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.     

Glycomics by ion mobility tandem mass spectrometry of chondroitin sulfate disaccharide domain in biglycan

Biglycan (BGN), a small leucine-rich repeat proteoglycan, is involved in a variety of pathological processes including malignant transformation, for which the upregulation of BGN was found related to cancer cell invasiveness. Because the functions of BGN are mediated by its chondroitin/dermatan sulfate (CS/DS) chains through the sulfates, the determination of CS/DS structure and sulfation pattern is of major importance. In this study, we have implemented an advanced glycomics method based on ion mobility separation (IMS) mass spectrometry (MS) and tandem MS (MS/MS) to characterize the CS disaccharide domains in BGN. The high separation efficiency and sensitivity of this technique allowed the discrimination of five distinct CS disaccharide motifs, of which four irregulated in their sulfation pattern. For the first time, trisulfated unsaturated and bisulfated saturated disaccharides were found in BGN, the latter species documenting the non-reducing end of the chains. The structural investigation by IMS MS/MS disclosed that in one or both of the CS/DS chains, the non-reducing end is 3-O-sulfated GlcA in a rather rare bisulfated motif having the structure 3-O-sulfated GlcA-4-O-sulfated GalNAc. Considering the role played by BGN in cancer cell spreading, the influence on this process of the newly identified sequences will be investigated in the future.     

Ultra-fast screening of free fatty acids in human plasma using ion mobility mass spectrometry

Free fatty acids are involved in many metabolic regulations in the human body. In this work, an ultra-fast screening method was developed for the analysis of free fatty acids using trapped ion mobility spectrometry coupled with mass spectrometry. Thirty-three free fatty acids possessing different unsaturation degrees and different carbon chain lengths were baseline separated and characterized within milliseconds. Saturated, monounsaturated, and polyunsaturated free fatty acids showed different linearities between collision cross-section values and m/z. The establishment of correlations between structures and collision cross-section values provided additional qualitative information and made it possible to determine free fatty acids which were out of the standards pool but possessed the confirmed linearity. The gas-phase separation made the quantitative analysis reliable and repeatable at a much lower time cost than chromatographic methods. The sensitivity was comparable to and even better than the reported results. The method was validated and applied to profiling free fatty acids in human plasma. Saturated free fatty acids abundance in the fasting state was found to be lower than that in the postprandial state, while unsaturated species abundance was found higher. The method was fast and robust with minimum sample pretreatment, so it was promising in the high-throughput screening of free fatty acids.     

Time-of-flight ion mobility spectrometry and differential mobility spectrometry: A comparative study of their efficiency in the analysis of halogenated compounds

The ion mobilities of halogenated aromatics which are of interest in environmental chemistry and process monitoring were characterized with field-deployable ion mobility spectrometers and differential mobility spectrometers. The dependence of mobility of gas-phase ions formed by atmospheric-pressure photoionization (APPI) on the electric field was determined for a number of structural isomers. The structure of the product ions formed was identified by investigations using the coupling of ion mobility spectrometry with mass spectrometry (APPI-IMS-MS) and APPI-MS. In contrast to conventional time-of-flight ion mobility spectrometry (IMS) with constant linear voltage gradients in drift tubes, differential mobility spectrometry (DMS) employs the field dependence of ion mobility. Depending on the position of substituents, differences in field dependence were established for the isomeric compounds in contrast to conventional IMS in which comparable reduced mobility values were detected for the isomers investigated. These findings permit the differentiation between most of the investigated isomeric aromatics with a different constitution using DMS.     

Separation of catechin epimers by complexation using ion mobility mass spectrometry

Ion mobility coupled with mass spectrometry provides a fast and repeatable method to separate catechin epimers by previous complexation with selected chiral modifiers and transition metals. Several combinations with chiral ligands such as D‐ and L‐amino acids and/or additional metal cations, chiral crown ethers, tartaric acid and heptakis(2,6‐di‐O‐methyl)‐β‐cyclodextrin were screened for their ability to affect the separation efficiency. The clusters having the form of [2M + D‐amino acid + Cu²⁺ ? 3H]^? (M stands for (?)‐epicatechin or (+)‐catechin) showed improvement in stereodifferentiation between two epimeric catechins in comparison to the analysis of pure epimers, where no separation was observed or the separation was hampered by the formation of mixed dimer complexes. Among various examined D‐amino acids only those possessing hydrophobic side chains induced the improvement of separation efficiency. The best peak‐to‐peak resolution (R_p–p) was determined to be 0.71 for [2M + D‐Leucine + Cu²⁺ ? 3H]^? clusters. Copyright © 2015 John Wiley & Sons, Ltd.     

Identification and separation of saxitoxins using hydrophilic interaction liquid chromatography coupled to traveling wave ion mobility‐mass spectrometry

The aim of this work was to develop a reliable and efficient analytical method to characterise and differentiate saxitoxin analogues (STX), including sulphated (gonyautoxins, GTX) and non‐sulphated analogues. For this purpose, hydrophilic interaction liquid chromatography (HILIC) was used to separate sulphated analogues. We also resorted to ion mobility spectrometry to differentiate the STX analogues because this technique adds a new dimension of separation based on ion gas phase conformation. Positive and negative ionisation modes were used for gonyautoxins while positive ionisation mode was used for non‐sulphated analogues. Subsequently, the coupling of these three complementary techniques, HILIC‐IM‐MS, permitted the separation and identification of STX analogues; isomer differentiation was achieved in HILIC dimension while non‐sulphated analogues were separated in the IM‐MS dimension. Additional structural characteristics concerning the conformation of STXs could be obtained using IM‐MS measurements. Thus, the collision cross sections (CCS) of STXs are reported for the first time in the positive ionisation mode. These experimental CCSs correlated well with the calculated CCS values using the trajectory method. Copyright © 2015 John Wiley & Sons, Ltd.     

Discriminating alkylbenzene isomers with tandem mass spectrometry using a dielectric barrier discharge ionization source

Soft ambient ionization sources generate reactive species that interact with analyte molecules to form intact molecular ions, which allows rapid, sensitive, and direct identification of the molecular mass. We used a dielectric barrier discharge ionization (DBDI) source with nitrogen at atmospheric pressure to detect alkylated aromatic hydrocarbon isomers (C₈H₁₀ or C₉H₁₂). Intact molecular ions [M]^•+ were detected at 2.4 kV_pp, but at increased voltage (3.4 kV_pp), [M + N]⁺ ions were formed, which could be used to differentiate regioisomers by collision-induced dissociation (CID). At 2.4 kV_pp, alkylbenzene isomers with different alkyl-substituents could be identified by additional product ions: ethylbenzene and -toluene formed [M-2H]⁺, isopropylbenzene formed abundant [M-H]⁺, and propylbenzene formed abundant C₇H₇⁺. At an operating voltage of 3.4 kV_pp, fragmentation of [M + N]⁺ by CID led to neutral loss of HCN and CH₃CN, which corresponded to steric hindrance for excited state N-atoms approaching the aromatic ring (C-H). The ratio of HCN to CH₃N loss (interday relative standard deviation [RSD] < 20%) was distinct for ethylbenzene and ethyltoluene isomers. The greater the number of alkyl-substituents (C-CH₃) and the more sterically hindered (meta > para > ortho) the aromatic core, the greater the loss of CH₃CN relative to HCN was.     

Characterization of polysorbate 85, a nonionic surfactant,by liquid chromatography vs. ion mobility separation coupled with tandem mass spectrometry

Liquid chromatography (LC) and ion mobility (IM) separation have been coupled with mass spectrometry (MS) and tandem mass spectrometry (MS²) to characterize a commercially important nonionic surfactant, polysorbate 85. The constituents of this amphiphilic blend contained a sorbitan or isosorbide core that was chain extended with poly(ethylene oxide) (PEO) and partially esterified at the PEO termini with oleic acid or, to a lesser extent, other fatty acids. Using interactive LC in reverse-phase mode, the oligomers of the surfactant were separated according to their hydrophobicity/hydrophilicity balance. On the other hand, IM spectrometry dispersed the surfactant oligomers by their charge and collision cross section (i.e. size/shape). With either separation method, an increased number of fatty ester groups and/or lack of the polar sorbitan (or isosorbide) core led to higher retention/drift times, enabling the separation of isobaric species or species with superimposed isotope patterns, so that their ester content could be conclusively identified by MS². LC–MS and IM–MS permitted the detection of several byproducts besides the major PEO-sorbitan oleate oligomers. LC–MS provides the separation resolution needed for quantitative determination of the degree of esterification. IM–MS, which minimizes analysis time and solvent use, is ideally suitable for a fast, qualitative survey of samples differing in their minor constituents or impurities.     

Liquid chromatography–tandem mass spectrometry of porphyrins and porphyrinogens in biological materials: separation and identification of interfering poly(ethylene) glycol by travelling wave ion mobility spectrometry/tandem mass spectrometry

Biological and clinical samples for porphyrin and porphyrinogen analyses by liquid chromatography–tandem mass spectrometry (LC‐MS/MS) are often contaminated with poly(ethylene)glycol (PEG), which complicates the interpretation of mass spectra and characterisation of new porphyrin metabolites. Two contaminating PEG molecules (m/z 833 and m/z 835) were completely separated from uroporphyrin I (m/z 831) by travelling wave ion mobility spectrometry and characterised by tandem mass spectrometry. One of the PEG species (m/z 835) also co‐eluted with uroporphyrinogen I (m/z 837) and was unresolvable by travelling wave ion mobility spectrometry/MS, therefore contaminating the MS/MS mass spectra owing to isotope distribution. These PEG species, with the [M + H]⁺ ions at m/z at 833 and/or m/z 835, co‐eluted with uroporphyrin I and uroporphyrinogen I by LC‐MS/MS and could be wrongly identified as uroporphomethenes. Copyright © 2013 John Wiley & Sons, Ltd.     

Atmospheric-pressure ionization studies and field dependence of ion mobilities of isomeric hydrocarbons using a miniature differential mobility spectrometer

The ionization pathways and ion mobility were determined for sets of structural isomeric and stereoisomeric non-polar hydrocarbons (saturated and unsaturated cyclic hydrocarbons and aromatic hydrocarbons) using a novel miniature differential mobility spectrometer with atmospheric-pressure photoionization (APPI) to assess how structural and stereochemical differences influence ion formation and ion mobility. The analytical results obtained using the differential mobility spectrometry (DMS) were compared with the reduced mobility values measured using conventional time-of-flight ion mobility spectrometry (IMS) with the same ionization technique.The majority of differences in DMS ion mobility spectra observed among isomeric cyclic hydrocarbons can be explained by the formation of different product ions. Comparable differences in ion formation were also observed using conventional IMS and by investigations using the coupling of ion mobility spectrometry with mass spectrometry (APPI-IMS-MS) and APPI-MS. Using DMS, isomeric aromatic hydrocarbons can in the majority of cases be distinguished by the different behavior of product ions in the strong asymmetric radio frequency (rf) electric field of the drift channel. The different peak position of product ions depending on the electric field amplitude permits the differentiation between most of the investigated isomeric aromatics with a different constitution; this stands in contrast to conventional IMS in which comparable reduced mobility values were detected for the isomeric aromatic compounds.     

Kinetic measurements of phosphoglucose isomerase and phosphomannose isomerase by direct analysis of phosphorylated aldose–ketose isomers using tandem mass spectrometry

A mass spectrometry based method for the direct determination of kinetic constants for phosphoglucose isomerase (PGI) and phosphomannose isomerase (PMI) is described. PGI catalyzes the interconversion between glucose-6-phosphate (Glc6P) and fructose-6-phosphate (Fru6P) and PMI performs the same function between mannose-6-phosphate (Man6P) and Fru6P. These two enzymes are essential in the pathways of glycolytic or oxidative metabolism of carbohydrates and have been considered as potential therapeutic targets. Traditionally, they are assayed either by spectrophotometric detection of Glc6P with one or more coupling enzymes or by a colorimetric detection of Fru6P. However, no suitable assay for Man6P has been developed yet to study the reaction of PMI in the direction from Fru6P to Man6P. In the work presented herein, a general assay for the isomeric substrate-product pair between Glc6P and Fru6P or between Man6P and Fru6P was developed, with the aim of directly studying the kinetics of PGI and PMI in both directions. The 6-phosphorylated aldose and ketose isomers were distinguished based on their corresponding tandem mass spectra (MS²) obtained on a quadrupole ion trap mass spectrometer, and a multicomponent quantification method was utilized to determine the composition of binary mixtures. Using this method, the conversion between Fru6P and Glc6P and that between Fru6P and Man6P are directly monitored. The equilibrium constants for the reversible reactions catalyzed by PGI and PMI are measured to be 0.3 and 1.1, respectively, and the kinetic parameters for both substrates of PGI and PMI are also determined. The values of K_M and V_max for Fru6P as substrate of PMI are reported to be 0.15 mM and 7.78 μmol/(min mg), respectively. All other kinetic parameters measured correlate well with those obtained using traditional methods, demonstrating the accuracy and reliability of this assay.     

Measurements of coeluting unlabeled and C-labeled polychlorinated biphenyl congeners with partially overlapping fragment profiles using a tandem mass spectrometry

¹³C-labeled compounds are often employed as surrogate or internal standards to monitor the performance of extraction and instrumental analysis procedures for their unlabeled counterparts. However, labeled and unlabeled counterparts most often coelute chromatographically with overlapping mass ion fragments, posing a challenge to the accurate quantification of these compounds. In the present study, an analytical scheme, using coeluting unlabeled and ¹³C-labeled polychlorinated biphenyl (PCB) congeners as the model compounds, was developed with a low-resolution tandem mass spectrometer (MS/MS) to determine the appropriate ranges of PCB congener concentrations that satisfy the no-interference condition. Interferences from unlabeled PCBs to quantitation of labeled counterparts could be minimized when ¹³C-labeled PCB congeners were quantified in the MS/MS mode within a certain concentration range. In addition, good agreements between the measured and theoretically predicted quantitation errors were observed for all labeled PCB congeners except PCB 180. The exception with labeled PCB 180 was mainly attributed to the occurrence of instrumental analytical uncertainty, as analytical error was also observed with absence of unlabeled PCB 180. These results indicate that MS/MS techniques can serve as a useful tool to minimize interferences with quantitation of isotopically labeled compounds from their unlabeled counterparts, which possess partially overlapping ion fragment profiles.     

Separation of isomeric disaccharides by traveling wave ion mobility mass spectrometry using CO2 as drift gas

The use of CO₂ as a massive and polarizable drift gas is shown to greatly improve peak‐to‐peak resolution (R_p‐p), as compared with N₂, for the separation of disaccharides in a Synapt G2 traveling wave ion mobility cell. Near or baseline R_p‐p was achieved for three pairs of sodiated molecules of disaccharide isomers, that is, cellobiose and sucrose (R_p‐p = 0.76), maltose and sucrose (R_p‐p = 1.04), and maltose and lactose (R_p‐p = 0.74). Ion mobility mass spectrometry using CO₂ as the drift gas offers therefore an attractive alternative for fast and efficient separation of isomeric disaccharides. Copyright © 2012 John Wiley & Sons, Ltd.     

Characterizing ion mobility and collision cross section of fatty acids using electrospray ion mobility mass spectrometry

This study investigated the ion mobility (IM) and the collision cross section (CCS) of fatty acids (FAs) using electrospray IM MS. The IM analysis of 18 FA ions showed intriguing differences among the saturated FAs, monounsaturated FAs, multi‐unsaturated FAs, and cis‐isomer/trans‐isomer with respect to the aliphatic tail chains. The length of aliphatic tail chain present in the ion structures had a strong influence on the differentiation of drift, while the number of double bond showed a weaker influence. The tiny drift differences between cis‐isomer and trans‐isomer were also observed. In the CCS measurements, two internal standards were involved in the mobility calibration and accuracy estimation. It insured our empirical CCS values were of high experimental precision (±0.35% or better) and accuracy (±0.25% or better). Moreover, the mass‐to‐charge ratio (m/z) – mobility plots obtained by ion mobility spectrometry with mass spectrometry analysis of FAs – was used to investigate the structural relationship between the molecules. Each series of FAs sharing a similar structure was aligned in the linear plot. Finally, the developed procedure was applied to the determination of FAs in rat adipose tissues, and it allowed the presence of 13 FAs to be confirmed with their exact masses and CCS values. These studies reveal the direct relationship between the behaviors in IM and the molecular structures and thus may provide further validations to the FA identification process. Copyright © 2015 John Wiley & Sons, Ltd.     

离子淌度质谱技术及其应用研究进展

离子迁移谱（ion mobility spectrometry,IMS）是利用离子迁移率K（离子碰撞截面）差异来实现不同离子的分离与测定,具有分析速度快、检测灵敏度高的优点,其与质谱联用在蛋白质组学、代谢组学、医药等领域已获得了广泛的应用. 随着分析对象复杂性的增加,对IMS的分辨能力也提出了更高要求. 行波离子迁移谱（travelling wave ion mobility spectrometry,TWIMS）采用时域连续的行波电场实现离子传输与分离,其分析通道的长度不受行波电压幅值的限制,理论上可以无限延长离子分析通道来提高分辨能力. 目前,TWIMS的分辨率最高可达1 860,对于分析存在多种同分异构体的复杂样品别具优势. 对TWIMS的原理及分辨能力的影响因素进行了介绍,进一步探讨了不同结构TWIMS仪器的特点、性能和应用,对TWIMS未来发展方向进行了展望.     

Characterization of volatile metabolites formed by molds on barley by mass and ion mobility spectrometry

The contamination of barley by molds on the field or in storage leads to the spoilage of grain and the production of mycotoxins, which causes major economic losses in malting facilities and breweries. Therefore, on‐site detection of hidden fungus contaminations in grain storages based on the detection of volatile marker compounds is of high interest. In this work, the volatile metabolites of 10 different fungus species are identified by gas chromatography (GC) combined with two complementary mass spectrometric methods, namely, electron impact (EI) and chemical ionization at atmospheric pressure (APCI)‐mass spectrometry (MS). The APCI source utilizes soft X‐radiation, which enables the selective protonation of the volatile metabolites largely without side reactions. Nearly 80 volatile or semivolatile compounds from different substance classes, namely, alcohols, aldehydes, ketones, carboxylic acids, esters, substituted aromatic compounds, alkenes, terpenes, oxidized terpenes, sesquiterpenes, and oxidized sesquiterpenes, could be identified. The profiles of volatile and semivolatile metabolites of the different fungus species are characteristic of them and allow their safe differentiation. The application of the same GC parameters and APCI source allows a simple method transfer from MS to ion mobility spectrometry (IMS), which permits on‐site analyses of grain stores. Characterization of IMS yields limits of detection very similar to those of APCI‐MS. Accordingly, more than 90% of the volatile metabolites found by APCI‐MS were also detected in IMS. In addition to different fungus genera, different species of one fungus genus could also be differentiated by GC‐IMS.     

高效液相色谱-电喷雾离子阱质谱联用法测定金银花中绿原酸和咖啡酸

建立了高效液相色谱-电喷雾离子阱质谱法(HPLC-ESI-ITMSn)同时测定了金银花中绿原酸和咖啡酸活性成分的分析方法。样品用体积分数80%乙醇回流提取,用Zorbax Eclipse XDB-C18柱分离,以体积分数0.5%甲酸的乙腈-甲酸水溶液为梯度流动相,以保留时间和质荷比对分离出的组分予以定性确证,用峰面积进行定量。绿原酸和咖啡酸的线性范围均为10.0~1000.0μg/L,检出限(以信噪比为3计)均为2.0μg/L。样品的加标平均回收率为92.7%~98.7%,相对标准偏差为1.4%~2.7%。该方法适用于其它复杂体系中绿原酸和咖啡酸的分析。     

Comparison of differential mobility spectrometry and mass spectrometry for gas chromatographic detection of ignitable liquids from fire debris using projected difference resolution

The significance of forensic arson analysis accelerates the applications of new technologies in this area. Based on the previously reported application of differential mobility spectrometry (DMS) as a detection method for gas chromatography (GC) in arson analysis, the performances of DMS and mass spectrometry (MS) were compared using a novel chemometric tool, projected difference resolutions (PDRs). The PDR results show that one-way mass spectra data exhibit higher resolution than DMS data, while total ion chromatograms from GC–DMS show higher resolution than that from GC/MS for differentiating seven kinds of ignitable liquids. Combining the information from both chromatography and spectra, two-way data always have higher resolution than one-way data for these two detection methods, and GC/MS would exhibit better performance than GC–DMS according to the minimum resolution value. To verify the PDR results, a fuzzy rule-building expert system was applied for classifying these seven kinds of ignitable liquids from fire debris based on GC–DMS and GC/MS data, respectively. The prediction accuracies were consistent with PDR results, which proved that PDR is a powerful tool in comparing the performances of different analysis methods for pattern recognition. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Size dependence of the folding of multiply charged sodium cationized polylactides revealed by ion mobility mass spectrometry and molecular modelling

Ion mobility spectrometry coupled with mass spectrometry was used to experimentally determine the three-dimensional structure of multiply charged sodium cationized polylactides (PLA). In particular, the experiments were conducted to evaluate the influence of the charge state and the size on the gas-phase conformation of cationized PLA. The measured collision cross sections were then compared to calculated values obtained by computational chemistry methods. The most striking feature was the experimental and theoretical observation of a breaking point in the quasilinear relationship between the average collision cross sections and the number of monomer units for the triply charged cations. This breaking point was theoretically demonstrated, for the doubly and triply charged cations, to be associated with a significant folding of the polymer chains around the cationizing agents. The occurrence of such breaking points could be exploited to correlate the charge state of the most intense ion series observed upon electrospray ionization with the number-average molecular mass of a polymer.     

Enhancement of mass spectrometry performance for proteomic analyses using highfield asymmetric waveform ion mobility spectrometry (FAIMS)

Remarkable advances in mass spectrometry sensitivity and resolution have been accomplished over the past two decades to enhance the depth and coverage of proteome analyses. As these technological developments expanded the detection capability of mass spectrometers, they also revealed an increasing complexity of low abundance peptides, solvent clusters and sample contaminants that can confound protein identification. Separation techniques that are complementary and can be used in combination with liquid chromatography are often sought to improve mass spectrometry sensitivity for proteomics applications. In this context, high‐field asymmetric waveform ion mobility spectrometry (FAIMS), a form of ion mobility that exploits ion separation at low and high electric fields, has shown significant advantages by focusing and separating multiply charged peptide ions from singly charged interferences. This paper examines the analytical benefits of FAIMS in proteomics to separate co‐eluting peptide isomers and to enhance peptide detection and quantitative measurements of protein digests via native peptides (label‐free) or isotopically labeled peptides from metabolic labeling or chemical tagging experiments. Copyright © 2015 John Wiley & Sons, Ltd.