Study on the properties of the S3-state by mass spectrometry in the filamentous cyanobacterium Oscillatoria chalybea

In the present paper we analyzed the properties of the S₃-state in the filamentous cyanobacterium Oscillatoria chalybea by mass spectrometry. In this organism a substantial O₂-signal due to a single flash is observed even after extensive dark adaptation (20 min). This signal can be measured by mass spectrometry as well as amperometrically on an oxygen electrode and is not due to an interference of respiratory and photosynthetic electron transport in the prokaryotic membrane. The mass spectrometric analysis shows that, if S₃ is generated by two flashes in a medium containing only H₂¹⁶O, addition of H₂¹⁸O and subsequent firing of a third flash yields O₂ evolution labelled with ¹⁸O. It appears that the isotopic composition of the O₂ evolved corresponds to the isotopic composition of the water in the suspension. This experiment shows that water oxidation does not proceed via an oxygen precursor or water derivatives bound to the S₃-state. This conclusion has been reached shortly before ours by Radmer and Ollinger [15] in the reverse marker experiment. From our study with O. chalybea it appears that freshly generated S₃ can be distinguished from metastable S₃ by the mass spectrometric method. It looks as if in contrast to freshly generated S₃ metastable S₃ contained bound unexchangeable water or an oxidized water derivative.     

Synthesis and Mass Spectrometric Fragmentation Characteristics of Imidazole Ribosides–Analogs of Intermediates of Purine De Novo Synthetic Pathway

Two inherited deficiencies have been described in purine de novo synthesis pathway. Both the defects are diagnosed by detecting ribosides—dephosphorylated substrates of the enzymes—in patient's urine. We describe here a synthesis and mass spectrometric fragmentation of ribosides potentially of diagnostic importance for defects in the second part of the pathway. All the species, except 5-amino-4-imidazolesuccinocarboxamideriboside can be synthesized from the commercially available 5-amino-4-imidazolecarboxamideriboside by chemical methods. Fragmentation spectra of the compounds were obtained by the ion trap mass spectrometry. During fragmentation an opening of the imidazole ring was not observed for any of the compounds but loss of its substituents in the form of small molecules (NH₃, CO₂, CO) is the major route of fragmentation. The ribose moiety cleaves off molecule(s) of water, undergoes a cross-ring cleavage or breaks away as a whole.     

The yeast metabolome addressed by electrospray ionization mass spectrometry: Initiation of a mass spectral library and its applications for metabolic footprinting by direct infusion mass spectrometry

Mass spectrometry (MS) has been a major driver for metabolomics, and gas chromatography (GC)-MS has been one of the primary techniques used for microbial metabolomics. The use of liquid chromatography (LC)-MS has however been limited, but electrospray ionization (ESI) is very well suited for ionization of microbial metabolites without any previous derivatization needed. To address the capabilities of ESI-MS in detecting the metabolome of Saccharomyces cerevisiae, the in silico metabolome of this organism was used as a template to present a theoretical metabolome. This showed that in combination with the specificity of MS up to 84% of the metabolites can be identified in a high mass accuracy ESI-spectrum. A total of 66 metabolites were systematically analyzed by positive and negative ESI-MS/MS with the aim of initiating a spectral library for ESI of microbial metabolites. This systematic analysis gave insight into the ionization and fragmentation characteristics of the different metabolites. With this insight, a small study of metabolic footprinting with ESI-MS demonstrated that biological information can be extracted from footprinting spectra. Statistical analysis of the footprinting data revealed discriminating ions, which could be assigned using the in silico metabolome. By this approach metabolic footprinting can advance from a classification method that is used to derive biological information based on guilt-by-association, to a tool for extraction of metabolic differences, which can guide new targeted biological experiments. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Peak intensity prediction in MALDI-TOF mass spectrometry: A machine learning study to support quantitative proteomics

Background  

Mass spectrometry is a key technique in proteomics and can be used to analyze complex samples quickly. One key problem with the mass spectrometric analysis of peptides and proteins, however, is the fact that absolute quantification is severely hampered by the unclear relationship between the observed peak intensity and the peptide concentration in the sample. While there are numerous approaches to circumvent this problem experimentally (e.g. labeling techniques), reliable prediction of the peak intensities from peptide sequences could provide a peptide-specific correction factor. Thus, it would be a valuable tool towards label-free absolute quantification.     

Protein identification from two-dimensional gel electrophoresis analysis of Klebsiella pneumoniae by combined use of mass spectrometry data and raw genome sequences

Separation of proteins by two-dimensional gel electrophoresis (2-DE) coupled with identification of proteins through peptide mass fingerprinting (PMF) by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is the widely used technique for proteomic analysis. This approach relies, however, on the presence of the proteins studied in public-accessible protein databases or the availability of annotated genome sequences of an organism. In this work, we investigated the reliability of using raw genome sequences for identifying proteins by PMF without the need of additional information such as amino acid sequences. The method is demonstrated for proteomic analysis of Klebsiella pneumoniae grown anaerobically on glycerol. For 197 spots excised from 2-DE gels and submitted for mass spectrometric analysis 164 spots were clearly identified as 122 individual proteins. 95% of the 164 spots can be successfully identified merely by using peptide mass fingerprints and a strain-specific protein database (ProtKpn) constructed from the raw genome sequences of K. pneumoniae. Cross-species protein searching in the public databases mainly resulted in the identification of 57% of the 66 high expressed protein spots in comparison to 97% by using the ProtKpn database. 10 dha regulon related proteins that are essential for the initial enzymatic steps of anaerobic glycerol metabolism were successfully identified using the ProtKpn database, whereas none of them could be identified by cross-species searching. In conclusion, the use of strain-specific protein database constructed from raw genome sequences makes it possible to reliably identify most of the proteins from 2-DE analysis simply through peptide mass fingerprinting.     

Characterization of Oligonucleotide Sequence Isomers in Mixtures Using HPLC/MS

Abstract

A method is described for the analysis of mixtures containing sequence isomers of oligonucleotides. The approach consists of an electrospray ionization mass spectrometric analysis in direct combination with HPLC separation. Mass spectrometry can provide sequence information based on the fragmentation patterns of oligonucleotides allowing the simultaneous characterization of sequence isomers. An example is shown for the characterization of a mixture of dCAGT, dCGTA, dTCAG, dAGTC and dTCGA.     

Searching sequence databases via de novo peptide sequencing by tandem mass spectrometry

There are many computer programs that can match tandem mass spectra of peptides to database-derived sequences; however, situations can arise where mass spectral data cannot be correlated with any database sequence. In such cases, sequences can be automatically deduced de novo, without recourse to sequence databases, and the resulting peptide sequences can be used to perform homologous nonexact searches of sequence databases. This article describes details on how to implement both a de novo sequencing program called “Lutefisk,” and a version of FASTA that has been modified to account for sequence ambiguities inherent in tandem mass spectrometry data.     

Combining recombinant ribonuclease U2 and protein phosphatase for RNA modification mapping by liquid chromatography–mass spectrometry

Ribonuclease (RNase) mapping of modified nucleosides onto RNA sequences is limited by RNase availability. A codon-optimized gene for RNase U2, a purine selective RNase with preference for adenosine, has been designed for overexpression using Escherichia coli as the host. Optimal expression conditions were identified enabling generation of milligram-scale quantities of active RNase U2. RNase U2 digestion products were found to terminate in both 2′,3′-cyclic phosphates and 3′-linear phosphates. To generate a homogeneous 3′-linear phosphate set of products, an enzymatic approach was investigated. Bacteriophage lambda protein phosphatase was identified as the optimal enzyme for hydrolyzing cyclic phosphates from RNase U2 products. The compatibility of this enzymatic approach with liquid chromatography–tandem mass spectrometry (LC–MS/MS) RNA modification mapping was then demonstrated. RNase U2 digestion followed by subsequent phosphatase treatment generated nearly 100% 3′-phosphate-containing products that could be characterized by LC–MS/MS. In addition, bacteriophage lambda protein phosphatase can be used to introduce ¹⁸O labels within the 3′-phosphate of digestion products when incubated in the presence of H₂¹⁸O, allowing prior isotope labeling methods for mass spectrometry to include digestion products from RNase U2.     

Low mass cutoff evasion with qz value optimization in ion trap

An ion trap is a powerful analyzer because of its high resolution, high sensitivity, and multistage mass analysis (MSⁿ) capabilities. Multiple fragmentation analysis provides useful information regarding peptide sequence and biomolecular structure; however, this approach is limited by an inherent low mass cutoff (LMCO) derived from collision-induced dissociation (CID). To avoid the LMCO for application of an ion trap to iTRAQ, we optimized the q_z value, which is a parameter that is proportional to the applied fundamental AC radio frequency voltage of a tandem mass spectrometry (MS/MS) event. Considering that many ion trap MS analyses employ CID as the MS/MS method, this method can be a practical one without any instrumental changes.     

ICC-CLASS: isotopically-coded cleavable crosslinking analysis software suite

Background  

Successful application of crosslinking combined with mass spectrometry for studying proteins and protein complexes requires specifically-designed crosslinking reagents, experimental techniques, and data analysis software. Using isotopically-coded ("heavy and light") versions of the crosslinker and cleavable crosslinking reagents is analytically advantageous for mass spectrometric applications and provides a "handle" that can be used to distinguish crosslinked peptides of different types, and to increase the confidence of the identification of the crosslinks.     

Production of goniodomin A by the planktonic, chain-forming dinoflagellate Alexandrium monilatum (Howell) Balech isolated from the Gulf Coast of the United States

The chain-forming dinoflagellate Alexandrium monilatum has been reported to be associated with widespread discolored water and increased fish mortality in the Mississippi Sound and off the eastern and western coasts of Florida. Previous studies over the last 60–70 years have determined that A. monilatum produces a harmful substance(s) that is predominantly contained in the cell mass as exhibited by apparent increased toxicity when the organism cytolyses. The current research in our lab corroborated earlier research demonstrating that A. monilatum produces a lipophilic toxin, unlike its Alexandrium relatives noted for their production of saxitoxin-like toxins. Using sophisticated chemical, chromatographic, and analytical techniques, we have successfully purified and identified the molecular structure of the toxin produced by A. monilatum. We utilized a 500 MHz NMR to carry out a number of experiments (i.e., ¹H, ¹³C, COSY, HSQC, and HMBC) to unambiguously determine the molecular structure of the toxin. In addition, we report mass analysis of the toxin utilizing electrospray ionization-mass spectrometry (ESI-MS), matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS), and Q-TOF mass spectral techniques. The toxin is representative of a polyether macrolide with an empirical formula of C₄₃H₆₀O₁₂. This toxic compound is shown to be identical to a Japanese tidepool toxin identified as goniodomin A, which is produced by another Alexandrium species.     

In silico fragmentation for computer assisted identification of metabolite mass spectra

Background  

Mass spectrometry has become the analytical method of choice in metabolomics research. The identification of unknown compounds is the main bottleneck. In addition to the precursor mass, tandem MS spectra carry informative fragment peaks, but the coverage of spectral libraries of measured reference compounds are far from covering the complete chemical space. Compound libraries such as PubChem or KEGG describe a larger number of compounds, which can be used to compare their in silico fragmentation with spectra of unknown metabolites.     

Mass spectrometric and chemical stability of the Asp-Pro bond in herpes simplex virus epitope peptides compared with X-Pro bonds of related sequences.

The mass spectrometric analysis of the immunodominant epitope region (273-284) of herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) showed a favoured fission at the Asp-Pro peptide bond. The fast atom bombardment collision induced dissociation (FAB-CID) study of closely related X-Pro peptides documented that neither the length nor the amino acid composition of the peptide has a significant influence on this preferential cleavage. At the same time the DP bond proved to be sensitive to acidic conditions in the course of peptide synthesis. These observations prompted us to compare the chemical and mass spectrometric stability of a new set of nonapeptides related to the 273-284 epitope region of gD, i.e. SALLEDPVG and SALLEXPVG peptides, where X = A, K, I, S, F, E or D, respectively. The chemical stability of these peptides during acidic hydrolysis was investigated by electrospray ionization mass spectrometry (ESI-MS) and the products were identified by ESI-MS and on-line high performance liquid chromatography-mass spectrometry (HPLC-MS). The mass spectrometric fragmentation and bond stability of the untreated peptide samples were also studied using ESI-MS and liquid secondary ion mass spectrometry (LSIMS). Both the chemical hydrolysis and the mass spectrometric fragmentation showed that the Asp-Pro bond could easily be cleaved, while the KP bond proved to be stable under both circumstances. On the other hand, the XP bond (X = A, I, S, F or E) fragmented easily under the mass spectrometric conditions, but was not sensitive to the acidolysis.     

A novel informatics concept for high-throughput shotgun lipidomics based on the molecular fragmentation query language

Shotgun lipidome profiling relies on direct mass spectrometric analysis of total lipid extracts from cells, tissues or organisms and is a powerful tool to elucidate the molecular composition of lipidomes. We present a novel informatics concept of the molecular fragmentation query language implemented within the LipidXplorer open source software kit that supports accurate quantification of individual species of any ionizable lipid class in shotgun spectra acquired on any mass spectrometry platform.     

Transformation of prednisolone to a 20β-hydroxy prednisolone compound by <Emphasis Type="Italic">Streptomyces roseochromogenes</Emphasis> TS79

Prednisolone represents an important compound in pharmaceutical preparations. To obtain more bioactive prednisolone derivatives, the microbial transformation of prednisolone was carried out. The steroid products were assigned by an interpretation of their spectral data using mass spectrometry and proton nuclear magnetic resonance (¹H NMR) analyses. The product was assigned the chemical structure of 11β, 17α, 20β, 21-tetrahydroxypregna-1,4-diene-3-one (named as 20β-hydroxy prednisolone). The conversion of prednisolone to 20β-hydroxy prednisolone by Streptomyces roseochromogenes TS79 was different from a previous study on the microbial transformation of steroid by this organism, which usually generates a 16α-hydroxy steroid product. The different reaction parameters for maximum conversion of prednisolone were optimized. The analysis revealed that the optimum values of the tested variables were 7.5 mg/ml prednisolone dissolved in DMSO and added to the 24-h pre-culture fermentation culture containing 0.05% MgSO₄ and incubated for 24 h. A conversion of 95.1% of prednisolone was observed, which has the potential to be used in industrial production.     

Computational prediction of proteotypic peptides for quantitative proteomics

Mass spectrometry-based quantitative proteomics has become an important component of biological and clinical research. Although such analyses typically assume that a protein's peptide fragments are observed with equal likelihood, only a few so-called 'proteotypic' peptides are repeatedly and consistently identified for any given protein present in a mixture. Using >600,000 peptide identifications generated by four proteomic platforms, we empirically identified >16,000 proteotypic peptides for 4,030 distinct yeast proteins. Characteristic physicochemical properties of these peptides were used to develop a computational tool that can predict proteotypic peptides for any protein from any organism, for a given platform, with >85% cumulative accuracy. Possible applications of proteotypic peptides include validation of protein identifications, absolute quantification of proteins, annotation of coding sequences in genomes, and characterization of the physical principles governing key elements of mass spectrometric workflows (e.g., digestion, chromatography, ionization and fragmentation).     

Isolation and characterization of conjugated gibberellins in maturing seeds of Sechium edule

Endosperm and cotyledons of Sechium edule at different stages of seed development were found to contain three novel GA conjugates. 3-propyl- or 3-acetyl- GA_4/7 were characterized by mass spectral analysis and were found to be biologically active in contrast to synthetic GA_4/7 propyl esters. An unusual GA glucoside showing biological activity was also isolated from both tissues and characterized by mass spectrometry and NMR analysis as 16,17-dihydro-16-hydroxy-GA₁₅ alcohol glucoside. The functional significance of this novel GA conjugate is discussed.     

基于数据非依赖采集的蛋白质组质谱数据解析方法研究进展

数据非依赖采集（DIA）是蛋白质组学领域近年来快速发展的质谱采集技术，其通过无偏碎裂隔离窗口内的所有母离子采集二级谱图，理论上可实现蛋白质样品的深度覆盖，同时具有高通量、高重现性和高灵敏度的优点。现有的DIA数据采集方法可以分为全窗口碎裂方法、隔离窗口序列碎裂方法和四维DIA数据采集方法（4D-DIA）3大类。针对DIA数据的不同特点，主要数据解析方法包括谱库搜索方法、蛋白质序列库直接搜索方法、伪二级谱图鉴定方法和从头测序方法4大类。解析得到的肽段鉴定结果需要进行可信度评估，包括使用机器学习方法的重排序和对报告结果集合的假发现率估计两个步骤，实现对数据解析结果的质控。本文对DIA数据的采集方法、数据解析方法及软件和鉴定结果可信度评估方法进行了整理和综述，并展望了未来的发展方向。     

Carbohydrate and amino acid metabolism of Spironucleus vortens

The metabolism of Spironucleus vortens, a parasitic, diplomonad flagellate related to Giardia intestinalis, was investigated using a combination of membrane inlet mass spectrometry, ¹H NMR, ¹³C NMR, bioscreen continuous growth monitoring, and ion exchange chromatography. The products of glucose-fuelled and endogenous metabolism were identified by ¹H NMR and ¹³C NMR as ethanol, acetate, alanine and lactate. Mass spectrometric monitoring of gas metabolism in buffered cell suspensions showed that glucose and ethanol could be used by S. vortens as energy-generating substrates, but bioscreen automated monitoring of growth in culture medium, as well as NMR analyses, suggested that neither of these compounds are the substrates of choice for this organism. Ion-exchange chromatographic analyses of free amino-acid and amino-acid hydrolysate of growth medium revealed that, despite the availability of large pools of free amino-acids in the medium, S. vortens hydrolysed large amounts of proteins during growth. The organism produced alanine and aspartate, and utilised lysine, arginine, leucine, cysteine and urea. However, mass spectrometric and bioscreen investigations showed that addition of the utilised amino acids to diluted culture medium did not induce any significant increase in metabolic or growth rates. Moreover, as no significant amounts of ornithine were produced, and addition of arginine under aerobic conditions did not generate NO production, there was no evidence of the presence of an energy-generating, arginine dihydrolase pathway in S. vortens under in vitro conditions.