表面修饰探针纳升电喷雾质谱脂质组学对大型溞和蚤状溞的快速鉴别

采用表面修饰探针敞开式纳升电喷雾质谱技术建立了脂质组学方法, 以实现溞属(Daphnia)生物大型溞和蚤状溞的快速鉴别. 采用微尺度表面修饰探针直接从Daphnia体内萃取和富集出脂质化合物, 然后在常压敞开条件下进行纳升电喷雾质谱分析, 利用高分辨率质谱仪获得脂质指纹图谱并进行脂质种类和结构鉴定. 采用该方法获得了大型溞和蚤状溞的脂质指纹图谱, 并研究了大型溞和蚤状溞体内的脂质组成特征. 通过结合化学计量学手段实现了大型溞和蚤状溞的快速区分, 并鉴定了相关的生物标志物. 结果表明, 所建立的微萃取探针纳升电喷雾质谱脂质组学方法具有分析速度快、 灵敏度高的优点, 可用于Daphnia属生物的快速鉴定和识别.     

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。     

Software tool for mining liquid chromatography/multi‐stage mass spectrometry data for comprehensive glycerophospholipid profiling

Electrospray ionization mass spectrometry (ESI‐MS) has emerged as an indispensable tool in the field of lipidomics. Despite the growing interest in lipid analysis, there are only a few software tools available for data evaluation, as compared for example to proteomics applications. This makes comprehensive lipid analysis a complex challenge. Thus, a computational tool for harnessing the raw data from liquid chromatography/mass spectrometry (LC/MS) experiments was developed in this study and is available from the authors on request. The Profiler‐Merger‐Viewer tool is a software package for automatic processing of raw‐data from data‐dependent experiments, measured by high‐performance liquid chromatography hyphenated to electrospray ionization hybrid linear ion trap Fourier transform mass spectrometry (FTICR‐MS and Orbitrap) in single and multi‐stage mode. The software contains three parts: processing of the raw data by Profiler for lipid identification, summarizing of replicate measurements by Merger and visualization of all relevant data (chromatograms as well as mass spectra) for validation of the results by Viewer. The tool is easily accessible, since it is implemented in Java and uses Microsoft Excel (XLS) as output format. The motivation was to develop a tool which supports and accelerates the manual data evaluation (identification and relative quantification) significantly but does not make a complete data analysis within a black‐box system. The software's mode of operation, usage and options will be demonstrated on the basis of a lipid extract of baker's yeast (S. cerevisiae). In this study, we focused on three important representatives of lipids: glycerophospholipids, lyso‐glycerophospholipids and free fatty acids. Copyright © 2010 John Wiley & Sons, Ltd.     

Precise and global identification of phospholipid molecular species by an Orbitrap mass spectrometer and automated search engine Lipid Search

In the present research, we have established a new lipidomics approach for the comprehensive and precise identification of molecular species in a crude lipid mixture using a LTQ Orbitrap mass spectrometer (MS) and reverse-phase liquid chromatography (RPLC) combination with our newly developed lipid search engine “Lipid Search”. LTQ Orbitrap provides high mass accuracy MS spectra by Fourier-transform (FT) mass spectrometer mode and can perform rapid MSⁿ by ion trap (IT) mass spectrometer mode. In this study, the negative ion mode was selected to detect fragment ions from phospholipids, such as fatty acid anions, by MS2 or MS3. We selected the specific detection approach by neutral loss survey-dependent MS3, for the identification of molecular species of phosphatidylcholine, sphingomyelin and phosphatidylserine. Identification of molecular species was performed by using both the high mass accuracy of the mass spectrometric data obtained from FT mode and structural data obtained from fragments in IT mode. Some alkylacyl and alkenylacyl species have the same m/z value as molecular-related ions and fragment ions, thus, direct acid hydrolysis analysis was performed to identify alkylacyl and alkenylacyl species, and then the RPLC–LTQ Orbitrap method was applied. As a result, 290 species from mouse liver and 248 species from mouse brain were identified within six different classes of phospholipid, only those in manually detected and confirmed. Most of all manually detected mass peaks were also automatically detected by “Lipid Search”. Adding to differences in molecular species in different classes of phospholipids, many characteristic differences in molecular species were detected in mouse liver and brain. More variable number of saturated and monounsaturated fatty acid-containing molecular species were detected in mouse brain than liver.     

Reshaping Lipid Biochemistry by Pushing Barriers in Structural Lipidomics

Lipidomics is a rapidly growing field with numerous examples showing the importance of lipid molecules throughout biology. It has also shed light onto the vast and complex functions performed by many lipids that possess an immense diversity in molecular structures. Mass spectrometry (MS) is the tool of choice for analyzing lipids and has been the key catalyst driving the field forward. However, MS does not yet permit true molecular lipidomics wherein the identification and quantification of lipids having defined molecular structures can be routinely achieved. Here we describe recent advances in MS‐based lipidomics that allow access to higher levels of molecular information in lipidomics experiments. These advances will form a key piece of the puzzle as the field moves towards systems characterization of lipids at the molecular level.     

Mass‐spectrometry‐based lipidomics

Lipids, which have a core function in energy storage, signalling and biofilm structures, play important roles in a variety of cellular processes because of the great diversity of their structural and physiochemical properties. Lipidomics is the large‐scale profiling and quantification of biogenic lipid molecules, the comprehensive study of their pathways and the interpretation of their physiological significance based on analytical chemistry and statistical analysis. Lipidomics will not only provide insight into the physiological functions of lipid molecules but will also provide an approach to discovering important biomarkers for diagnosis or treatment of human diseases. Mass‐spectrometry‐based analytical techniques are currently the most widely used and most effective tools for lipid profiling and quantification. In this review, the field of mass‐spectrometry‐based lipidomics was discussed. Recent progress in all essential steps in lipidomics was carefully discussed in this review, including lipid extraction strategies, separation techniques and mass‐spectrometry‐based analytical and quantitative methods in lipidomics. We also focused on novel resolution strategies for difficult problems in determining C=C bond positions in lipidomics. Finally, new technologies that were developed in recent years including single‐cell lipidomics, flux‐based lipidomics and multiomics technologies were also reviewed.     

A simple and universal method for the separation and identification of phospholipid molecular species

One of the major challenges in lipidomics is to obtain as much information about the lipidome as possible. Here, we present a simple yet universal high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) method to separate molecular species of all phospholipid classes in one single run. The method is sensitive, robust and allows lipid profiling using full scan mass spectrometry, as well as lipid class specific scanning in positive and negative ionisation mode. This allows high-throughput processing of samples for lipidomics, even if different types of MS analysis are required. Excellent separation of isobaric and even isomeric species is achieved, and original levels of lyso-lipids can be determined without interference from lyso-lipids formed from diacyl species by source fragmentation. As examples of application of this method, more than 400 phospholipid species were identified and quantified in crude phospholipid extracts from rat liver and the parasitic helminth Schistosoma mansoni.     

Structural characterization of ether lipids from the archaeon Sulfolobus islandicus by high‐resolution shotgun lipidomics

The molecular structures, biosynthetic pathways and physiological functions of membrane lipids produced by organisms in the domain Archaea are poorly characterized as compared with that of counterparts in Bacteria and Eukaryota. Here we report on the use of high‐resolution shotgun lipidomics to characterize, for the first time, the lipid complement of the archaeon Sulfolobus islandicus. To support the identification of lipids in S. islandicus, we first compiled a database of ether lipid species previously ascribed to Archaea. Next, we analyzed the lipid complement of S. islandicus by high‐resolution Fourier transform mass spectrometry using an ion trap‐orbitrap mass spectrometer. This analysis identified five clusters of molecular ions that matched ether lipids in the database with sub‐ppm mass accuracy. To structurally characterize and validate the identities of the potential lipid species, we performed structural analysis using multistage activation on the ion trap‐orbitrap instrument as well as tandem mass analysis using a quadrupole time‐of‐flight machine. Our analysis identified four ether lipid species previously reported in Archaea, and one ether lipid species that had not been described before. This uncharacterized lipid species features two head group structures composed of a trisaccharide residue carrying an uncommon sulfono group (?SO₃) and an inositol phosphate group. Both head groups are linked to a glycerol dialkyl glycerol tetraether core structure having isoprenoid chains with a total of 80 carbon atoms and 4 cyclopentane moieties. The shotgun lipidomics approach deployed here defines a novel workflow for exploratory lipid profiling of Archaea. Copyright © 2015 John Wiley & Sons, Ltd.     

Mobility-Modulated Sequential Dissociation Analysis Enables Structural Lipidomics in Mass Spectrometry Imaging

Spatial lipidomics based on mass spectrometry imaging (MSI) is a powerful tool for fundamental biology studies and biomarker discovery. But the structure-resolving capability of MSI is limited because of the lack of multiplexed tandem mass spectrometry (MS/MS) method, primarily due to the small sample amount available from each pixel and the poor ion usage in MS/MS analysis. Here, we report a mobility-modulated sequential dissociation (MMSD) strategy for multiplex MS/MS imaging of distinct lipids from biological tissues. With ion mobility-enabled data-independent acquisition and automated spectrum deconvolution, MS/MS spectra of a large number of lipid species from each tissue pixel are acquired, at no expense of imaging speed. MMSD imaging is highlighted by MS/MS imaging of 24 structurally distinct lipids in the mouse brain and the revealing of the correlation of a structurally distinct phosphatidylethanolamine isomer (PE 18 : 1_18 : 1) from a human hepatocellular carcinoma (HCC) tissue. Mapping of structurally distinct lipid isomers is now enabled and spatial lipidomics becomes feasible for MSI.     

Shotgun lipidomics on a LTQ Orbitrap mass spectrometer by successive switching between acquisition polarity modes

Top–down shotgun lipidomics relies on direct infusion of total lipid extracts into a high‐resolution tandem mass spectrometer and implies that individual lipids are recognized by their accurately determined m/z. Lipid ionization efficiency and detection specificity strongly depend on the acquisition polarity, and therefore it is beneficial to analyze lipid mixtures in both positive and negative modes. Hybrid LTQ Orbitrap mass spectrometers are widely applied in top–down lipidomics; however, rapid polarity switching was previously unfeasible because of the severe and immediate degradation of mass accuracy. Here, we report on a method to rapidly acquire high‐resolution spectra in both polarity modes with sub‐ppm mass accuracy and demonstrate that it not only simplifies and accelerates shotgun lipidomics analyses but also improves the lipidome coverage because more lipid classes and more individual species within each class are recognized. In this way, shotgun analysis of total lipid extracts of human blood plasma enabled to quantify 222 species from 15 major lipid classes within 7 min acquisition cycle. Copyright © 2012 John Wiley & Sons, Ltd.     

Analysis of human plasma lipids and soybean lecithin by means of high‐performance thin‐layer chromatography and matrix‐assisted laser desorption/ionization mass spectrometry

An improved analytical strategy for the analysis of complex lipid mixtures using matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) in combination with high‐performance thin‐layer chromatography (HPTLC) is reported. Positive ion MALDI RTOF MS was applied as a rapid screening tool for the various neutral (e.g. triacylglycerols) and polar (e.g. glycerophospholipids and ‐sphingolipids) lipid classes derived from crude lipid extracts of e.g. human plasma as well as soybean lecithin. Finally, MALDI seamless post‐source decay (PSD) product ion analysis was performed in order to obtain further structural information (head‐ and acyl‐group identification) of selected lipid species and structure verification. A Coomassie Brilliant Blue R‐250 staining protocol for lipids on HPTLC plates was evaluated and was found to be fully compatible with subsequent MALDI‐MS. Lipids were analyzed after elution from the HPTLC phase material of the selected band (corresponding to certain lipid classes) by using the proper organic solvent mixture or in few cases directly from the HPTLC plates (a type of on‐line HPTLC/MALDI‐MS coupling). More than 70 distinct lipid species from seven different lipid classes in the range between m/z 500 and 1500 could be identified from the lipid extracts of human plasma and soybean lecithin, respectively. The general high sensitivity of MALDI‐MS detection allowed the analysis of even minor lipid classes from only very small volumes of human plasma (50 µL). The combination of HPTLC, Coomassie staining and positive ion MALDI curved field RTOF‐MS represents a straightforward strategy during lipidomics studies of food and clinically relevant human lipid samples. Copyright © 2009 John Wiley & Sons, Ltd.     

Algorithm for processing raw mass spectrometric data to identify and quantitate complex lipid molecular species in mixtures by data-dependent scanning and fragment ion database searching

We developed the Lipid Qualitative/Quantitative Analysis (LipidQA) software platform to identify and quantitate complex lipid molecular species in biological mixtures. LipidQA can process raw electronic data files from the TSQ-7000 triple stage quadrupole and LTQ linear ion trap mass spectrometers from Thermo-Finnigan and the Q-TOF hybrid quadrupole/time-of-flight instrument from Waters-Micromass and could readily be modified to accommodate data from others. The program processes multiple spectra in a few seconds and includes a deisotoping algorithm that increases the accuracy of structural identification and quantitation. Identification is achieved by comparing MS(2) spectra obtained in a data-dependent manner to a library of reference spectra of complex lipids that we have acquired or constructed from established fragmentation rules. The current form of the algorithm can process data acquired in negative or positive ion mode for glycerophospholipid species of all major head-group classes.     

Computer-assisted interpretation of atmospheric pressure chemical ionization mass spectra of triacylglycerols

Current lipidomics approaches require simple and rapid algorithms enabling the interpretation of mass spectra of lipids. Most lipids are complex mixtures of related components in which the composition of the aliphatic fatty acid chains varies from one molecule to the next. Triacylglycerols (TAGs) are an example of such a lipid class. Fatty acid chains are the only parts of the molecule to change from one species to another. Fatty acids, and consequently also TAGs, can be characterized by two parameters; the number of carbon atoms and the number of double bonds. All calculations reflecting relations among ions in the spectra can be easily made using these parameters. An algorithm for the automated interpretation of TAGs from atmospheric pressure chemical ionization mass spectra (TriglyAPCI) is presented in this paper. The algorithm first identifies diacylglycerol fragments and molecular adducts. In the next step, relations among the ions are searched and possible TAG structures are suggested. Individual features of the algorithm are described in detail and the software performance is demonstrated for the liquid chromatography/mass spectrometric (LC/MS) analysis of TAGs isolated from the termite Prorhinotermes canalifrons.     

Rapid analysis of flavonoids based on spectral library development in positive ionization mode using LC-HR-ESI-MS/MS

Natural product screening in plants has always been a difficult task due to the complex nature of the plant material and diverse structures of the compounds present in them. Flavonoids are important and diverse class of plant secondary metabolites with numerous medicinal activities. The present study focuses on the development of a high-resolution tandem mass spectral library for the rapid and authentic identification of common flavonoids. A total of forty flavonoid standards belong to class flavones, isoflavones, flavanones, flavanols and anthocyanins were pooled into two solutions applying logP-based strategy. The flavonoids were analyzed using LC-QTOF-MS high-resolution mass spectrometer with optimization of different instrumental parameters to achieve good sensitivity. The library was built by incorporating names, molecular formulae, exact masses, and MS, and MS/MS spectra of analyzed flavonoids using Bruker Library Editor tool. The fragmentation pattern observed for the standard compounds were compared to the fragments reported in the literature. To assess the practical implications, an extract of tea sample was analyzed and screened using the developed library, which resulted in the identification of three common flavonoids based on their HR-ESI-MS/MS spectral features. The established LC-HR-MS/MS method can be used for the targeted identification of flavonoids in complex samples like food material from different botanical families.     

A shotgun tandem mass spectrometric analysis of phospholipids with normal-phase and/or reverse-phase liquid chromatography/electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry is used in lipidomics studies. The present research established a top-down liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) shotgun analysis method for phospholipids (PLs) using a normal-phase column or a C30 reverse-phase column with the data-dependent MS/MS scanning mode. A normal-phase column can separate most of the major different classes of PLs. By using LC/ESI-MS/MS with a normal-phase column, approximately 50 molecular species were identified in a PL mixture from rat liver. When the reverse-phase column was used, the PLs could be separated depending on their hydrophobicity, essentially the length of their fatty acyl chains and the number of unsaturated bonds in them. The LC/ESI-MS/MS method using a C30 reverse-phase column was applied to phosphatidylcholine (PC) and phosphatidylethanolamine (PE) mixtures as test samples. Molecular species with the same molecular mass but with different pairs of fatty acyl chains were separately identified. As a result, about 60 PC and 50 PE species were identified. PLs from rat liver were subjected to LC/ESI-MS/MS using the C30 reverse-phase column and about 110 molecular species were identified. Off-line two-dimensional LC/ESI-MS/MS with the normal-phase and C30 reverse-phase columns allowed more accurate identification of molecular species by using one-dimensional C30 reverse-phase LC/ESI-MS/MS analysis of the collected fractions.     

De novo analysis of electron impact mass spectra using fragmentation trees

The automated fragmentation analysis of high resolution EI mass spectra based on a fragmentation tree algorithm is introduced. Fragmentation trees are constructed from EI spectra by automated signal extraction and evaluation. These trees explain relevant fragmentation reactions and assign molecular formulas to fragments. The method enables the identification of the molecular ion and the molecular formula of a metabolite if the molecular ion is present in the spectrum. These identifications are independent of existing library knowledge and, thus, support assignment and structural elucidation of unknown compounds. The method works even if the molecular ion is of very low abundance or hidden under contaminants with higher masses. We apply the algorithm to a selection of 50 derivatized and underivatized metabolites and demonstrate that in 78% of cases the molecular ion can be correctly assigned. The automatically constructed fragmentation trees correspond very well to published mechanisms and allow the assignment of specific relevant fragments and fragmentation pathways even in the most complex EI-spectra in our dataset. This method will be very helpful in the automated analysis of metabolites that are not included in common libraries and it thus has the potential to support the explorative character of metabolomics studies.     

Separation and identification of neutral cereal lipids by normal phase high-performance liquid chromatography,using evaporative light-scattering and electrospray mass spectrometry for detection

A novel method was developed for the analysis of molecular species in neutral lipid classes, using separation by normal phase high-performance liquid chromatography, followed by detection by evaporative light-scattering and electrospray ionization tandem mass spectrometry. Monoacid standards, i.e. sterol esters, triacylglycerols, fatty acids, diacylglycerols, free sterols and monoacylglycerols, were separated to baseline on microbore 3 μm-silica gel columns. Complete or partial separation of molecular species in each lipid class permitted identification by automatic tandem mass spectrometry of ammonium adducts, produced via positive electrospray ionization. After optimization of the method, separation and identification of molecular species of various lipid classes was comprehensively tested by analysis of neutral lipids from the free lipid extract of maize flour.     

CHASE, a charge-assisted sequencing algorithm for automated homology-based protein identifications with matrix-assisted laser desorption/ionization time-of-flight post-source decay fragmentation data

We describe CHASE, a novel algorithm for automated de novo sequencing based on the mass spectrometric (MS) fragmentation analysis of tryptic peptides. This algorithm is used for protein identification from sequence similarity criteria and consists of four steps: (1) derivatization of tryptic peptides at the N-terminus with a negatively charged reagent; (2) post-source decay (PSD) fragmentation analysis of peptides; (3) interpretation of the mass peaks with the CHASE algorithm and reconstruction of the amino acid sequence; (4) transfer of these data to software for protein identifications based on sequence homology (Basic Local Alignment Search Tool, BLAST). This procedure deduced the correct amino acid sequence of tryptic peptide samples and also was able to deduce the correct sequence from difficult mass patterns and identify the amino acid sequence. This allows complete automation of the process starting from MS fragmentation of complex peptide mixtures at low concentration (e.g. from silver-stained gel bands) to identification of the protein. We also show that if PSD data are collected in a single spectrum (instead of the segmented mode offered by conventional matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) instrumentation), the complete workflow from MS-PSD data acquisition to similarity-based identification can be completely automated. This strategy may be applied to proteomic studies for protein identification based on automated de novo sequencing instead of MS or tandem MS patterns. We describe the Charge Assisted Sequencing Engine (CHASE) algorithm, the working protocol, the performance of the algorithm on spectra from MALDI-TOFMS and the data comparison between a TOF and a TOF-TOF instrument.     

A new lipidomics approach by thin-layer chromatography-blot-matrix-assisted laser desorption/ionization imaging mass spectrometry for analyzing detailed patterns of phospholipid molecular species

Thin-layer chromatography (TLC) is a highly established convenient technique for lipid separation and partial characterization of neutral and acidic glycosphingolipids (GSLs) and phospholipids, in mixtures. Meanwhile, imaging mass spectrometry (IMS) is a promising tool for lipidomics. However, some lipid classes are detected more sensitively than others, which can lead to suppression effects when complex mixtures are analyzed. Therefore to analyze complex lipid mixtures, a precise separation into the individual lipid classes is necessary. Here we present our highly sensitive and convenient analytical technology that combines TLC and IMS, namely the TLC-Blot–MALDI-IMS method, to visualize whole lipids and individual molecular species with high sensitivity compared with common staining methods. This method allows for easy visualization of all lipids with a linear range of approximately one order of magnitude and precision <16% RSD, making it useful for differential display analysis of lipids.     

Simultaneous quantification and identification using <Superscript>18</Superscript>O labeling with an ion trap mass spectrometer and the analysis software application “ZoomQuant”

Stable isotope labeling with (18)O is a promising technique for obtaining both qualitative and quantitative information from a single differential protein expression experiment. The small 4 Da mass shift produced by incorporation of two molecules of (18)O, and the lack of available methods for automated quantification of large data sets has limited the use of this approach with electrospray ionization-ion trap (ESI-IT) mass spectrometers. In this paper, we describe a method of acquiring ESI-IT mass spectrometric data that provides accurate calculation of relative ratios of peptides that have been differentially labeled using(18)O. The method utilizes zoom scans to provide high resolution data. This allows for accurate calculation of (18)O/(16)O ratios for peptides even when as much as 50% of a (18)O labeled peptide is present as the singly labeled species. The use of zoom scan data also provides sufficient resolution for calculating accurate ratios for peptides of +3 and lower charge states. Sequence coverage is comparable to that obtained with data acquisition modes that use only MS and MS/MS scans. We have employed a newly developed analysis software tool, ZoomQuant, which allows for the automated analysis of large data sets. We show that the combination of zoom scan data acquisition and analysis using ZoomQuant provides calculation of isotopic ratios accurate to approximately 21%. This compares well with data produced from (18)O labeling experiments using time of flight (TOF) and Fourier transform-ion cyclotron resonance (FT-ICR) MS instruments.