Paternò-Büchi(PB)反应与串联质谱结合实现不饱和脂质精确结构解析

脂质在一系列关键生命过程中发挥着重要作用。结构决定功能,因此脂质结构解析与功能研究一直受到研究者的关注。尽管质谱已成为脂质分析的最常用技术之一,但脂质的精细结构解析仍面临诸多挑战。为解决上述问题,将Paternò-Büchi(PB)光化学反应与串联质谱相结合,实现了不饱和脂质中碳碳双键■的定位以及不饱和脂质■位置异构体的定量分析。此外,该技术可与鸟枪法、液相色谱-质谱和质谱成像等技术联用,用于复杂生物样品中脂质的分析。     

常压电离质谱在脂质精确结构解析中的研究进展

脂类化合物的结构和功能与细胞生理过程密切相关,脂质结构的精确解析对探索脂质的生物学功能起着至关重要的作用。常压电离质谱技术的出现为常压敞开环境条件下的原位、实时、直接、快速生物脂质分析和组学研究提供了重要的手段。随着常压电离质谱技术在脂质分析和脂质组学研究的不断深入,对脂质结构特别是碳碳双键(C‖C)位置的精细解析就显得尤为重要。该文详细阐述了基于Paternò-Büchi反应、环氧化、臭氧诱导解离、紫外光解离的常压电离质谱技术,以及这些技术在脂质C‖C位置精确鉴定及异构体区分的应用,并展望了常压电离质谱在脂质化合物精确结构解析方面的发展趋势。     

十二碳单烯-1-乙酸酯双键位置异构体的质谱研究

本文报道了十二碳单烯-1-乙酸酯双键位置异构体质谱的特征,应用质量分析离子动能谱(MIKES)研究离子的断裂机理,结果表明: 支配此类化合物基本断裂方式的主要是游离基中心引发的各种类型H的重排,双键位移和电荷转移, 离子在断裂前已形成结构上能快速相互转化的异构化离子的混合物, 因此这类双键位置异构体的质谱非常相似.     

化学计量学方法在脂质组学数据解析中的应用

脂质组学是依赖于分析技术而发展的一门新兴学科,用于全面表征与基因调控、蛋白表达、脂质代谢密切相关的脂质分子,揭示脂质在各种生命活动中的作用机制和代谢途径网络。随着质谱及其联用技术进一步发展和完善,脂质组学逐渐向快速、自动化和高通量的方向发展,而大规模的脂质组数据分析已成为脂质组学研究领域的一大难点。化学计量学主要应用于脂质组学中的基线校正和背景扣除、信号峰识别、同位素分布解析、统计分析等过程,因此,基于化学计量学方法的脂质组学数据自动化解析策略成为研究者关心的热点。该文对近年来化学计量学在脂质组学数据解析中的应用进行了综述,并对基于化学计量学的脂质组学数据解析的未来发展进行了展望。     

甾醇TMS衍生物EI源质谱规律研究

甾醇是广泛存在于动植物界的一类重要天然产物.海洋微藻中C3羟基甾醇的结构多样主要体现在4位有无甲基存在,甾核上的双键数目和位置,侧链上的双键位置及侧链上烷基化程度和位置.早在60年代就有关于甾醇乙酸酯质谱特殊碎片的报道[1-4].     

化学修饰在质谱中的应用 I:长链不饱和脂肪酸的双键定位: 2-烯基取代杂环化合物的质谱

作者提出了一种用"远端基团修饰法"来确定双键位置的新途径.长链不饱和脂肪酸环化为相应的2-烯基杂环化合物后,这些杂环母核的强大的正荷定域作用使产物的质谱的结构专一性大为提高,并能直接提供有关双键位置的信息.本文报道了微量衍生化方法与八类代表性杂环衍生物与四种苯并唑衍生物的质谱比较.     

化学修饰在质谱中的应用 Ⅰ.长链不饱和脂肪酸的双键定位——2-烯基取代杂环化合物的质谱

作者提出了一种用“远端基团修饰法”来确定双键位置的新途径.长链不饱和脂肪酸环化为相应的2-烯基杂环化合物后,这些杂环母核的强大的正荷定域作用使产物的质谱的结构专一性大为提高,并能直接提供有关双键位置的信息.本文报道了微量衍生化方法与八类代表性杂环衍生物与四种苯并噁唑衍生物的质谱比较.     

功能脂质组质谱分析

近年来,鉴于脂质在疾病发生发展过程中的重要作用,功能脂质组研究受到广泛关注.质谱技术是功能脂质组分析的主要手段,本文将介绍用于功能脂质组分析的质谱技术,概述国内功能脂质组质谱分析的研究进展,并提出问题和展望,以期可以在现有方法上进行改进提高,用以发现更多的功能脂质,进而用于发展疾病生物标志物及治疗靶标以及研究疾病机理等,并期待推进功能脂质组学研究的发展.     

相似分析/质谱法鉴别二十种脂肪族双烯-1-醇及乙酸酯

本文报道了二十个脂肪族双烯-1-醇和乙酸酯EI-质谱的特征, 并提出了相似分析法鉴别这些共轭双烯、孤立双烯的双键位置及几何异构体的质谱。     

离子迁移质谱技术及其在脂质分析中的应用

脂质在细胞和生物组织中发挥着重要作用,传统的脂质组学分析方法虽可以实现脂质鉴定,但脂质结构的多样性和相似性仍为脂质组学研究中全脂质分析提出了巨大的挑战。离子迁移谱是根据离子在缓冲气体或电力场中的迁移特性对其进行分离的技术,将离子迁移谱与传统脂质分析方法联用不仅有助于复杂脂质的分离,同时还可对脂质异构体进行分析鉴定,提高脂质分析的分辨率和选择性。该文主要对不同离子迁移质谱技术及其在脂质分析中的应用进行综述,并对其未来发展方向进行了展望。     

Electron‐induced dissociation (EID) for structure characterization of glycerophosphatidylcholine: determination of double‐bond positions and localization of acyl chains

Glycerophospholipids are a highly abundant and diverse collection of biologically relevant lipids, and distinction between isomeric and isobaric species is a fundamental aspect for confident identification. The ability to confidently assign a unique structure to a glycerophospholipid of interest is dependent on determining the number and location of the points of unsaturation and assignment of acyl chain position. The use of high‐energy electrons (>20 eV) to induce gas‐phase dissociation of intact precursor ions results in diagnostic product ions for localizing double‐bond positions and determining acyl chain assignment. We describe a high‐resolution, tandem mass spectrometry method for structure characterization of glycerophospholipids using electron‐induced dissociation (EID). Furthermore, the inclusion of nomenclature to systematically assign bond cleavage sites with acyl chain position and double‐bond location enables a uniform platform for lipid identification. The EID methodology detailed here combines novel application of an electron‐based dissociation technique with high‐resolution mass spectrometry that facilitates a new experimental approach for lipid biomarker discovery and validation. Copyright © 2015 John Wiley & Sons, Ltd.     

On‐Demand Electrochemical Epoxidation in Nano‐Electrospray Ionization Mass Spectrometry to Locate Carbon–Carbon Double Bonds

Reported here is the first on‐demand electrochemical epoxidation incorporated into the standard nano‐electrospray ionization mass spectrometry (nanoESI‐MS) workflow for double‐bond identification. The capability lies in a novel tunable electro‐epoxidation of double bonds, where onset of the reaction can be controlled by simply tuning the spray voltage. On‐demand formation of mono‐/multiple epoxides is achieved at different voltages. The electro‐epoxidized products are then fragmented by tandem MS to generate diagnostic ions, indicating the double bond position(s). The process is completed within seconds, holding great potential for high‐throughput analysis. The rapid switch‐on/off electro‐epoxidation of a single sample, the low sample consumption, the demonstrated applicability to complex lipids containing multiple double bonds, and the advantage of not requiring extra apparatus make this method attractive for use in lipid‐related biological studies.     

Using ambient ozone for assignment of double bond position in unsaturated lipids

Unsaturated lipids deposited onto a range of materials are observed to react with the low concentrations of ozone present in normal laboratory air. Parent lipids and ozonolysis cleavage products are both detected directly from surfaces by desorption electrospray ionisation mass spectrometry (DESI-MS) with the resulting mass spectra providing clear evidence of the double bond position within these molecules. This serendipitous process has been coupled with thin-layer chromatography (TLC) to provide a simple but powerful approach for the detailed structural elucidation of lipids present in complex biological extracts. Lipid extracts from human lens were deposited onto normal phase TLC plates and then developed to separate components according to lipid class. Exposure of the developed plates to laboratory air for ca. 1 h prior to DESI-MS analysis gave rise to ozonolysis products allowing for the unambiguous identification of double bond positions in even low abundant, unsaturated lipids. In particular, the co-localization of intact unsaturated lactosylceramides (LacCer) with products from their oxidative cleavage provide the first evidence for the presence of three isomeric LacCer (d18:0/24:1) species in the ocular lens lipidome, i.e., variants with double bonds at the n-9, n-7 and n-5 positions.     

Versatile lipid profiling by liquid chromatography–high resolution mass spectrometry using all ion fragmentation and polarity switching. Preliminary application for serum samples phenotyping related to canine mammary cancer

Lipids represent an extended class of substances characterized by such high variety and complexity that makes their unified analyses by liquid chromatography coupled to either high resolution or tandem mass spectrometry (LC–HRMS or LC–MS/MS) a real challenge. In the present study, a new versatile methodology associating ultra high performance liquid chromatography coupled to high resolution tandem mass spectrometry (UHPLC–HRMS/MS) have been developed for a comprehensive analysis of lipids. The use of polarity switching and “all ion fragmentation” (AIF) have been two action levels particularly exploited to finally permit the detection and identification of a multi-class and multi-analyte extended range of lipids in a single run. For identification purposes, both higher energy collision dissociation (HCD) and in-source CID (collision induced dissociation) fragmentation were evaluated in order to obtain information about the precursor and product ions in the same spectra. This approach provides both class-specific and lipid-specific fragments, enhancing lipid identification. Finally, the developed method was applied for differential phenotyping of serum samples collected from pet dogs developing spontaneous malignant mammary tumors and health controls. A biological signature associated with the presence of cancer was then successfully revealed from this lipidome analysis, which required to be further investigated and confirmed at larger scale.     

Localization of cyclopropyl groups and alkenes within glycerophospholipids using gas-phase ion/ion chemistry

Shotgun lipid analysis using electrospray ionization tandem mass spectrometry (ESI-MS/MS) is a common approach for the identification and characterization of glycerophohspholipids GPs. ESI-MS/MS, with the aid of collision-induced dissociation (CID), enables the characterization of GP species at the headgroup and fatty acyl sum compositional levels. However, important structural features that are often present, such as carbon–carbon double bond(s) and cyclopropane ring(s), can be difficult to determine. Here, we report the use of gas-phase charge inversion reactions that, in combination with CID, allow for more detailed structural elucidation of GPs. CID of a singly deprotonated GP, [GP − H]⁻, generates FA anions, [FA − H]⁻. The fatty acid anions can then react with doubly charged cationic magnesium tris-phenanthroline complex, [Mg(Phen)₃]²⁺, to form charge inverted complex cations of the form [FA − H + MgPhen₂]⁺. CID of the complex generates product ion spectral patterns that allow for the identification of carbon–carbon double bond position(s) as well as the sites of cyclopropyl position(s) in unsaturated lipids. This approach to determining both double bond and cyclopropane positions is demonstrated with GPs for the first time using standards and is applied to lipids extracted from Escherichia coli.     

On-Demand Electrochemical Epoxidation in Nano-Electrospray Ionization Mass Spectrometry to Locate Carbon–Carbon Double Bonds

Reported here is the first on-demand electrochemical epoxidation incorporated into the standard nano-electrospray ionization mass spectrometry (nanoESI-MS) workflow for double-bond identification. The capability lies in a novel tunable electro-epoxidation of double bonds, where onset of the reaction can be controlled by simply tuning the spray voltage. On-demand formation of mono-/multiple epoxides is achieved at different voltages. The electro-epoxidized products are then fragmented by tandem MS to generate diagnostic ions, indicating the double bond position(s). The process is completed within seconds, holding great potential for high-throughput analysis. The rapid switch-on/off electro-epoxidation of a single sample, the low sample consumption, the demonstrated applicability to complex lipids containing multiple double bonds, and the advantage of not requiring extra apparatus make this method attractive for use in lipid-related biological studies.     

Pinpointing Double Bonds in Lipids by Paternò‐Büchi Reactions and Mass Spectrometry

The positions of double bonds in lipids play critical roles in their biochemical and biophysical properties. In this study, by coupling Paternò–Büchi (P‐B) reaction with tandem mass spectrometry, we developed a novel method that can achieve confident, fast, and sensitive determination of double bond locations within various types of lipids. The P‐B reaction is facilitated by UV irradiation of a nanoelectrospray plume entraining lipids and acetone. Tandem mass spectrometry of the on‐line reaction products via collision activation leads to the rupture of oxetane rings and the formation of diagnostic ions specific to the double bond location.     

Phenyl- and cyclopentylimino derivatization for double bond location in unsaturated C<Subscript>37</Subscript>-C<Subscript>40</Subscript> alkenones by GC-MS

A method for the identification of double bond locations in polyunsaturated long chain alkenones adapted to nanogram amounts as currently analyzed by gas chromatography coupled to mass spectrometry (GC-MS) has been developed. The method is based on interpretation of the electron impact mass spectra of the imino derivatives of the carbonyl groups using either cyclopentyl or phenyl substitutents. Other complementary derivatization methods such as elaidization and hydrogenation have also been used for structural characterization of these compounds. This application has led to the identification of a novel homologous series of di-, tri-, and tetraunsaturated ketones with carbon number chain lengths between 37 and 40 in coastal hypersaline sediments. The novel series identified shows a distribution in which the double bond position between different homologs is established by reference to the distance from the carbonyl group whereas the previously known alkenones were constituted by unsaturated homologs with double bonds located at defined distances of the terminal methyl. This difference points out to a dissimilar, but still unknown, biogenic precursor of these novel alkenones.     

Analysis of cancer cell lipids using matrix‐assisted laser desorption/ionization 15‐T Fourier transform ion cyclotron resonance mass spectrometry

A combination of methodologies using the extremely high mass accuracy and resolution of 15‐T Fourier transform ion cyclotron resonance (FT‐ICR) mass spectrometry (MS) was introduced for the identification of intact cancer cell phospholipids. Lipids from a malignant glioma cell line were initially analyzed at a resolution of >200 000 and identified by setting the mass tolerance to ±1 mDa using matrix‐assisted laser desorption/ionization (MALDI) 15‐T FT‐ICR MS in positive ion mode. In most cases, a database search of potential lipid candidates using the exact masses of the lipids yielded only one possible chemical composition. Extremely high mass accuracy (<0.1 ppm) was then attained by using previously identified lipids as internal standards. This, combined with an extremely high resolution (>800 000), yielded well‐resolved isotopic fine structures allowing for the identification of lipids by MALDI 15‐T FT‐ICR MS without using tandem mass spectrometric (MS/MS) analysis. Using this method, a total of 38 unique lipids were successfully identified. Copyright © 2012 John Wiley & Sons, Ltd.     

Identification of lysophosphatidylcholine (LPC) and platelet activating factor (PAF) from PC12 cells and mouse cortex using liquid chromatography/multi-stage mass spectrometry (LC/MS3)

Lipids play essential roles in cellular structural support, energy storage and signal transduction. Recently, mass spectrometry (MS) has been used to produce three-dimensional maps that elucidate the lipid composition of complex cellular lysates. The identification of individual lipids within these maps is slow and requires the synthesis and spiking of each candidate lipid. We present a novel MS-based technique that rapidly elucidates the atomic connectivity of the fatty acid/alcohol substituent on the sn-1 position of several different families of glycerophosphocholine-containing lipids within the confines of a chromatographic separation. Sodiated lipid species were fragmented to produce radical cations which lost successive methylene groups upon further collisional activation to reveal the identity of the parent molecule. This approach was demonstrated to be effective on isobaric members of the lysophosphatidylcholine (LPC) and platelet activating factor (PAF) families of glycerophospholipids. We demonstrate the application of this technique to unambiguously identify these species within complex cellular lysates and tissue extracts.