Quantitative trait loci analysis of seed‐specialized metabolites reveals seed‐specific flavonols and differential regulation of glycoalkaloid content in tomato

Given the potential health benefits (and adverse effects), of polyphenolic and steroidal glycoalkaloids in the diet there is a growing interest in fully elucidating the genetic control of their levels in foodstuffs. Here we carried out profiling of the specialized metabolites in the seeds of the Solanum pennellii introgression lines identifying 338 putative metabolite quantitative trait loci (mQTL) for flavonoids, steroidal glycoalkaloids and further specialized metabolites. Two putative mQTL for flavonols and one for steroidal glycoalkaloids were cross‐validated by evaluation of the metabolite content of recombinants harboring smaller introgression in the corresponding QTL interval or by analysis of lines from an independently derived backcross inbred line population. The steroidal glycoalkaloid mQTL was localized to a chromosomal region spanning 14 genes, including a previously defined steroidal glycoalkaloid gene cluster. The flavonoid mQTL was further validated via the use of transient and stable overexpression of the Solyc12g098600 and Solyc12g096870 genes, which encode seed‐specific uridine 5′‐diphosphate‐glycosyltransferases. The results are discussed in the context of our understanding of the accumulation of polyphenols and steroidal glycoalkaloids, and how this knowledge may be incorporated into breeding strategies aimed at improving nutritional aspects of plants as well as in fortifying them against abiotic stress.     

Comprehensive mass spectrometry‐guided phenotyping of plant specialized metabolites reveals metabolic diversity in the cosmopolitan plant family Rhamnaceae

Plants produce a myriad of specialized metabolites to overcome their sessile habit and combat biotic as well as abiotic stresses. Evolution has shaped the diversity of specialized metabolites, which then drives many other aspects of plant biodiversity. However, until recently, large‐scale studies investigating the diversity of specialized metabolites in an evolutionary context have been limited by the impossibility of identifying chemical structures of hundreds to thousands of compounds in a time‐feasible manner. Here we introduce a workflow for large‐scale, semi‐automated annotation of specialized metabolites and apply it to over 1000 metabolites of the cosmopolitan plant family Rhamnaceae. We enhance the putative annotation coverage dramatically, from 2.5% based on spectral library matches alone to 42.6% of total MS/MS molecular features, extending annotations from well‐known plant compound classes into dark plant metabolomics. To gain insights into substructural diversity within this plant family, we also extract patterns of co‐occurring fragments and neutral losses, so‐called Mass2Motifs, from the dataset; for example, only the Ziziphoid clade developed the triterpenoid biosynthetic pathway, whereas the Rhamnoid clade predominantly developed diversity in flavonoid glycosides, including 7‐O‐methyltransferase activity. Our workflow provides the foundations for the automated, high‐throughput chemical identification of massive metabolite spaces, and we expect it to revolutionize our understanding of plant chemoevolutionary mechanisms.     

质谱流式技术用于单细胞检测

质谱流式技术(mass cytometry)是利用质谱原理对单细胞进行多参数检测的流式技术,能够在单细胞水平实现超过50种标志物的同时测量,显著增强了对细胞生长进程和复杂细胞系统的评估能力。该文简要介绍了质谱流式技术的基本工作原理,并从金属元素标记、质量分析器、高维单细胞数据处理等方面展开论述,阐明设计新型金属元素标签和选择飞行时间质谱的必要性,归纳分析高维单细胞数据的算法并总结各种算法的优点和局限性。     

三种前处理在基质辅助激光解析电离飞行时间质谱鉴定假丝酵母菌属中的应用比较

目的 比较3种前处理方法在基质辅助激光解析电离飞行时间质谱(MALDI TOF MS)鉴定假丝酵母菌属中的结果可靠性。 方法 以ITS测序鉴定结果为金标准,对临床分离的66株假丝酵母分别采用传统直涂法、改良直涂法和甲酸-乙腈蛋白提取法进行前处理,MALDI TOF MS鉴定,比较3种方法的Biotyper Log值,分析质谱图的差异。 结果 传统直涂法、改良直涂法和甲酸-乙腈提取法对66株假丝酵母的属水平鉴定率分别为48.5%、50.0%和97.0%,Biotyper Log均值分别为1.628、1.674和2.010,其中甲酸-乙腈提取法对66株假丝酵母的种水平鉴定率为53.0%。甲酸-乙腈提取法得到的质谱图比另2种方法的质谱图离子峰更加密集,图像更复杂,鉴定结果可信度更高。 结论 甲酸-乙腈蛋白提取法对假丝酵母菌属的鉴定成功率和可靠性明显高于传统直涂法和改良直涂法,对临床假丝酵母菌病的准确诊断具有重要价值。     

Mass spectrometry of hydrogen/deuterium exchange in 70S ribosomal proteins from E. coli

The 70S ribosome from Escherichia coli is a supermacro complex (MW: 2.7MDa) comprising three RNA molecules and more than 50 proteins. We have for the first time successfully analyzed the flexibility of 70S ribosomal proteins in solution by detecting the hydrogen/deuterium exchange with mass spectrometry. Based on the deuterium incorporation map of the X-ray structure obtained at the time of each exchange, we demonstrate the structure-flexibility-function relationship of ribosome focusing on the deuterium incorporation of the proteins binding ligands (tRNA, mRNA, and elongation factor) and the relation with structural assembly processes.     

Global analysis of metabolites in rat and human urine based on gas chromatography/time-of-flight mass spectrometry

Sediment in urine may contain low-molecular-weight compounds that should be included in the analysis. To date, no systematic investigation has addressed this issue. We investigated three primary factors that influence the extraction efficiency of metabolites during preparation of urine samples for metabolomic research: centrifugation, pH, and extraction solvents. Obtained with the use of gas chromatography/time-of-flight mass spectrometry (GC/TOFMS) technique and principal component analysis (PCA), our results indicate that (1) conventional centrifugation causes an apparent loss of some metabolites, indicating that urine samples for metabolomic research should not be centrifuged before procedures are undertaken to recover the metabolites; (2) pH adjustment has a large impact on the recovery of metabolites and is therefore not encouraged; (3) with design of experiment analysis, methanol and water yield the optimal extraction efficiency. Differences between rat and human urine were observed and are discussed. Ninety-nine metabolites identified in rat and human urine are presented. An efficient protocol is proposed for the pretreatment of urine samples.     

Unveiling the spatial distribution of aflatoxin B1 and plant defense metabolites in maize using AP-SMALDI mass spectrometry imaging

In order to cope with the presence of unfavorable compounds, plants can biotransform xenobiotics, translocate both parent compounds and metabolites, and perform compartmentation and segregation at the cellular or tissue level. Such a scenario also applies to mycotoxins, fungal secondary metabolites with a pre-eminent role in plant infection. In this work, we aimed to describe the effect of the interplay between Zea mays (maize) and aflatoxin B1 (AFB1) at the tissue and organ level. To address this challenge, we used atmospheric pressure scanning microprobe matrix-assisted laser desorption/ionization mass spectrometry imaging (AP-SMALDI MSI) to investigate the biotransformation, localization and subsequent effects of AFB1 on primary and secondary metabolism of healthy maize plants, both in situ and from a metabolomics standpoint. High spatial resolution (5 µm) provided fine localization of AFB1, which was located within the root intercellular spaces, and co-localized with its phase-I metabolite aflatoxin M2. We provided a parallel visualization of maize metabolic changes, induced in different organs and tissues by an accumulation of AFB1. According to our untargeted metabolomics investigation, anthocyanin biosynthesis and chlorophyll metabolism in roots are most affected. The biosynthesis of these metabolites appears to be inhibited by AFB1 accumulation. On the other hand, metabolites found in above-ground organs suggest that the presence of AFB1 may also activate the biochemical response in the absence of an actual fungal infection; indeed, several plant secondary metabolites known for their antimicrobial or antioxidant activities were localized in the outer tissues, such as phenylpropanoids, benzoxazinoids, phytohormones and lipids.     

基质辅助激光解吸电离飞行时间质谱技术用于常见益生菌鉴定的初步研究

目的建立基质辅助激光解吸电离飞行时间质谱(MADLI-TOF MS)技术鉴定常见益生菌的实验方法并对MADLI-TOF MS技术的适用性进行初步评价。方法对MADLI-TOF MS技术鉴定常见益生菌过程中各影响因素进行考察,筛选出最佳的实验条件。利用19株供试菌株所得的蛋白指纹图谱对MADLI-TOF MS技术的适用性进行研究。结果建立了MADLI-TOF MS技术鉴定常见益生菌的最佳实验方法。初步证明MADLI-TOF MS技术具备在属、种、亚种以及菌株水平上鉴定常见益生菌的能力。结论建立的实验方法稳定性高、重复性好,可以作为MADLI-TOF MS技术鉴定常见益生菌的参考方法。MADLI-TOF MS技术可以作为常见益生菌鉴定的方法之一。     

Mass spectrometry analysis of HIV-1 Vif reveals an increase in ordered structure upon oligomerization in regions necessary for viral infectivity

HIV-1 Vif, an accessory protein in the viral genome, performs an important role in viral pathogenesis by facilitating the degradation of APOBEC3G, an endogenous cellular inhibitor of HIV-1 replication. In this study, intrinsically disordered regions are predicted in HIV-1 Vif using sequence-based algorithms. Intrinsic disorder may explain why traditional structure determination of HIV-1 Vif has been elusive, making structure-based drug design impossible. To characterize HIV-1 Vif's structural topology and to map the domains involved in oligomerization we used chemical cross-linking, proteolysis, and mass spectrometry. Cross-linking showed evidence of monomer, dimer, and trimer species via denaturing gel analysis and an additional tetramer via western blot analysis. We identified 47 unique linear peptides and 24 (13 intramolecular; 11 intermolecular) noncontiguous, cross-linked peptides, among the noncross-linked monomer, cross-linked monomer, cross-linked dimer, and cross-linked trimer samples. Almost complete peptide coverage of the N-terminus is observed in all samples analyzed, however reduced peptide coverage in the C-terminal region is observed in the dimer and trimer samples. These differences in peptide coverage or "protections" between dimer and trimer indicate specific differences in packing between the two oligomeric forms. Intramolecular cross-links within the monomer suggest that the N-terminus is likely folded into a compact domain, while the C-terminus remains intrinsically disordered. Upon oligomerization, as evidenced by the intermolecular cross-links, the C-terminus of one Vif protein becomes ordered by wrapping back on the N-terminal domain of another. In addition, the majority of the intramolecular cross-links map to regions that have been previously reported to be necessary for viral infectivity. Thus, this data suggests HIV-1 Vif is in a dynamic equilibrium between the various oligomers potentially allowing it to interact with other binding partners.     

Hydrogen-exchange mass spectrometry reveals activation-induced changes in the conformational mobility of p38alpha MAP kinase

Hydrogen-deuterium exchange measurements represent a powerful approach to investigating changes in conformation and conformational mobility in proteins. Here, we examine p38α MAP kinase (MAPK) by hydrogen-exchange (HX) mass spectrometry to determine whether changes in conformational mobility may be induced by kinase phosphorylation and activation. Factors influencing sequence coverage in the HX mass spectrometry experiment, which show that varying sampling depths, instruments, and peptide search strategies yield the highest coverage of exchangeable amides, are examined. Patterns of regional deuteration in p38α are consistent with tertiary structure and similar to deuteration patterns previously determined for extracellular-signal-regulated kinase (ERK) 2, indicating that MAPKs are conserved with respect to the extent of local amide HX. Activation of p38α alters HX in five regions, which are interpreted by comparing X-ray structures of unphosphorylated p38α and X-ray structures of phosphorylated p38γ. Conformational differences account for altered HX within the activation lip, the P + 1 site, and the active site. In contrast, HX alterations are ascribed to activation-induced effects on conformational mobility, within substrate-docking sites (αF-αG, β7-β8), the C-terminal core (αE), and the N-terminal core region (β4-β5, αL16, αC). Activation also decreases HX in a 3-10 helix at the C-terminal extension of p38α. Although this helix in ERK2 forms a dimerization interface that becomes protected from HX upon activation, analytical ultracentrifugation shows that this does not occur in p38α because both unphosphorylated and diphosphorylated forms are monomeric. Finally, HX patterns in monophosphorylated p38α are similar to those in unphosphorylated kinase, indicating that the major activation lip remodeling events occur only after diphosphorylation. Importantly, patterns of activation-induced HX show differences between p38α and ERK2 despite their similarities in overall deuteration, suggesting that although MAPKs are closely related with respect to primary sequence and tertiary structure, they have distinct mechanisms for dynamic control of enzyme function.     

Investigating the Effects of Chemical Gradients on Performance and Reliability within Perovskite Solar Cells with TOF‐SIMS

Time‐of‐flight secondary‐ion mass spectrometry (TOF‐SIMS), a powerful analytical technique sensitive to all components of perovskite solar cell (PSC) materials, can differentiate between the various organic species within a PSC absorber or a complete device stack. The ability to probe chemical gradients through the depth of a device (both organic and inorganic), with down to 100 nm lateral resolution, can lead to unique insights into the relationships between chemistry in the absorber bulk, at grain boundaries, and at interfaces as well as how they relate to changes in performance and/or stability. In this review, the technique is described; then, from the literature, several examples of how TOF‐SIMS have been used to provide unique insight into PSC absorbers and devices are covered. Finally, the common artifacts that can be introduced if the data are improperly collected, as well as methods to mitigate these artifacts are discussed.     

Lipoproteomics I: mapping of proteins in low-density lipoprotein using two-dimensional gel electrophoresis and mass spectrometry

The molecular mechanisms underlying the relationship between low-density lipoprotein (LDL) and the risk of atherosclerosis are not clear. Therefore, detailed information about the protein composition of LDL may contribute to reveal its role in atherogenesis and the mechanisms that lead to coronary disease in humans. Here, we sought to map the proteins in human LDL by a proteomic approach. LDL was isolated by two-step discontinuous density-gradient ultracentrifugation and the proteins were separated with two-dimensional gel electrophoresis and identified with peptide mass fingerprinting, using matrix assisted laser desorption/ionization-time of flight-mass spectrometry and with amino acid sequencing using electrospray ionization tandem mass spectrometry. These procedures identified apo B-100, apo C-II, apo C-III (three isoforms), apo E (four isoforms), apo A-I (two isoforms), apo A-IV, apo J and apo M (three isoforms not previously described). In addition, three proteins that have not previously been identified in LDL were found: serum amyloid A-IV (two isoforms), calgranulin A, and lysozyme C. The identities of apo M, calgranulin A, and lysozyme C were confirmed by sequence information obtained after collision-induced dissociation fragmentation of peptides characteristic for these proteins. Moreover, the presence of lysozyme C was further corroborated by demonstrating enriched hydrolytic activity in LDL against Micrococcus lysodeikticus. These results indicate that in addition to the dominating apo B-100, LDL contains a number of other apolipoproteins, many of which occur in different isoforms. The demonstration, for the first time, that LDL contains calgranulin A and lysozyme C raises the possibility that LDL proteins may play hitherto unknown role(s) in immune and inflammatory reactions of the arterial wall.     

Structural identification of methyl protodioscin metabolites in rats' urine and their antiproliferative activities against human tumor cell lines

Methyl protodioscin (MPD), a furostanol saponin, is a preclinical drug shown potent antiproliferative activities against most cell lines from leukemia and solid tumors. The metabolites of MPD in rats' urine after single oral doses of 80 mg/kg were investigated in this research. Ten metabolites were isolated and purified by liquid-liquid extraction, open-column chromatography, medium-pressure liquid chromatography, and preparative high-performance liquid chromatography. The structural identification of the metabolites was carried out by high resolution mass spectra, NMR spectroscopic methods including (1)H NMR, (13)C NMR and 2D NMR, as well as chemical ways. The 10 metabolites were elucidated to be dioscin (M-1), pregna-5,16-dien-3beta-ol-20-one-O-alpha-l-rhamnopyranosyl-(1-->2)-[alpha-l-rhamnopyranosyl-(1-->4)]-beta-d-glucopyranoside (M-2), diosgenin (M-3), protobioside (M-4), methyl protobioside (M-5), 26-O-beta-d-glucopyrannosyl(25R)-furan-5-ene-3beta, 22alpha, 26-trihydroxy-3-O-alpha-l-rhamnopyranosyl-(1-->4)-beta-d-glucopyranoside(M-6),26-O-beta-d-glucopyranosyl(25R)-furan-5-ene-3beta,26-dihydroxy-22-methoxy-3-O-alpha-l-rhamnopyranosyl-(1-->4)-beta-d-glucopyranoside (M-7), prosapogenin A of dioscin (M-8), prosapogenin B of dioscin (M-9), and diosgenin-3-O-beta-d-glucopyranoside (M-10), respectively. M-1 was the main urinary metabolite of MPD in rats. Some metabolites showed potent antiproliferative activities against HepG2, NCI-H460, MCF-7 and HeLa cell lines in vitro.     

基质辅助激光解吸电离飞行时间质谱技术作为常见益生菌菌种鉴定辅助工具的研究

目的评价基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)技术用于常见益生菌菌株鉴定及潜在益生菌菌株筛选的可行性。方法利用16S rDNA序列分析在方法学上对MALDI-TOF MS技术的鉴定能力进行研究;通过MALDI-TOF MS技术对现有保藏菌株的鉴定结果研究MALDI-TOF MS技术的鉴定准确性及优越性。结果 MALDI-TOF MS技术具备较16S rDNA序列分析更高的菌株鉴定能力;MALDI-TOF MS技术的鉴定结果准确、稳定。结论 MALDI-TOF MS技术可以作为准确、快速、廉价及可高通量操作的菌株鉴定方法应用于常见益生菌菌株的鉴定及潜在益生菌菌株的筛选。     

Lipid mapping of colonic mucosa by cluster TOF-SIMS imaging and multivariate analysis in cftr knockout mice

The cftr knockout mouse model of cystic fibrosis (CF) shows intestinal obstruction; malabsorption and inflammation; and a fatty acid imbalance in intestinal mucosa. We performed a lipid mapping of colon sections from CF and control (WT) mice by cluster time of flight secondary-ion mass spectrometry (TOF-SIMS) imaging to localize lipid alterations. Data were processed either manually or by multivariate statistical methods. TOF-SIMS analysis showed a particular localization for cholesteryl sulfate at the epithelial border, C16:1 fatty acid in Lieberkühn glands, and C18:0 fatty acid in lamina propria and submucosa. Significant increases in vitamin E (vE) and C16:0 fatty acid in the epithelial border of CF colon were detected. Principal component analysis (PCA) and partitioning clustering allowed us to characterize different structural regions of colonic mucosa according to variations in C14:0, C16:0, C16:1, C18:0, C18:1, C18:2, C20:3, C20:4, and C22:6 fatty acids; phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol glycerolipids; cholesterol; vitamin E; and cholesteryl sulfate. PCA on spectra from Lieberkühn glands led to separation of CF and WT individuals. This study shows for the first time the spatial distribution of lipids in colonic mucosa and suggests TOF-SIMS plus multivariate analyses as a powerful tool to investigate disease-related tissue spatial lipid signatures.     

MALDI-TOF MS技术在临床病原真菌鉴定中的应用进展

基质辅助激光解吸电离飞行时间质谱技术（MALDI-TOF-MS）目前是一种快速而可靠的微生物鉴定方法.随着可鉴定真菌谱的完善,MALDI-TOF MS技术已逐步应用于临床常见致病酵母菌、酵母样真菌和丝状菌的鉴定中,本文将就此做一综述.     

Evidence of different G-quadruplex DNA binding with biogenic polyamines probed by electrospray ionization-quadrupole time of flight mass spectrometry,circular dichroism and atomic force microscopy

The binding properties of five G-quadruplex oligonucleotides (humtel24, k-ras32, c-myc22, c-kit1 and c-kit2) with polyamines have been investigated by electrospray ionization-quadrupole time of flight mass spectrometry, circular dichroism, melting temperature, atomic force microscopy (AFM) and molecular simulation. The MS results demonstrated that the polyamines and G-quadruplex DNA can form complexes with high affinity, and one molecule of G-quadruplex DNA can combine several molecules (1–5) of polyamines. The binding affinities of the polyamines to DNA were in the order of spermine > spermidine > putrescine. After binding with polyamines, the conformations of the G-quadruplex DNA were significantly changed, and spermine can induce the configurations of k-ras32 and c-kit1 to deviate from their G-quadruplex structures at high concentrations. In the presence of K⁺, the conformations of G-quadruplex DNA were stabilized, while polyamines can also induced alterations of their configurations. Melting temperature experiments suggested that the T_m of the DNA–polyamine complexes obviously increased both in the absence and presence of K⁺. The AFM results indicated that polyamines can induce aggregation of G-quadruplex DNA. Above results illustrated that the polyamines bound with the phosphate backbone and the base-pairs of G-quadruplex structures. Combining with the molecular simulation, the binding mode of the G-quadruplex DNA and polyamines were discussed. The results obtained would be beneficial for understanding the biological and physiological functions of polyamines and provide useful information for development of antitumor drugs.     

A new method to improve sensitivity and resolution in matrix-assisted laser desorption/ionization time of flight mass spectrometry

High detection sensitivity and resolution are two critical parameters for recording good peptide mass fingerprints (PMF) of low abundance proteins. This paper reports a mass spectrometry (MS) sample preparation technique that could improve sensitivity and resolution. By coating the MS steel target with a thin layer of pentadecafluorooctamido propyltrimethoxysilane, which was both polar and nonpolar solvent repellent, the transferred sample droplets on its surface were significantly smaller. As a result, the analyte of the peptide mixture became more concentrated and homogeneous, which helped to improve the sensitivity. The advantages of a modified MS target were documented by mass spectra improvement of attomole level standard peptides and silver-stained proteins from polyacrylamide gels. The mass signal of angiotensin II at 100 attomole was difficult to record on the conventional support, whereas it was easily detected on the modified one. The PMF of cytochrome C was also better recorded on the modified support, in terms of both signal-to-noise ratio and the number of detected peptides. When silver-stained proteins from two-dimensional electrophoresis gels were analyzed, in most cases more satisfactory peptide mass spectra were obtained from the modified support. Searching protein databases with more mass data from the improved PMFs, several unknown proteins were successfully identified.     

New preparation techniques for molecular and in‐situ analysis of ancient organic micro‐ and nanostructures

Organic microfossils preserved in three dimensions in transparent mineral matrices such as cherts/quartzites, phosphates, or carbonates are best studied in petrographic thin sections. Moreover, microscale mass spectrometry techniques commonly require flat, polished surfaces to minimize analytical bias. However, contamination by epoxy resin in traditional petrographic sections is problematic for the geochemical study of the kerogen in these microfossils and more generally for the in situ analysis of fossil organic matter. Here, we show that epoxy contamination has a molecular signature that is difficult to distinguish from kerogen with time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS). This contamination appears pervasive in organic microstructures embedded in micro‐ to nano‐crystalline carbonate. To solve this problem, a new semi‐thin section preparation protocol without resin medium was developed for micro‐ to nanoscale in situ investigation of insoluble organic matter. We show that these sections are suited for microscopic observation of Proterozoic microfossils in cherts. ToF‐SIMS reveals that these sections are free of pollution after final removal of a <10 nm layer of contamination using low‐dose ion sputtering. ToF‐SIMS maps of fragments from aliphatic and aromatic molecules and organic sulfur are correlated with the spatial distribution of organic microlaminae in a Jurassic stromatolite. Hydrocarbon‐derived ions also appeared correlated with kerogenous microstructures in Archean cherts. These developments in analytical procedures should help future investigations of organic matter and in particular, microfossils, by allowing the spatial correlation of microscopy, spectroscopy, precise isotopic microanalyses, and novel molecular microanalyses such as ToF‐SIMS.