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.     

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).     

Localized Sampling Enables Monitoring of Cell State via Inline Electrospray Ionization Mass Spectrometry

Nascent advanced therapies, including regenerative medicine and cell and gene therapies, rely on the production of cells in bioreactors that are highly heterogeneous in both space and time. Unfortunately, advanced therapies have failed to reach a wide patient population due to unreliable manufacturing processes that result in batch variability and cost prohibitive production. This can be attributed largely to a void in existing process analytical technologies (PATs) capable of characterizing the secreted critical quality attribute (CQA) biomolecules that correlate with the final product quality. The Dynamic Sampling Platform (DSP) is a PAT for cell bioreactor monitoring that can be coupled to a suite of sensor techniques to provide real-time feedback on spatial and temporal CQA content in situ. In this study, DSP is coupled with electrospray ionization mass spectrometry and direct-from-culture sampling to obtain measures of CQA content in bulk media and the cell microenvironment throughout the entire cell culture process (≈3 weeks). Post hoc analysis of this real-time data reveals that sampling from the microenvironment enables cell state monitoring (e.g., confluence, differentiation). These results demonstrate that an effective PAT should incorporate both spatial and temporal resolution to serve as an effective input for feedback control in biomanufacturing.     

Characterization of a New Abscisic Acid-conjugated Metabolite in Crude Plant Extract by Desorption Chemical Ionization and Secondary Ion Mass Spectrometry

A new conjugate of hydroxyabscisic acid, tentatively named MeHMG-HOABA, along with a known conjugate, β-hydroxy-/?-methylglutarylhydroxyabscisic acid (HMG-HOABA), were iso-lated from immature seeds of Robinia pseudacacia and determined. Evidence for the occurrence of MeHMG-HOABA as a natural metabolite, and not as an artifact, was provided by desorption chemical ionization (DCI) and secondary ion mass spectrometry (SIMS) in conjunction with the technique of linked scanning at constant B/E. The mass spectrometric technique allows the detection and characterization of the conjugates to be analysed even in a crude plant extract.     

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.     

Identification of Clinically Relevant Fungi and Prototheca Species by rRNA Gene Sequencing and Multilocus PCR Coupled with Electrospray Ionization Mass Spectrometry

Background

Multilocus PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) is a new strategy for pathogen identification, but information about its application in fungal identification remains sparse.

Methods

One-hundred and twelve strains and isolates of clinically important fungi and Prototheca species were subjected to both rRNA gene sequencing and PCR/ESI-MS. Three regions of the rRNA gene were used as targets for sequencing: the 5′ end of the large subunit rRNA gene (D1/D2 region), and the internal transcribed spacers 1 and 2 (ITS1 and ITS2 regions). Microbial identification (Micro ID), acquired by combining results of phenotypic methods and rRNA gene sequencing, was used to evaluate the results of PCR/ESI-MS.

Results

For identification of yeasts and filamentous fungi, combined sequencing of the three regions had the best performance (species-level identification rate of 93.8% and 81.8% respectively). The highest species-level identification rate was achieved by sequencing of D1/D2 for yeasts (92.2%) and ITS2 for filamentous fungi (75.8%). The two Prototheca species could be identified to species level by D1/D2 sequencing but not by ITS1 or ITS2. For the 102 strains and isolates within the coverage of PCR/ESI-MS identification, 87.3% (89/102) achieved species-level identification, 100% (89/89) of which were concordant to Micro ID on species/complex level. The species-level identification rates for yeasts and filamentous fungi were 93.9% (62/66) and 75% (27/36) respectively.

Conclusions

rRNA gene sequencing provides accurate identification information, with the best results obtained by a combination of ITS1, ITS2 and D1/D2 sequencing. Our preliminary data indicated that PCR/ESI-MS method also provides a rapid and accurate identification for many clinical relevant fungi.     

Focus on Metabolism: Alteration of Plant Primary Metabolism in Response to Insect Herbivory

Plants in nature, which are continuously challenged by diverse insect herbivores, produce constitutive and inducible defenses to reduce insect damage and preserve their own fitness. In addition to inducing pathways that are directly responsible for the production of toxic and deterrent compounds, insect herbivory causes numerous changes in plant primary metabolism. Whereas the functions of defensive metabolites such as alkaloids, terpenes, and glucosinolates have been studied extensively, the fitness benefits of changes in photosynthesis, carbon transport, and nitrogen allocation remain less well understood. Adding to the complexity of the observed responses, the feeding habits of different insect herbivores can significantly influence the induced changes in plant primary metabolism. In this review, we summarize experimental data addressing the significance of insect feeding habits, as related to herbivore-induced changes in plant primary metabolism. Where possible, we link these physiological changes with current understanding of their underlying molecular mechanisms. Finally, we discuss the potential fitness benefits that host plants receive from altering their primary metabolism in response to insect herbivory.Plants in nature are subject to attack by a wide variety of phytophagous insects. Nevertheless, the world is green, and most plants are resistant to most individual species of insect herbivores. To a large extent, this resistance is due to an array of toxic and deterrent small molecules and proteins that can prevent nonadapted insects from feeding. Although many plant defenses are produced constitutively, others are inducible (i.e. defense-related metabolites and proteins that are normally present at low levels become more abundant in response to insect feeding). Inducible defense systems, which allow more energy to be directed toward growth and reproduction in the absence of insect herbivory, represent a form of resource conservation. Well-studied examples of inducible plant defenses include the production of nicotine in tobacco (Nicotiana tabacum; Baldwin et al., 1998), protease inhibitors in tomato (Solanum lycopersicum; Ryan, 2000), benzoxazinoids in maize (Zea mays; Oikawa et al., 2004), and glucosinolates in Arabidopsis (Arabidopsis thaliana; Mewis et al., 2005). Additionally, herbivore-induced plant responses can include the production of physical defenses such as trichomes or thickened cell walls that can make insect feeding more difficult. Some plant defensive metabolites are highly abundant, suggesting that their biosynthesis can have a significant effect on overall plant metabolism. For instance, benzoxazinoids can constitute 1% to 2% of the total dry matter of some Poaceae (Zúñiga et al., 1983), and up to 6% of the nitrogen in herbivore-induced Nicotiana attenuata can be devoted to nicotine production (Baldwin et al., 1998).In addition to the herbivore-induced production of physical and chemical defenses, numerous changes in plant primary metabolism occur in response to insect herbivory. Among other observed effects, these can include either elevated or suppressed photosynthetic efficiency, remobilization of carbon and nitrogen resources, and altered plant growth rate. However, although the defensive value of induced toxins such as nicotine, terpenes, benzoxazinoids, and glucosinolates is clear, it is sometimes more difficult to elucidate the function of herbivore-induced changes in plant primary metabolism. Insects may also manipulate plant primary metabolism for their own benefit, making it challenging to determine whether the observed changes are actually a plant defensive response.Here, we describe commonly observed changes in plant primary metabolism, focusing on carbohydrates and nitrogen, and discuss their possible functions in plant defense against insect herbivory. There are large differences among published studies involving different plant-herbivore combinations, and no universal patterns in the herbivory-induced changes in plant primary metabolism. Therefore, we also discuss how the potential benefits can depend on the tissue that is being attacked, the extent of the tissue damage, and the type of insect herbivore that is involved in the interaction.     

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.     

Imaging Matrix Assisted Laser Desorption Ionization Mass Spectrometry: a technique to map plant metabolites within tissues at high spatial resolution

Imaging Matrix Assisted Laser Desorption Ionization Mass Spectrometry provides a new and powerful tool to analyse the distribution of metabolites within plant tissues. The two matrices alpha-cyano-4-hydroxycinnamic acid (alpha-CHCA) and 9-aminoacridine provide a useful combination that allows the measurement of amino acids, sugars, and phosphorylated metabolites. Results are presented showing that representatives of these metabolites are unevenly distributed in wheat seeds at different stages of development and under temperature stress.     

Imaging Mass Spectrometry Reveals Acyl-Chain- and Region-Specific Sphingolipid Metabolism in the Kidneys of Sphingomyelin Synthase 2-Deficient Mice

Obesity was reported to cause kidney injury by excessive accumulation of sphingolipids such as sphingomyelin and ceramide. Sphingomyelin synthase 2 (SMS2) is an important enzyme for hepatic sphingolipid homeostasis and its dysfunction is considered to result in fatty liver disease. The expression of SMS2 is also high in the kidneys. However, the contribution of SMS2 on renal sphingolipid metabolism remains unclear. Imaging mass spectrometry is a powerful tool to visualize the distribution and provide quantitative data on lipids in tissue sections. Thus, in this study, we analyzed the effects of SMS2 deficiency on the distribution and concentration of sphingomyelins in the liver and kidneys of mice fed with a normal-diet or a high-fat-diet using imaging mass spectrometry and liquid chromatography/electrospray ionization-tandem mass spectrometry. Our study revealed that high-fat-diet increased C18–C22 sphingomyelins, but decreased C24-sphingomyelins, in the liver and kidneys of wild-type mice. By contrast, SMS2 deficiency decreased C18–C24 sphingomyelins in the liver. Although a similar trend was observed in the whole-kidneys, the effects were minor. Interestingly, imaging mass spectrometry revealed that sphingomyelin localization was specific to each acyl-chain length in the kidneys. Further, SMS2 deficiency mainly decreased C22-sphingomyelin in the renal medulla and C24-sphingomyelins in the renal cortex. Thus, imaging mass spectrometry can provide visual assessment of the contribution of SMS2 on acyl-chain- and region-specific sphingomyelin metabolism in the kidneys.     

Electrospray Ionization-Mass Spectrometry and Tandem Mass Spectrometry Reveal Self-Association and Metal-Ion Binding of Hydrophobic Peptides: A Study of the Gramicidin Dimer

Gramicidin is a membrane pentadecapeptide that acts as a channel, allowing the passage of monovalent metal ions and assisting in bacterial cell death. The active form is a noncovalently bound dimer. One means to study the self-assembly of this peptide has been to compare the state of the peptide in various solvents ranging from hydrophilic (e.g., trifluoroethanol) to hydrophobic (e.g., n-propanol). In this article, we report the use of electrospray mass spectrometry to study the self-association of gramicidin in various organic and mixed solvents that are introduced directly into the mass spectrometer. The dimer (both homo and hetero) can survive the introduction into the gas phase, and the amount in the gas phase increases with the decreasing dielectric constant of the solvent, reflecting solution-phase behavior. Tandem mass spectrometry data reveal that the stability of dimer in the gas phase decreases with increasing metal ion size, strongly suggesting that the metal ion binds inside the dimer between the monomers.     

Focus on Metabolism: Something Old,Something New: Conserved Enzymes and the Evolution of Novelty in Plant Specialized Metabolism

Plants produce hundreds of thousands of small molecules known as specialized metabolites, many of which are of economic and ecological importance. This remarkable variety is a consequence of the diversity and rapid evolution of specialized metabolic pathways. These novel biosynthetic pathways originate via gene duplication or by functional divergence of existing genes, and they subsequently evolve through selection and/or drift. Studies over the past two decades revealed that diverse specialized metabolic pathways have resulted from the incorporation of primary metabolic enzymes. We discuss examples of enzyme recruitment from primary metabolism and the variety of paths taken by duplicated primary metabolic enzymes toward integration into specialized metabolism. These examples provide insight into processes by which plant specialized metabolic pathways evolve and suggest approaches to discover enzymes of previously uncharacterized metabolic networks.The plant kingdom collectively produces hundreds of thousands of low molecular weight organic molecules traditionally known as secondary metabolites, some of which have been shown to play roles in abiotic and biotic stress responses (e.g. herbivory defense), beneficial insect interactions (e.g. pollinator attraction), and communication with other plant and nonplant species (e.g. allelopathy and legume-rhizobia interactions; Saito and Matsuda, 2010; Pichersky and Lewinsohn, 2011; Wink, 2011). These metabolites have been widely used throughout the course of human history as medicines, spices, perfumes, cosmetics, and pest-control agents as well as in religious and cultural rituals. For the past 150 years, there has been a strong focus on documenting the chemical diversity of secondary metabolites in the plant kingdom, leading to the discovery of diverse classes of compounds such as terpenes, flavonoids, alkaloids, phenylpropanoids, glucosinolates, and polyketides. These secondary compounds were historically differentiated from products of primary metabolism, such as sugars, amino acids, nucleic acids, and fatty acids, as being nonessential for plant survival (Sachs, 1874; Kossel, 1891; Hartmann, 2008). However, by the 1980s, important functional roles began to be elucidated for metabolites previously classified as secondary, such as the phenolics (e.g. plant-microbe interactions and UV-B light protection; Bolton et al., 1986; Peters et al., 1986; Li et al., 1993; Landry et al., 1995), alkaloids (defense against herbivory; Steppuhn et al., 2004), and terpenes (defense against herbivory, antimicrobial activities, and volatile pollinator attractants; Papadopoulou et al., 1999; Schiestl and Ayasse, 2001; Erbilgin et al., 2006; Nieuwenhuizen et al., 2009). This change in our understanding of their roles has led to the coining of a new term, specialized metabolites, for these compounds, both to acknowledge their importance and to reflect the fact that many of them are phylogenetically restricted (Pichersky et al., 2006; Pichersky and Lewinsohn, 2011).Although the structural diversity of specialized metabolites far exceeds that of primary metabolites, all specialized metabolite classes are ultimately derived from primary metabolic precursors (Wink, 2011). For example, phenylpropanoids are derived from the amino acid Phe (Vogt, 2010), while the biosynthetic blocks of terpenes, isopentenyl diphosphate and dimethylallyl diphosphate, originate from mevalonate, a sterol precursor, and alternatively from methylerythritol phosphate, which is derived from glycolytic pathway precursors (Kirby and Keasling, 2009). Nitrogen-containing alkaloids are derived from a variety of primary metabolites, including amino acids and purine nucleosides (Facchini, 2001). Over the past 20 years, an increasing number of specialized metabolic enzymes have also been found to have their origins in primary metabolic pathways (Weng, 2014). Such shifts in enzyme function are made possible primarily by the process of gene duplication, which is very common in plants.     

Enantiospecific Analysis of 8‐Prenylnaringenin in Biological Fluids by Liquid‐Chromatography‐Electrospray Ionization Mass Spectrometry: Application to Preclinical Pharmacokinetic Investigations

8‐Prenylnaringenin (8PN) is a naturally occurring bioactive chiral prenylflavonoid found most commonly in the female flowers of hops (Humulus lupulus L.). A stereospecific method of analysis for 8PN in biological fluids is necessary to study the pharmacokinetic disposition of each enantiomer. A novel and simple liquid chromatographic‐electrospray ionization‐mass spectrometry (LC‐ESI‐MS) method was developed for the simultaneous determination of R‐ and S‐8PN in rat serum and urine. Carbamazepine was used as the internal standard (IS). Enantiomeric resolution of 8PN was achieved on a Chiralpak^® AD‐RH column with an isocratic mobile phase consisting of 2‐propanol and 10 mM ammonium formate (pH 8.5) (40:60, v/v) and a flow rate of 0.7 mL/min. Detection was achieved using negative selective ion monitoring (SIM) of 8PN at m/z 339.15 for both enantiomers and positive SIM m/z at 237.15 for the IS. The calibration curves for urine were linear over a range of 0.01–75 µg/mL and 0.05–75 µg/mL for serum with a limit of quantification of 0.05 µg/mL in serum and 0.01 µg/mL in urine. The method was successfully validated showing that it was sensitive, reproducible, and accurate for enantiospecific quantification of 8PN in biological matrices. The assay was successfully applied to a preliminary study of 8PN enantiomers in rat. Chirality 26:419–426, 2014. © 2014 Wiley Periodicals, Inc.     

Focus on Metabolism: Functional Divergence of Diterpene Syntheses in the Medicinal Plant Salvia miltiorrhiza

The medicinal plant Salvia miltiorrhiza produces various tanshinone diterpenoids that have pharmacological activities such as vasorelaxation against ischemia reperfusion injury and antiarrhythmic effects. Their biosynthesis is initiated from the general diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate by sequential reactions catalyzed by copalyl diphosphate synthase (CPS) and kaurene synthase-like cyclases. Here, we report characterization of these enzymatic families from S. miltiorrhiza, which has led to the identification of unique pathways, including roles for separate CPSs in tanshinone production in roots versus aerial tissues (SmCPS1 and SmCPS2, respectively) as well as the unique production of ent-13-epi-manoyl oxide by SmCPS4 and S. miltiorrhiza kaurene synthase-like2 in floral sepals. The conserved SmCPS5 is involved in gibberellin plant hormone biosynthesis. Down-regulation of SmCPS1 by RNA interference resulted in substantial reduction of tanshinones, and metabolomics analysis revealed 21 potential intermediates, indicating a complex network for tanshinone metabolism defined by certain key biosynthetic steps. Notably, the correlation between conservation pattern and stereochemical product outcome of the CPSs observed here suggests a degree of correlation that, especially when combined with the identity of certain key residues, may be predictive. Accordingly, this study provides molecular insights into the evolutionary diversification of functional diterpenoids in plants.Salvia miltiorrhiza, a Lamiaceae species known as red sage or tanshen, is a traditional Chinese medicinal herb that is described in Shen Nong Ben Cao Jing, the oldest classical Chinese herbal book, which dates from between 25 and 220 C.E. The lipophilic pigments from the reddish root and rhizome consist of abietane quinone diterpenoids (Nakao and Fukushima, 1934), largely tanshinone IIA, cryptotanshinone, and tanshinone I (Zhong et al., 2009). These are highly bioactive. For example, tanshinone IIA exerts vasorelaxative activity, has antiarrhythmic effects, provides protection against ischemia reperfusion injury (Zhou et al., 2005; Gao et al., 2008; Sun et al., 2008), and exhibits anticancer activities (Efferth et al., 2008; Lee et al., 2008; Wang et al., 2008; Gong et al., 2011). In addition, tanshinones have been reported to have a broad spectrum of antimicrobial activities against various plant pathogens, including rice (Oryza sativa) blast fungus Magnaporthe oryzae (Zhao et al., 2011). Although tanshinones are mainly accumulated in the roots, trace amounts of tanshinones have been detected in aerial organs as well (Hang et al., 2008).Diterpenoid biosynthesis is initiated by diterpene synthases (diTPSs), which catalyze cyclization and/or rearrangement of the general acyclic precursor (E,E,E)-geranylgeranyl diphosphate (GGPP) to form various hydrocarbon backbone structures that are precursors to more specific families of diterpenoids (Zi et al., 2014). Previous work has indicated that tanshinone biosynthesis is initiated by cyclization of GGPP to copalyl diphosphate (CPP) by a CPP synthase (SmCPS1) and subsequent further cyclization to the abietane miltiradiene by a kaurene synthase-like cyclase (SmKSL1), so named for its homology to the ent-kaurene synthases (KSs) required for GA plant hormone biosynthesis (Gao et al., 2009). Miltiradiene is a precursor to at least cryptotanshinone (Guo et al., 2013), and RNA interference (RNAi) knockdown of SmCPS1 expression reduces tanshinone production, at least in hairy root cultures (Cheng et al., 2014). The identification of SmCPS1 and SmKSL1 has been followed by that of many related diTPSs from other Lamiaceae plant species (Caniard et al., 2012; Sallaud et al., 2012; Schalk et al., 2012; Brückner et al., 2014; Pateraki et al., 2014). These largely exhibit analogous activity, particularly the CPSs, which produce CPP or the stereochemically related 8α-hydroxy-labd-13E-en-15-yl diphosphate (LDPP) rather than the enantiomeric (ent) CPP relevant to GA biosynthesis.To further investigate diterpenoid biosynthesis in S. miltiorrhiza, we report here a more thorough characterization of its diTPS family. A previously reported whole-genome shotgun sequencing survey (Ma et al., 2012) has indicated that there are at least five CPSs, although only two KSL genes in S. miltiorrhiza (Supplemental Table S1). Intriguingly, based on a combination of biochemical and genetic (RNAi gene silencing) evidence, we find that these diTPSs nevertheless account for at least four different diterpenoid biosynthetic pathways, each dependent on a unique CPS, with the KS presumably involved in GA biosynthesis seeming to be responsible for alternative diterpenoid metabolism as well. In addition, our studies clarify the evolutionary basis for the observed functional diversity, with investigation of gene structure, positive selection, molecular docking, and mutational analysis used to explore the driving force for the functional divergence of these diTPSs. Moreover, we report metabolomic analysis, also carried out with SmCPS1 RNAi lines, which enables prediction of the downstream steps in tanshinone biosynthesis.     

Analysis of Polymerase Chain Reaction-Amplified DNA Products by Mass Spectrometry Using Matrix-Assisted Laser Desorption and Electrospray: Current Status

Recent advances in molecular biology are making it possible to diagnose genetic diseases and identify pathogens through the analysis of DNA. As clinical applications for molecular diagnosis increase, rapid, reliable methods for determination of DNA size will be needed. Mass spectrometry offers the potential of analyzing amplified DNA quickly and reliably, without the need for gel-based separation and sample labeling steps that are conventionally employed. Both electrospray ionization and matrix-assisted laser desorption/ionization have been evaluated for the size analysis of DNA using both synthetic oligonucleotides and PCR-amplified samples corresponding to bases 1626 to 1701 of the cystic fibrosis transmembrane conductance regulator gene. Both technologies have been demonstrated to have mass range and sensitivity required for the analysis of PCR-amplified DNA in this size range using minimal sample preparation. Steps required to incorporate either ionization technique into a reliable analytical scheme for the rapid, routine analysis of DNA are outlined.     

电喷雾萃取电离技术在蛋白质分析中的应用及展望

电喷雾萃取电离技术(extractive electrospray ionization,EESI)是一种能灵敏地电离固体、液体、气体、黏性样品等复杂基体中痕量大分子和小分子的新兴软电离技术．在简要介绍EESI原理的基础上,着重综述其在蛋白质分析中的应用．与商品化质谱仪器配置的电喷雾电离源(electrospray ionization,ESI)不同,EESI能够在常压条件下最大程度地保留蛋白质在样品中的原始构象,并获得大量具有生物活性的蛋白质离子．由此可见,EESI及类似的技术在蛋白质芯片制备、高分辨率氢/氘交换质谱(蛋白质结构分析)、蛋白质计量等方面具有良好的应用前景．本文也对其发展趋势进行了展望．     

MALDI Mass Spectrometry Imaging: A Novel Tool for the Identification and Classification of Amyloidosis

Amyloidosis is a group of diseases caused by extracellular accumulation of fibrillar polypeptide aggregates. So far, diagnosis is performed by Congo red staining of tissue sections in combination with polarization microscopy. Subsequent identification of the causative protein by immunohistochemistry harbors some difficulties regarding sensitivity and specificity. Mass spectrometry based approaches have been demonstrated to constitute a reliable method to supplement typing of amyloidosis, but still depend on Congo red staining. In the present study, we used matrix‐assisted laser desorption/ionization mass spectrometry imaging coupled with ion mobility separation (MALDI‐IMS MSI) to investigate amyloid deposits in formalin‐fixed and paraffin‐embedded tissue samples. Utilizing a novel peptide filter method, we found a universal peptide signature for amyloidoses. Furthermore, differences in the peptide composition of ALλ and ATTR amyloid were revealed and used to build a reliable classification model. Integrating the peptide filter in MALDI‐IMS MSI analysis, we developed a bioinformatics workflow facilitating the identification and classification of amyloidosis in a less time and sample‐consuming experimental setup. Our findings demonstrate also the feasibility to investigate the amyloid's protein composition, thus paving the way to establish classification models for the diverse types of amyloidoses and to shed further light on the complex process of amyloidogenesis.     

Comparison of Non-covalent Interactions Between a Series of <Emphasis Type=Italic>N</Emphasis>-Phosphoryl Dipeptide or Methyl Esters and Protein by Electrospray Ionization Mass Spectrometry

The non-covalent interaction between a series of N-phosphoryl dipeptides (or methyl esters) (DPP) and protein was studied by ESI-MS and UV–vis spectrometer. The function of different groups in DPP and binding sites of protein were investigated. The results revealed that hydroxyl and aromatic ring in DPP were both important group for the interaction, and aromatic ring had double functions on the interaction. In addition, the molecular size, flexibility and steric hindrance showed obvious effects on the interaction, while, the chirality, sequence and length of carbon chains (changing 1–2C) of amino acid residue in DPP showed little effects on the interaction under the experimental conditions. Phosphoryl oligopeptides having extended structure, good molecular flexibility and smaller spatial hindrance could contract the protein conformation in solution. The aromatic, basic, acid and amide amino acid residues of protein may be the main binding sites and contributed to the survival of the complexes.