Analysis of Antiretroviral Nucleosides by Electrospray Ionization Mass Spectrometry and Collision Induced Dissociation

Abstract

Antiretroviral nucleoside drugs used against the human immunodeficiency virus (HIV) infection have been analyzed using negative ion electrospray ionization (ESI) mass spectrometry and collision-induced dissociation (CID-MS/MS). Mass fragmentation of azidothymidine (AZT), didanosine (ddI), dideoxycytidine (ddC) and dideoxythiacytidine (3TC) were obtained at different cone voltages and collision energies. Fragmentation of purines and pyrimidines occurred by different pathways. For purines (ddI), the fragmentation was similar to those found in endogenous nucleosides; mainly the pseudo molecular ion is present (M-H) and a cleavage through the glycosidic bond forming (B) was observed. For pyrimidines (AZT, ddC, 3TC), the fragmentation pathways were different from endogenous nucleosides; for AZT, the fragmentation occurred primarily through the elimination of the azido group in the 3′-position (M-H₂-N₃), whereas ddC and 3TC presented more complex fragmentation patterns. For ddC, fragmentation appeared to be dominated by a retro Diels-Alder mechanism (M-CONH). For 3TC, the sulfur atom in the sugar moiety provided greater stability to the charge, producing fragments where the charge resided initially in the dideoxyribose (M-C₂O₂H₆).     

Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological tissues at the hundreds to tens of microns scale. When performed under ambient conditions, sample pre-treatment becomes unnecessary, thus simplifying the protocol while maintaining the high quality of information obtained. Desorption electrospray ionization (DESI) is a spray-based ambient MSI technique that allows for the direct sampling of surfaces in the open air, even in vivo. When used with a software-controlled sample stage, the sample is rastered underneath the DESI ionization probe, and through the time domain, m/z information is correlated with the chemical species'' spatial distribution. The fidelity of the DESI-MSI output depends on the source orientation and positioning with respect to the sample surface and mass spectrometer inlet. Herein, we review how to prepare tissue sections for DESI imaging and additional experimental conditions that directly affect image quality. Specifically, we describe the protocol for the imaging of rat brain tissue sections by DESI-MSI.     

毛冬青中三萜皂苷类化合物的提取分离及电喷雾多级质谱快速分析研究

建立电喷雾多级质谱结合硅胶柱层析快速、全面分析中药毛冬青中三萜皂苷成分的方法。毛冬青干燥根70%甲醇提取物,先用乙酸乙酯萃取,再用正丁醇萃取。取正丁醇萃取物经硅胶柱层析,以甲醇-氯仿-水(1∶9∶0.1→2∶8∶0.2→3.5∶6.5∶1→1∶1∶0.3,V/V)溶剂系统进行梯度洗脱。通过TLC比较,将毛冬青皂苷粗提物分成3个组分。通过全扫描电喷雾多级质谱对每个组分分别进行分析,并结合文献报道,确定皂苷的结构。确定了毛冬青中11个已知皂苷的结构,对1个未知皂苷的结构进行了推测。此方法具有操作简单、成本低、效率高等优点。     

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry (SESI-MS)

Secondary electrospray ionization mass spectrometry (SESI-MS) is a method developed for the rapid detection of volatile compounds, without the need for sample pretreatment. The method was first described by Fenn and colleagues¹ and has been applied to the detection of illicit drugs² and explosives^3-4, the characterization of skin volatiles⁵, and the analysis of breath^6-7.SESI ionization occurs by proton transfer reactions between the electrospray solution and the volatile analyte, and is therefore suitable for the analysis of hetero-organic molecules, just as in traditional electrospray ionization (ESI). However, unlike standard ESI, the proton transfer process of SESI occurs in the vapor phase rather than in solution (Fig. 1), and therefore SESI is best suited for detecting organic volatiles and aerosols.We are expanding the use of SESI-MS to the detection of bacterial volatiles as a method for bacterial identification and characterization⁸. We have demonstrated that SESI-MS volatile fingerprinting, combined with a statistical analysis method, can be used to differentiate bacterial genera, species, and mixed cultures in a variety of growth media.⁸ Here we provide the steps for obtaining bacterial volatile fingerprints using SESI-MS, including the instrumental parameters that should be optimized to ensure robust bacterial identification and characterization.     

Electrospray Ionization Mass Spectrometric Study of the Multiple Intracellular Monomeric and Polymeric Hemoglobins of Glycera dibranchiata

The intracellular hemoglobin (Hb) of the marine polychaete Glycera dibranchiata is comprised of two groups of globins differing in their primary structures and state of aggregation. About six electrophoretically and chromatographically distinct monomeric Hbs which have Leu as the distal residue, and an equal number of polymeric Hbs which have the usual distal His, have been identified to date. Deconvolution of the electrospray ionization mass spectra (ESI-MS) of the Hbs and of their carbamidomethylated, reduced, and reduced/carbamidomethylated forms, using a maximum entropy-based approach (MaxEnt), showed the presence of at least 18 peaks attributable to monomer Hbs (14,500–15,200 Da) and an approximately equal number of polymer Hb peaks (15,500–16,400 Da). Although the ratio of the monomer to polymer components in pooled Hb preparations remained constant at 60:40, Hb from individuals had generally less than 6 monomer and 6 polymer components; 2 of the 19 individuals appeared to be deficient in polymer Hbs. Taking into account possible fragmentations of the known monomeric and polymeric globin sequences, we estimate conservatively that there are 10 monomeric and an equal number of polymeric Hbs, the majority comprising a single free Cys. Surprisingly, the calculated mass of the sequence deduced from the high-resolution monomer Hb crystal structures does not correspond to any of the observed masses. ESI-MS of the monomer Hb crystal revealed 11 components, of which 5, accounting for 67% of total, were related to the three major sequences GMG2–4. These findings underline the need for routine mass spectrometric characterization of all protein preparations. The complete resolution of the Glycera Hb ESI-MS using MaxEnt processing illustrates the power of this method to resolve complex protein mixtures.     

N-Terminal Acetylation of Two Major Latex Proteins from Arabidopsis thaliana Using Electrospray Ionization Tandem Mass Spectrometry

The primary structure of two proteins named major latex protein in Arabidopsis thaliana were characterized by matrix-assisted laser desorption/ionization time of flight mass spectrometer and Nano-electrospray ionization tandem mass spectrometry (nanoESI-MS/MS) after two-dimensional gel electrophoresis separation. We revealed that the two proteins with the same N termini and the N-terminal alanine were acetylated after methionine cleavage by fragmentation of three doubly charged peptides using a quadrupole-time of flight 2 tandem mass spectrometer. It was worth noting that one peptide with sodium addition and acetylation was sequenced. It is usually difficult to analyze the peptide sequence of sodium adduct due to the 22-Da increment. The two proteins are highly homologous, and both their N-terminal and C-terminal peptides were sequenced. Of the two proteins, gi|15236568 (spot A) appears only in the seeding stage and flower organ, but gi|15236566 (spot B) appears throughout the whole life of A. thaliana. The biological mechanism of the two proteins and the function of N-terminal acetylation remain to be elucidated. This study showed that ESI-MS/MS was a powerful tool for the characterization of N-terminal acetylation of proteins.     

A Comparative Study on Structural Elucidation of Sialyl Oligosaccharides by Mass Spectrometry with Fast Atom Bombardment,Electrospray Ionization,and Matrix Assisted Laser Desorption/Ionization

To establish a new protocol for sensitive detection and structural characterization of sialyl oligosaccharides, their sensitivities and structural information from mass spectrometry and tandem mass spectrometry with FAB-, ESI-, and MALDI were evaluated in detail. Among these ionization methods, FAB-MS and FAB-MS/MS gave reproducible and predictable spectra carrying information on sequence and branching of sialyl oligosaccharides after derivatization with 2-aminopyridine (PA). With both positive and negative ion modes, their structural elucidation promises to be straightforward, MS/MS specta being measurable at as low as 200 pmol. Thus, this method consitutes a powerful tool for sensitive detection and structural characterization of limited quantities of sialyl oligosaccharides by FAB-MS and FAB-MS/MS.     

Electrospray Ionization Mass Spectrometric Study of the Multiple Intracellular Monomeric and Polymeric Hemoglobins of Glycera dibranchiata

The intracellular hemoglobin (Hb) of the marine polychaete Glycera dibranchiata is comprised of two groups of globins differing in their primary structures and state of aggregation. About six electrophoretically and chromatographically distinct monomeric Hbs which have Leu as the distal residue, and an equal number of polymeric Hbs which have the usual distal His, have been identified to date. Deconvolution of the electrospray ionization mass spectra (ESI-MS) of the Hbs and of their carbamidomethylated, reduced, and reduced/carbamidomethylated forms, using a maximum entropy-based approach (MaxEnt), showed the presence of at least 18 peaks attributable to monomer Hbs (14,500–15,200 Da) and an approximately equal number of polymer Hb peaks (15,500–16,400 Da). Although the ratio of the monomer to polymer components in pooled Hb preparations remained constant at 60:40, Hb from individuals had generally less than 6 monomer and 6 polymer components; 2 of the 19 individuals appeared to be deficient in polymer Hbs. Taking into account possible fragmentations of the known monomeric and polymeric globin sequences, we estimate conservatively that there are 10 monomeric and an equal number of polymeric Hbs, the majority comprising a single free Cys. Surprisingly, the calculated mass of the sequence deduced from the high-resolution monomer Hb crystal structures does not correspond to any of the observed masses. ESI-MS of the monomer Hb crystal revealed 11 components, of which 5, accounting for 67% of total, were related to the three major sequences GMG2–4. These findings underline the need for routine mass spectrometric characterization of all protein preparations. The complete resolution of the Glycera Hb ESI-MS using MaxEnt processing illustrates the power of this method to resolve complex protein mixtures.     

Stability Enhancement for Peptide Analysis by Electrospray Using the Triple Quadrupole Mass Spectrometer

Electrospray ionization sources, used with triple quadrupole mass spectrometers from PE/Sciex (API III+), Micromass (Quattro II), and Finnigan (TSQ 7000), were modified with a 35-gauge stainless steel needle. The dimensions of the needle were 63 μm i.d. by 145 μm o.d. with variable length, depending on the specific instrument. This modification led to enhanced signal stability, improved signal/noise ratios, and lowered sample consumption for a wide range of peptides. Stable baselines were observed with flow rates in the range of 50 nL/min to 5 μL/min. An alternative design, based on a metal wire housed within a fused silica capillary, led to the most stable signals of all during infusion, but caused excessive peak broadening with capillary chromatography. The Finnigan interface was further modified with an external postcolumn addition tee, used in conjunction with capillary liquid chromatography columns of 30 and 50 μm internal diameter. The best results with the modified Finnigan interface were acquired using the 50-μm column at a flow rate of 150 to 200 nL/min.     

基于外翻肠囊模型大鼠吸收的中药毛冬青三萜皂苷类成分电喷雾多级质谱快速分析研究

建立快速发现毛冬青三萜总皂苷主要吸收成分的外翻肠囊模型法,确定毛冬青总皂苷在体吸收的活性成分。采用大鼠外翻肠囊模型,收集毛冬青总皂苷给药后的肠囊液样品,采用电喷雾多级质谱快速、全面分析毛冬青三萜皂苷类成分在小肠的吸收。通过全扫描电喷雾多级质谱对肠囊液样品进行分析,并结合文献报道,确定皂苷的结构。在肠囊中检测到5个三萜皂苷,依次为:ilexoside O(5)、ilexoside K(4)、ilexoside J(3)、ilexoside E(8)和pedunculoside(13)。电喷雾多级质谱检测条件下,发现毛冬青总皂苷肠吸收的主要成分是三萜皂苷类成分,肠吸收的其它成分有待于进一步研究。     

Electrospray Mass Spectrometry and Multiple Sequence Analysis of Synthetic Peptide Libraries

The determination of the composition and purity of synthetic combinatorial libraries of free peptides requires analytical techniques that are especially suited for the challenging task of mixture analysis. Qualitative and semi-quantitive information on the composition of complex mixtures can be obtained by electrospray mass spectrometry with a maximum of reliability, accuracy, and fastness. Especially in combination with tandem mass spectrometry, contaminations with by-products (e.g., peptides with uncleaved side-chain protecting groups) can be easily revealed. Multiple sequence analysis based on Edman degradation allows a quantitative determination of the composition of libraries as well as the determination of coupling efficiencies during the synthesis of a library.     

Artifact-Free Quantification of Free 3-Chlorotyrosine, 3-Bromotyrosine, and 3-Nitrotyrosine in Human Plasma by Electron Capture–Negative Chemical Ionization Gas Chromatography Mass Spectrometry and Liquid Chromatography–Electrospray Ionization Tandem Mass Spectrometry

Halogenation and nitration of biomolecules have been proposed as key mechanisms of host defense against bacteria, fungi, and viruses. Reactive oxidants also have the potential to damage host tissue, and they have been implicated in disease. In the current studies, we describe specific, sensitive, and quantitative methods for detecting three stable markers of oxidative damage: 3-chlorotyrosine, 3-bromotyrosine, and 3-nitrotyrosine. Our results indicate that electron capture-negative chemical ionization-gas chromatography/mass spectrometry (EC-NCI GC/MS) is 100-fold more sensitive than liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-MS/MS) for analyzing authentic 3-chlorotyrosine, 3-bromotyrosine, and 3-nitrotyrosine. Using an isotopomer of tyrosine to evaluate artifactual production of the analytes during sample preparation and analysis, we found that artifact generation was negligible with either technique. However, LC-MS/MS proved cumbersome for analyzing multiple samples because it required 1.5 h of run and equilibration time per analysis. In contrast, EC-NCI GC/MS required only 5 min of run time per analysis. Using EC-NCI GC/MS, we were able to detect and quantify attomole levels of free 3-chlorotyrosine, 3-bromotyrosine, and 3-nitrotyrosine in human plasma. Our results indicate that EC-NCI GC/MS is a sensitive and specific method for quantifying free 3-chlorotyrosine, 3-bromotyrosine, and 3-nitrotyrosine in biological fluids in a single, rapid analysis and that it avoids generating any of the analytes ex vivo.     

MALDI-TOF-MS对寡核苷酸的序列分析

通过对几种基质测定含不同碱基的寡核苷酸的灵敏度及精确度的比较,发现用混合基质α－氰基４－羟基肉桂酸（α－Ｃｙａｎｏ）／３－羟基吡啶羧酸（３ＨＰＡ）用于基质辅助激光解吸附电离飞行时间质谱中测定脱氧寡核苷酸,不仅能得到较好的分子离子峰,而且一些金属离子的加合物峰能得到有效的抑制,提高了测定的灵敏度。用３′－和５′－外切酶对脱氧寡核苷酸１２－ｍｅｒ（５′－ＡＴＧＣＡＴＡＴＧＣＡＴ－３′）进行部分降解,再进行ＭＡＬＤＩ－ＴＯＦ－ＭＳ分析,得到了完整的寡核苷酸的序列。     

Focus on Metabolism: Spatially Resolved Plant Metabolomics: Some Potentials and Limitations of Laser-Ablation Electrospray Ionization Mass Spectrometry Metabolite Imaging

Laser-ablation electrospray ionization (LAESI)-mass spectrometry imaging has been applied to contrasting plant organs to assess its potential as a procedure for performing in vivo metabolomics in plants. In a proof-of-concept experiment, purple/white segmented Phalaenopsis spp. petals were first analyzed using standard liquid chromatography-mass spectrometry analyses of separate extracts made specifically from the purple and white regions. Discriminatory compounds were defined and putatively annotated. LAESI analyses were then performed on living tissues, and these metabolites were then relocalized within the LAESI-generated data sets of similar tissues. Maps were made to illustrate their locations across the petals. Results revealed that, as expected, anthocyanins always mapped to the purple regions. Certain other (nonvisible) polyphenols were observed to colocalize with the anthocyanins, whereas others were found specifically within the white tissues. In a contrasting example, control and Cladosporium fulvum-infected tomato (Solanum lycopersicum) leaves were subjected to the same procedures, and it could be observed that the alkaloid tomatine has clear heterogeneous distribution across the tomato leaf lamina. Furthermore, LAESI analyses revealed perturbations in alkaloid content following pathogen infection. These results show the clear potential of LAESI-based imaging approaches as a convenient and rapid way to perform metabolomics analyses on living tissues. However, a range of limitations and factors have also been identified that must be taken into consideration when interpreting LAESI-derived data. Such aspects deserve further evaluation before this approach can be applied in a routine manner.Plants are a tremendously rich source of a myriad of structurally and chemically diverse metabolites (Rao and Ravishankar, 2002; D’Auria and Gershenzon, 2005). Many of these metabolites have a (partly) known function in the plant, although our knowledge of the vast majority of plant secondary metabolites is still sparse, or even nonexistent (Rao and Ravishankar, 2002; D’Auria and Gershenzon, 2005; Fernie, 2007). Plant metabolites are also of considerable importance in a crop context. Indeed, most plant species that have undergone domestication have become crops specifically because they provide us with a source of chemicals. This is not only true for all of our food crops, but also for many other species of genera such as Pyrethrum (insecticides), Jasminium and Santalum (perfumes), Hevea (rubber), Nicotiana and Cannabis (drugs), Linum (oils), Artemisia and Taxus (pharmaceuticals), Cinnamomum (flavors), etc. However, despite the importance of plants as a source of exploitable and essential biochemicals, we often still have remarkably limited knowledge of the relevant biosynthetic pathways, the genetics behind the key enzymes, and indeed when, why, and where these metabolites are produced and stored within the plant in question (Fernie, 2007; Sumner et al., 2011; Kueger et al., 2012).The field of plant metabolomics has grown tremendously since its recent inception earlier this century (Fiehn et al., 2000; Fiehn, 2002). As an untargeted approach to gain a broad overview of the complexity of plant metabolic composition, the technology has, in a short time, made significant inroads into helping expand our knowledge of plant biochemistry (Kueger et al., 2012; Etalo et al., 2013; Hunerdosse and Nomura, 2014; Meret et al., 2014). Typically, rich metabolomics data sets already provide us with a valuable means to generate hypotheses relating to plant metabolism, which then become the focus of further, more direct investigation (Quanbeck et al., 2012). New technologies are being developed, and especially, new data-mining strategies are being designed to allow us to look deep into plant metabolism without having first to rely on preconceptions. However, there are significant limitations to the application of the technology, which still remain the topic of much research effort.Robust sampling approaches for plant biochemical analysis generally entail taking reliably measurable amounts of plant material that will yield detectable levels of the chemical components. Although for metabolomics analyses, samples of just 50 mg can often suffice, obtaining a reliable sample with minimum biological variation generally requires an initial pooling of materials from which a representative sample is then taken. We therefore treat plant tissue as being homogeneous, but this is clearly a gross oversimplification (Fernie, 2007). Plants have been considered to be composed of roughly 40 different cell types, and a plant organ such as a leaf will generally contain up to 15 different cell types (Martin et al., 2001). Different morphologies also parallel different biochemical composition. Even directly neighboring cells within an organ, for example, a leaf epidermis that often comprises pavement, guard, trichome, and glandular hair cells, are formed from cells already known to have distinctly different biochemistries. Making an extract, for any kind of metabolomics or standard biochemical analysis, therefore entails that we immediately lose most intercellular and intertissue resolution. However, our knowledge is growing in that, in addition to known or expected biochemical differences between cell types, metabolite accumulation across organs can be far from uniform; indeed, islands of higher and lower concentrations of particular metabolites have been observed. This is of course immediately visible when the metabolites concerned can be seen by the naked eye; anthocyanins, for example, are often found to be heterogeneously distributed across leaves, fruits, and flower petals, creating clear phenotypic patterns. The same may also be true of other compounds that are invisible to the human eye but that, in contrast, may still be detectable by insects (e.g. through their fluorescence capacity; see http://www.naturfotograf.com/UV_flowers_list.html; Gronquist et al., 2001).In an ideal situation, we would like to be able to look directly into a plant tissue and be able to analyze the biochemical composition at the single cell level. Some so-called metabolite imaging technologies, usually based on mass spectrometric detection (mass spectrometry imaging [MSI]), have recently been introduced as a step toward this optimistic goal. Included here are matrix-assisted laser desorption/ionization (MALDI)-MSI, direct analysis in real time, and desorption electrospray ionization approaches (Cody et al., 2005; Cornett et al., 2007; Ifa et al., 2010). Early examples of MALDI-MSI have shown not only how primary metabolites such as sugars can be strongly localized within plant organs (Rolletschek et al., 2011), but also how the heterogeneous distribution of glucosinolates in Arabidopsis (Arabidopsis thaliana) can potentially determine grazing behavior of caterpillars (Shroff et al., 2008). This technology continues to improve, and recent exciting developments have resulted in cellular and subcellular imaging of metabolites at a resolution of 5 to 9 µm using MALDI (Korte et al., 2015). However, some key practical limitations of MALDI-based approaches are centered around the need to initially have to pretreat/dehydrate the tissue prior to applying the required matrix solution and the requirement of applying a vacuum during the biochemical analysis. Recently, a new technology has been introduced, laser ablation electrospray ionization (LAESI), which can potentially overcome some of these limitations, given that measurements can be made on fresh, living tissue without the need for a vacuum, thus creating the potential for high-resolution in vivo metabolomics.Here, we report on a set of experiments performed to assess both the potential and limitations of using LAESI-based MSI approaches to perform metabolic mapping on living plant tissues. While identifying a number of technological challenges that still need to be tackled, we were able to show that it is possible to use LAESI-MSI to map metabolites directly onto their known location (in this case, by exploiting the visibility of anthocyanins) as well as localize invisible metabolites in the same tissue. Results have revealed that in plants, for both petal and leaf tissue, the distribution of metabolites can be highly heterogeneous, and that this heterogeneity is of potential relevance to our gaining a broader, more detailed understanding of the overall molecular organization and phenotypic features of plant tissues. Furthermore, knowledge of the nature and extent of this heterogeneity has particular relevance and importance when trying to understand how a plant functions as a system, interacting with its environment. We predict that a higher resolution understanding of plant biochemistry will lead to an increasingly discriminatory capacity in our ability to define more accurately the spatial complexity of plant molecular organization.     

Matrix Assisted Ionization Vacuum (MAIV), a New Ionization Method for Biological Materials Analysis Using Mass Spectrometry

The introduction of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) for the mass spectrometric analysis of peptides and proteins had a dramatic impact on biological science. We now report that a wide variety of compounds, including peptides, proteins, and protein complexes, are transported directly from a solid-state small molecule matrix to gas-phase ions when placed into the vacuum of a mass spectrometer without the use of high voltage, a laser, or added heat. This ionization process produces ions having charge states similar to ESI, making the method applicable for high performance mass spectrometers designed for atmospheric pressure ionization. We demonstrate highly sensitive ionization using intermediate pressure MALDI and modified ESI sources. This matrix and vacuum assisted soft ionization method is suitable for the direct surface analysis of biological materials, including tissue, via mass spectrometry.The conversion of large and nonvolatile compounds such as proteins into gas-phase ions is of immense fundamental and practical importance. The 2002 Nobel Prize in Chemistry was awarded for the accomplishment of this conversion via electrospray ionization (ESI)¹ (1) and matrix-assisted laser desorption/ionization (MALDI) (2) interfaced with mass spectrometry (MS) to obtain the molecular weights of proteins with high accuracy. These methods employ high voltage or a laser to form gaseous analyte ions from a wide variety of compounds in solution or a solid matrix, respectively.MALDI interfaced with a time-of-flight (TOF) mass spectrometer produces gas-phase analyte ions in vacuum and is the method of choice for the molecular imaging of biological surfaces. Ionization in vacuum provides excellent ion transmission (3), as well as good spatial resolution achieved using a focused laser beam. However, the analysis of protein complexes is very challenging with MALDI, requiring strategies such as first-shot phenomena (4) and chemical crosslinking (5). The necessity of a laser also makes MALDI less soft than ESI and produces background ions, which can hinder the analysis of small molecules (6, 7). MALDI is also of limited utility on high performance mass-to-charge (m/z) analyzers because of mass range issues related to the formation of singly charged ions, which also produce few fragment ions for structural characterization (8).Multiple charged ions produced directly from solution in ESI bring the m/z ratio within the range of high performance mass spectrometers, allowing the analysis of high-mass compounds. These instruments have advanced features for structural characterization, such as ion mobility spectrometry (IMS) for gas-phase separations (9–11), ultra-high mass resolution and mass accuracy (12–14), and advanced fragmentation such as electron transfer dissociation (ETD) (13, 14). However, ESI is limited for surface characterization, requiring approaches such as desorption-ESI (15) and laser ablation ESI (16), ionization methods that produce multiply charged ions but are not compatible with analyses of larger proteins or fragile complexes.A softer ionization approach is needed in order to observe fragile molecules and molecular complexes in living organisms at low levels directly from tissue and cell cultures, without extensive sample preparation, while retaining spatial information. Ideally, this approach would be compatible with mass spectrometers having advanced capabilities to aid structural characterization directly from surfaces. The new ionization method described here, in which molecules are transferred from solid-phase to gas-phase ions through the simple exposure of a material of interest in a suitable matrix to vacuum, is an advance toward this goal and is of fundamental interest.     

Study of Matrix Additives for Sensitive Analysis of Lipid A by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been widely used for structural characterization of bacterial endotoxins (lipid A). However, the mass spectrometric behavior of the lipid A molecule is highly dependent on the matrix. Furthermore, this dependence is strongly linked to phosphorylation patterns. Using lipid A from Escherichia coli O116 as a model system, we have investigated the effects of different matrices and comatrix compounds on the analysis of lipid A. In this paper, we report a highly sensitive matrix system for lipid A analysis, which consists of 5-chloro-2-mercaptobenzothiazole matrix and EDTA ammonium salt comatrix. This matrix system enhances the sensitivity of the analysis of diphosphorylated lipid A species by more than 100-fold and in addition provides tolerance to high concentrations of sodium dodecyl sulfate (SDS) and tolerance to sodium chloride and calcium chloride at 10 μM, 100 μM, and 10 μM concentrations. The method was further evaluated for analysis of lipid A species with different phosphorylation patterns and from different bacteria, including Helicobacter pylori, Salmonella enterica serovar Riogrande, and Francisella novicida.Lipopolysaccharide (LPS) is a major component of the outer membranes of Gram-negative bacteria (21). Typically, LPS molecules consist of a hydrophilic carbohydrate portion and a hydrophobic lipid A (or endotoxin). The lipid A molecule consists of a fatty acyl substituted β-d-GlcN-(1-6)-α-GlcN disaccharide unit that usually carries phosphate groups. Diphosphorylated lipid A is generally presumed to be phosphorylated at C-1 and C-4′ positions (9); however, lipid A moieties containing pyrophosphate (PP) groups have also been reported (13). The presence of phosphate groups in lipid A greatly affects the endotoxic properties of LPS (22). Deletion of either of these groups reduces an endotoxic activity of the resulting monophosphorylated LPS by approximately 100-fold (18). For example, monophosphorylated lipid A has been used as an adjuvant in a hepatitis B vaccine in Europe (1, 12).Mass spectrometry (MS) has been widely used to gain information about the heterogeneity, i.e., the number of different species of lipid A families and a distribution of fatty acids on each glucosamine residue (2, 3, 9, 16, 20, 23, 28, 29, 30, 32, 35, 36). Detailed structural information, including the phosphorylation pattern of lipid A, can be obtained by tandem mass spectrometry. Several matrices have been used for the analysis of lipid A using matrix-assisted laser desorption ionization-time of flight MS (MALDI-TOF MS), including 2,5-dihydroxybenzoic acid (DHB), 2,4,6-trihydroxyacetophenone (THAP), and 6-aza-2-thiothymine (ATT) (8). Although DHB has been widely used for peptide analysis, it produces uneven crystals and leads to poor spot-to-spot reproducibility (3, 6, 11). Furthermore, the low solubility in the solvent compatible with lipid A and nonuniformity in a matrix layer (crystals) can lead to variations in the ionization yield across the sample resulting in formation of “hot” (or “sweet”) spots (14). On the other hand, 5-chloro-2-mercaptobenzothiazole (CMBT) was found to offer excellent spot-to-spot reproducibility because of the homogeneous crystallization of the analyte/matrix mixture over the sample spot (33). CMBT is soluble in methanol-chloroform-water (4:4:1, vol:vol:vol), a solvent compatible with lipid A molecules, especially hexaacylated species. Thus, it has been widely used for lipid A analysis (4, 9, 23, 35, 33). Interestingly, different preparation procedures for analysis of lipid A species dictate a selection of the preferred matrix system (10). For example, lipid A prepared using a TRI Reagent-based procedure with a CMBT matrix was preferable for the detection of phosphoethanolamine modifications (35). On the other hand, the analysis of lipid A prepared using an LPS extraction kit-based procedure with DHB was preferable for the detection of aminoarabinose modification (10). In addition, divalent cations, such as Ca²⁺ or Mg²⁺, can bridge the phosphorylated negatively charged groups between neighboring LPS molecules to form aggregates (24). Thus, there is a need for technologies capable of characterizing lipid A from biologically relevant samples in an accurate, rapid, and highly sensitive manner. Here we attempt to establish an optimized MALDI MS matrix system for the sensitive analysis of lipid A, especially its diphosphorylated forms, including both pyrophosphorylated and bisphosphorylated species. We also propose to incorporate a complex reagent (additive or comatrix) for reducing the interference of cations (5, 7, 15).     

Analysis of the Saccharomyces Spindle Pole by Matrix-assisted Laser Desorption/Ionization (MALDI) Mass Spectrometry

A highly enriched spindle pole preparation was prepared from budding yeast and fractionated by SDS gel electrophoresis. Forty-five of the gel bands that appeared enriched in this fraction were analyzed by high-mass accuracy matrix-assisted laser desorption/ ionization (MALDI) peptide mass mapping combined with sequence database searching. This identified twelve of the known spindle pole components and an additional eleven gene products that had not previously been localized to the spindle pole. Immunoelectron microscopy localized eight of these components to different parts of the spindle. One of the gene products, Ndc80p, shows homology to human HEC protein (Chen, Y., D.J. Riley, P-L. Chen, and W-H. Lee. 1997. Mol. Cell Biol. 17:6049–6056) and temperature-sensitive mutants show defects in chromosome segregation. This is the first report of the identification of the components of a large cellular organelle by MALDI peptide mapping alone.     

Automatic Sampling and Analysis of Organics and Biomolecules by Capillary Action-Supported Contactless Atmospheric Pressure Ionization Mass Spectrometry

Contactless atmospheric pressure ionization (C-API) method has been recently developed for mass spectrometric analysis. A tapered capillary is used as both the sampling tube and spray emitter in C-API. No electric contact is required on the capillary tip during C-API mass spectrometric analysis. The simple design of the ionization method enables the automation of the C-API sampling system. In this study, we propose an automatic C-API sampling system consisting of a capillary (∼1 cm), an aluminium sample holder, and a movable XY stage for the mass spectrometric analysis of organics and biomolecules. The aluminium sample holder is controlled by the movable XY stage. The outlet of the C-API capillary is placed in front of the orifice of a mass spectrometer, whereas the sample well on the sample holder is moved underneath the capillary inlet. The sample droplet on the well can be readily infused into the C-API capillary through capillary action. When the sample solution reaches the capillary outlet, the sample spray is readily formed in the proximity of the mass spectrometer applied with a high electric field. The gas phase ions generated from the spray can be readily monitored by the mass spectrometer. We demonstrate that six samples can be analyzed in sequence within 3.5 min using this automatic C-API MS setup. Furthermore, the well containing the rinsing solvent is alternately arranged between the sample wells. Therefore, the C-API capillary could be readily flushed between runs. No carryover problems are observed during the analyses. The sample volume required for the C-API MS analysis is minimal, with less than 1 nL of the sample solution being sufficient for analysis. The feasibility of using this setup for quantitative analysis is also demonstrated.