

Uncovering matrix effects on lipid analyses in MALDI imaging mass spectrometry experiments

The specific matrix used in matrix‐assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) can have an effect on the molecules ionized from a tissue sample. The sensitivity for distinct classes of biomolecules can vary when employing different MALDI matrices. Here, we compare the intensities of various lipid subclasses measured by Fourier transform ion cyclotron resonance (FT‐ICR) IMS of murine liver tissue when using 9‐aminoacridine (9AA), 5‐chloro‐2‐mercaptobenzothiazole (CMBT), 1,5‐diaminonaphthalene (DAN), 2,5‐Dihydroxyacetophenone (DHA), and 2,5‐dihydroxybenzoic acid (DHB). Principal component analysis and receiver operating characteristic curve analysis revealed significant matrix effects on the relative signal intensities observed for different lipid subclasses and adducts. Comparison of spectral profiles and quantitative assessment of the number and intensity of species from each lipid subclass showed that each matrix produces unique lipid signals. In positive ion mode, matrix application methods played a role in the MALDI analysis for different cationic species. Comparisons of different methods for the application of DHA showed a significant increase in the intensity of sodiated and potassiated analytes when using an aerosol sprayer. In negative ion mode, lipid profiles generated using DAN were significantly different than all other matrices tested. This difference was found to be driven by modification of phosphatidylcholines during ionization that enables them to be detected in negative ion mode. These modified phosphatidylcholines are isomeric with common phosphatidylethanolamines confounding MALDI IMS analysis when using DAN. These results show an experimental basis of MALDI analyses when analyzing lipids from tissue and allow for more informed selection of MALDI matrices when performing lipid IMS experiments.     

A Caged In-Source Laser-Cleavable MALDI Matrix with High Vacuum Stability for Extended MALDI-MS Imaging

Insufficient vacuum stability of matrix chemicals is a major limitation in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) of large tissue sample cohorts. Here, we designed and synthesized the photo-cleavable caged molecule 4,5-dimethoxy-2-nitrobenzyl-2,5-dihydroxyacetophenone (DMNB-2,5-DHAP) and employed it for lipid MALDI-MSI of mouse brain tissue sections. DMNB-2,5-DHAP is vacuum-stable in a high vacuum MALDI ion source for at least 72 h. Investigation of the uncaging process suggested that the built-in laser (355 nm) in the MALDI ion source promoted the in situ generation of 2,5-DHAP. A caging group is used for the first time in designing a MALDI matrix that is vacuum-stable, uncaged upon laser irradiation during the measurement process, and that boosts lipid ion intensity with MALDI-2 laser-induced postionization.     

Repeat MALDI MS imaging of a single tissue section using multiple matrices and tissue washes

Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) techniques are continually being assessed with a view to improving the quality of information obtained from a given sample. A single tissue section will typically only be analyzed once by MALDI MSI and is then either used for histological staining or discarded. In this study, we explore the idea of repeat analysis of a single tissue section by MALDI MSI as a route toward improving sensitivity, structural characterization, and diversity of detected analyte classes. Repeat analysis of a single tissue section from a fresh frozen mouse brain is investigated with both α-cyano-4-hydroxycinnamic acid (CHCA) and para-nitroaniline (PNA). Repeat analysis is then applied to the acquisition of MALDI MSI and MALDI tandem mass spectrometry imaging employing collision induced dissociation (MS/MS imaging employing CID) from a formalin-fixed mouse brain section. Finally, both lipid and protein data are acquired from the same tissue section via repeat analysis utilizing CHCA, sinapinic acid (SA), and a tissue wash step. PNA was found to outperform CHCA as a matrix for repeat analysis; multiple lipids were identified using MS/MS imaging; both lipid and protein images were successfully acquired from a single tissue section.

A solvent‐free matrix application method for matrix‐assisted laser desorption/ionization imaging of small molecules

Matrix application continues to be a critical step in sample preparation for matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI). Imaging of small molecules such as drugs and metabolites is particularly problematic because the commonly used washing steps to remove salts are usually omitted as they may also remove the analyte, and analyte spreading is more likely with conventional wet matrix application methods. We have developed a method which uses the application of matrix as a dry, finely divided powder, here referred to as dry matrix application, for the imaging of drug compounds. This appears to offer a complementary method to wet matrix application for the MALDI‐MSI of small molecules, with the alternative matrix application techniques producing different ion profiles, and allows the visualization of compounds not observed using wet matrix application methods. We demonstrate its value in imaging clozapine from rat kidney and 4‐bromophenyl‐1,4‐diazabicyclo(3.2.2)nonane‐4‐carboxylic acid from rat brain. In addition, exposure of the dry matrix coated sample to a saturated moist atmosphere appears to enhance the visualization of a different set of molecules. Copyright © 2010 John Wiley & Sons, Ltd.     

Mass spectrometry imaging of triglycerides in biological tissues by laser desorption ionization from silicon nanopost arrays

Mass spectrometry imaging (MSI) is used increasingly to simultaneously detect a broad range of biomolecules while mapping their spatial distributions within biological tissue sections. Matrix‐assisted laser desorption ionization (MALDI) is recognized as the method‐of‐choice for MSI applications due in part to its broad molecular coverage. In spite of the remarkable advantages offered by MALDI, imaging of neutral lipids, such as triglycerides (TGs), from tissue has remained a significant challenge due to ion suppression of TGs by phospholipids, e.g. phosphatidylcholines (PCs). To help overcome this limitation, silicon nanopost array (NAPA) substrates were introduced to selectively ionize TGs from biological tissue sections. This matrix‐free laser desorption ionization (LDI) platform was previously shown to provide enhanced ionization of certain lipid classes, such as hexosylceramides (HexCers) and phosphatidylethanolamines (PEs) from mouse brain tissue. In this work, we present NAPA as an MSI platform offering enhanced ionization efficiency for TGs from biological tissues relative to MALDI, allowing it to serve as a complement to MALDI‐MSI. Analysis of a standard lipid mixture containing PC(18:1/18:1) and TG(16:0/16:0/16:0) by LDI from NAPA provided an ~49 and ~227‐fold higher signal for TG(16:0/16:0/16:0) relative to MALDI, when analyzed without and with the addition of a sodium acetate, respectively. In contrast, MALDI provided an ~757 and ~295‐fold higher signal for PC(18:1/18:1) compared with NAPA, without and with additional Na⁺. Averaged signal intensities for TGs from MSI of mouse lung and human skin tissues exhibited an ~105 and ~49‐fold increase, respectively, with LDI from NAPA compared with MALDI. With respect to PCs, MALDI provided an ~2 and ~19‐fold increase in signal intensity for mouse lung and human skin tissues, respectively, when compared with NAPA. The complementary coverage obtained by the two platforms demonstrates the utility of using both techniques to maximize the information obtained from lipid MS or MSI experiments.     

Imaging of lipids in rat heart by MALDI-MS with silver nanoparticles

Lipids are a major component of heart tissue and perform several important functions such as energy storage, signaling, and as building blocks of biological membranes. The heart lipidome is quite diverse consisting of glycerophospholipids such as phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), phosphatidylinositols (PIs), phosphatidylglycerols (PGs), cardiolipins (CLs), and glycerolipids, mainly triacylglycerols (TAGs). In this study, mass spectrometry imaging (MSI) enabled by matrix implantation of ionized silver nanoparticles (AgNP) was used to map several classes of lipids in heart tissue. The use of AgNP matrix implantation was motivated by our previous work showing that implantation doses of only 10¹⁴/cm² of 2 nm gold nanoparticulates into the first 10 nm of the near surface of the tissue enabled detection of most brain lipids (including neutral lipid species such as cerebrosides) more efficiently than traditional organic MALDI matrices. Herein, a similar implantation of 500 eV AgNP^? across the entire heart tissue section results in a quick, reproducible, solvent-free, uniform matrix concentration of 6 nm AgNP residing near the tissue surface. MALDI-MSI analysis of either positive or negative ions produce high-quality images of several heart lipid species. In negative ion mode, 24 lipid species [16 PEs, 4 PIs, 1 PG, 1 CL, 2 sphingomyelins (SMs)] were imaged. Positive ion images were also obtained from 29 lipid species (10 PCs, 5 PEs, 5 SMs, 9 TAGs) with the TAG species being heavily concentrated in vascular regions of the heart.