Multidimensional analysis of the insoluble sub-proteome of Oceanobacillus iheyensis HTE831, an alkaliphilic and halotolerant deep-sea bacterium isolated from the Iheya ridge

We report the first proteomic analysis of the insoluble sub-proteome of the alkaliphilic and halotolerant deep-sea bacterium Oceanobacillus iheyensis HTE831. A multidimensional gel-based and gel-free analysis was utilised and a total of 4352 peptides were initially identified by automated MS/MS identification software. Automated curation of this list using PROVALT reduced our peptide list to 467 uniquely identified peptides that resulted in the positive identification of 153 proteins. These identified proteins were functionally classified and physiochemically characterised. Of 26 proteins identified as hypothetical conserved, we have assigned function to all but four. A total of 41 proteins were predicted to possess signal peptides. In silico investigation of these proteins allowed us to identify three of the five bacterial classes of signal peptide, namely: (i) twin-arginine translocation; (ii) Sec-type and (iii) lipoprotein transport. Our proteomic strategy has also allowed us to identify, at neutral pH, a number of proteins described previously as belonging to two putative transport systems believed to be of importance in the alkaliphilic adaptation of O. iheyensis HTE831.     

电喷雾离子化/质谱法(ESI/MS)及其在生物大分子研究中的应用

电喷雾离子化／质谱法（ESI／MS）在各种有机化合物、多肽、蛋白质（含糖蛋白）、核苷酸、糖、脂类及合成高分子物质等分析领域获得了广泛的应用,本文系统介绍了ESI／MS的基本原理,其联用技术,及其在生物大分子研究,包括肽图谱测定,糖分析和核苷酸分析中的应用。     

Structural determination of C-glycosylflavones by mass spectrometry of their permethyl ethers: O-glycosyl-6-C-glycosylflavones

Permethylated O-glycosyl-C-glycosylflavones give well defined MS including an important molecular peak. Permethyl 6-C-glycosylflavones O-glycosylated on a phenolic hydroxyl group are easily distinguished from the isomeric permethyl 6-C-diholosylflavones. In both types, the position of the O-glycosidic bond can be deduced from the MS, eventually after acid hydrolysis. 2″-O-glycosyl-6-C-glycosylflavones can be differentiated from their 8-C isomers.     

Arbutin derivatives from the buds of Vaccinium dunalianum and their MS/MS spectrometric fragmentations

Four arbutin derivatives were isolated from the buds of Vaccinium dunalianum in which 4-hydroxyphenyl-6′-(3''-O-β-D-glucopyranosyl-4''-hydroxycinnamoyl)-O-β-D-glucopyranoside (1) was a new compound. The structure of the new compound was determined on the basis of NMR and HR-ESI-MS data. All the arbutin derivatives were subjected to the MS/MS analyses from which the MS/MS spectrometric fragmentations were summarized.     

A comparison of MS/MS‐based,stable‐isotope‐labeled,quantitation performance on ESI‐quadrupole TOF and MALDI‐TOF/TOF mass spectrometers

The peptide‐based quantitation accuracy and precision of LC‐ESI (QSTAR Elite) and LC‐MALDI (4800 MALDI TOF/TOF) were compared by analyzing identical Escherichia coli tryptic digests containing iTRAQ‐labeled peptides of defined abundances (1:1, 2.5:1, 5:1, and 10:1). Only 51.4% of QSTAR spectra were used for quantitation by ProteinPilot Software versus 66.7% of LC‐MALDI spectra. The average protein sequence coverages for LC‐ESI and LC‐MALDI were 24.0 and 18.2% (14.9 and 8.4 peptides per protein), respectively. The iTRAQ‐based expression ratios determined by ProteinPilot from the 57 467 ESI‐MS/MS and 26 085 MALDI‐MS/MS spectra were analyzed for measurement accuracy and reproducibility. When the relative abundances of peptides within a sample were increased from 1:1 to 10:1, the mean ratios calculated on both instruments differed by only 0.7–6.7% between platforms. In the 10:1 experiment, up to 64.7% of iTRAQ ratios from LC‐ESI MS/MS spectra failed S/N thresholds and were excluded from quantitation, while only 0.1% of the equivalent LC‐MALDI iTRAQ ratios were rejected. Re‐analysis of an archived LC‐MALDI sample set stored for 5 months generated 3715 MS/MS spectra for quantitation, compared with 3845 acquired originally, and the average ratios differed by only 3.1%. Overall, MS/MS‐based peptide quantitation performance of offline LC‐MALDI was comparable with on‐line LC‐ESI, which required threefold less time. However, offline LC‐MALDI allows the re‐analysis of archived HPLC‐separated samples.     

Sequential synthesis of chondroitin oligosaccharides by immobilized chondroitin polymerase mutants

Escherichia coli strain K4 expresses a chondroitin (CH)-polymerizing enzyme (K4CP) that contains two glycosyltransferase active domains. K4CP alternately transfers glucuronic acid (GlcA) and N-acetyl-galactosamine (GalNAc) residues using UDP-GlcA and UDP-GalNAc donors to the nonreducing end of a CH chain acceptor. Here we generated two K4CP point mutants substituted at the UDP-sugar binding motif (DXD) in the glycosyltransferase active domains, which showed either glycosyltransferase activity of the intact domain and retained comparable activity after immobilization onto agarose beads. The mutant enzyme-immobilized beads exhibited an addition of GlcA or GalNAc to GalNAc or GlcA residue at the nonreducing end of CH oligosaccharides and sequentially elongated pyridylamine-conjugated CH (PA-CH) chain by the alternate use. The sequential elongation up to 16-mer was successfully achieved as assessed by fluorescent detection on a gel filtration chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and MALDI potential lift tandem TOF mass spectrometry (MALDI-LIFT-TOF/TOF MS/MS) analyses in the negative reflection mode. This method provides exactly defined CH oligosaccharide derivatives, which are useful for studies on glycosaminoglycan functions.     

Simplified validation of borderline hits of database searches

Along with unequivocal hits produced by matching multiple MS/MS spectra to database sequences, LC-MS/MS analysis often yields a large number of hits of borderline statistical confidence. To simplify their validation, we propose to use rapid de novo interpretation of all acquired MS/MS spectra and, with the help of a simple software tool, display the candidate sequences together with each database search hit. We demonstrate that comparing hit database sequences and independent de novo interpretations of the same MS/MS spectra assists in rapid examination of ambiguous matches.     

Structures of the O-linked oligosaccharides of a complex glycoconjugate from Pseudallescheria boydii

Nonreducing O-linked oligosaccharides were obtained from the peptidorhamnomannan of mycelia of Pseudallescheria boydii by alkaline beta-elimination under reducing conditions. They were separated by gel filtration chromatography to give three oligosaccharide fractions. The major oligosaccharide from fraction 1 was characterized by a combination of techniques including electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI MS/MS), matrix-assisted laser desorption ionization mass spectrometry (MALDI MS), nuclear magnetic resonance (NMR), and methylation gas-liquid chromatography-mass spectrometry (GC-MS) analysis. It was branched, with a principal chain of alpha-Rhap-(1 --> 3)-alpha-Rhap-(1 --> 3)-alpha-Manp-(1 --> 2)-Man-ol substituted at O-6 of mannitol with an alpha-Glcp-(1 --> 4)-beta-Galp group. Species containing one and two additional alpha-Glcp-(1 --> 4) substituents in the rhamnose branch were also present. The major component of fraction 2 was a substructure of oligosaccharide-1, lacking a hexose from the Glc-Gal branch. Fraction 3 contained a mixture of smaller, unbranched, oligosaccharides. In hapten inhibition tests, fractions 1 and 2 blocked the reaction between peptidorhamnomannan (PRM) and rabbit anti-P. boydii mycelium hyperimmune serum by approximately 75%, whereas fraction 3 inhibited by approximately 55%.     

Topology and dynamics of melittin within the liposome revealed by a combination of mass spectrometry and chemical modification

The topology and dynamics of melittin within the liposome were investigated by a mass spectrometry coupled with acetylation. The MALDI-TOF MS and MALDI-QIT-TOF MS/MS analyses revealed that only N-terminal amine of melittin was dominantly acetylated in the presence of liposome although all of four primary amines were completely and rapidly acetylated in aqueous solution. This result indicates that melittin adopts the N-terminal-outside transmembrane topology within the liposome. The time course of acetylation followed the first-order kinetics at any examined temperatures (6-30 °C). The rate constant was less than that of the acetylation of melittin in aqueous solution. The activation energy for acetylation (74 kJ mol⁻¹) was comparable to that for dissociation of a lipid monomer from the membrane, suggesting a float-like longitudinal motion of melittin within the liposome. These results demonstrate that a mass spectrometry combined with chemical modification is very efficient way for clarifying the topology and dynamics of peptides bound to the membrane.     

Analysis of anabolic steroids in hair: Time courses in guinea pigs

Sensitive, specific, and reproducible methods for the quantitative determination of eight anabolic steroids in guinea pig hair have been developed using LC/MS/MS and GC/MS/MS. Methyltestosterone, stanozolol, methandienone, nandrolone, trenbolone, boldenone, methenolone and DHEA were administered intraperitoneally in guinea pigs. After the first injection, black hair segments were collected on shaved areas of skin. The analysis of these segments revealed the distribution of anabolic steroids in the guinea pig hair. The major components in hair are the parent anabolic steroids. The time courses of the concentrations of the steroids in hair (except methenolone, which does not deposit in hair) demonstrated that the peak concentrations were reached on days 2-4, except stanozolol, which peaked on day 10 after administration. The concentrations in hair appeared to be related to the physicochemical properties of the drug compound and to the dosage. These studies on the distribution of drugs in the hair shaft and on the time course of their concentration changes provide information relevant to the optimal time and method of collecting hair samples. Such studies also provide basic data that will be useful in the application of hair analysis in the control of doping and in the interpretation of results.     

Selective analysis of phenolic compounds in propolis by HPLC-MS/MS

Phenolic compounds (flavonoids and phenolic acid derivatives) are major active constituents of the resinous fraction of propolis, and also represent its allergenic principles. We have developed a chromatography electrospray ionisation tandem mass spectrometry (HPLC-ESI-MS/MS) method to characterise the polyphenolic fraction of propolis rapidly and quali-quantitatively. With precursor ion scanning, selective detection of caffeic esters was easily achieved, confirming the identification of prenyl caffeate, benzyl caffeate and phenylethyl caffeate by comparison with synthetic standards. The ionisation and fragmentation behaviour of the major propolis flavonoids was rationalised and applied to selected real samples. Taken together, the results of this study show that the introduction of precursor ion analysis leads to a significant improvement in the characterisation of the phenolic fraction of propolis, paving the way to the establishment of a better quality control for this important natural remedy. Copyright (c) 2007 John Wiley & Sons, Ltd.     

家蚕脂肪体蛋白质组学研究

通过高精度的双向电泳技术对家蚕末龄幼虫的脂肪体组织进行了研究,采用基质辅助质量飞行时间质谱对其中一些表达量较高的蛋白点进行了鉴定,并利用GPMAW软件结合家蚕基因组预测的蛋白质数据库构建了本地的肽质量指纹图谱数据库,对所得到的肽质量指纹图谱进行了分析。研究发现,经过双向凝胶电泳及其图象分析技术,银染可以分离出722个清晰蛋白点,这些蛋白质主要集中在分子量15～90kD区域,等电点pH4～8之间。MALDI-TOF-MS鉴定的41个蛋白点中都有较强的肽质量指纹信号峰,其中34个蛋白点得到了成功鉴定,其中包括了大量参与代谢的酶类、不同分子量的热激蛋白、重要的血液蛋白30K,Actin3等,这一结果对人们进一步认识家蚕脂肪体提供了有利的帮助。     

FragmentationAnalyzer: An open‐source tool to analyze MS/MS fragmentation data

A thorough understanding of the fragmentation processes in MS/MS can be a powerful tool in assessing the resulting peptide and protein identifications. We here present the freely available, open‐source FragmentationAnalyzer tool ( http://fragmentation‐analyzer.googlecode.com ) that makes it straightforward to analyze large MS/MS data sets for specific types of identified peptides, using a common set of peptide properties. This enables the detection of fragmentation pattern nuances related to specific instruments or due to the presence of post‐translational modifications.     

MALDI mass spectrometry in prostate cancer biomarker discovery

Matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry (MS) is a highly versatile and sensitive analytical technique, which is known for its soft ionisation of biomolecules such as peptides and proteins. Generally, MALDI MS analysis requires little sample preparation, and in some cases like MS profiling it can be automated through the use of robotic liquid-handling systems. For more than a decade now, MALDI MS has been extensively utilised in the search for biomarkers that could aid clinicians in diagnosis, prognosis, and treatment decision making. This review examines the various MALDI-based MS techniques like MS imaging, MS profiling and proteomics in-depth analysis where MALDI MS follows fractionation and separation methods such as gel electrophoresis, and how these have contributed to prostate cancer biomarker research. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.     

Advanced nanoscale separations and mass spectrometry for sensitive high-throughput proteomics

Recent developments in combined separations with mass spectrometry for sensitive and high-throughput proteomic analyses are reviewed herein. These developments primarily involve high-efficiency (separation peak capacities of ~10³) nanoscale liquid chromatography (flow rates extending down to approximately 20 nl/min at optimal liquid mobile-phase separation linear velocities through narrow packed capillaries) in combination with advanced mass spectrometry and in particular, high-sensitivity and high-resolution Fourier transform ion cyclotron resonance mass spectrometry. Such approaches enable analysis of low nanogram level proteomic samples (i.e., nanoscale proteomics) with individual protein identification sensitivity at the low zeptomole level. The resultant protein measurement dynamic range can approach 10⁶ for nanogram-sized proteomic samples, while more abundant proteins can be detected from subpicogram-sized (total) proteome samples. These qualities provide the foundation for proteomics studies of single or small populations of cells. The instrumental robustness required for automation and providing high-quality routine performance nanoscale proteomic analyses is also discussed.     

Development and validation of a high-throughput method for the quantitative analysis of d-amphetamine in rat blood using liquid chromatography/MS on a hybrid triple quadrupole-linear ion trap mass spectrometer and its application to a pharmacokinetic study

Amphetamines are a group of sympathomimetic drugs that exhibit strong central nervous system stimulant effects. d-Amphetamine ((+)-alpha-methylphenetylamine) is the parent drug in this class to which all others are structurally related. In drug discovery, d-amphetamine is extensively used either for the exploration of novel mechanisms involving the catecholaminergic system, or for the validation of new behavioural animal models. Due to this extensive use of d-amphetamine in drug research and its interest in toxicologic–forensic investigation, a specific and high-throughput method, with minimal sample preparation, is necessary for routine analysis of d-amphetamine in biological samples. We propose here a sensitive, specific and high-throughput bioanalytical method for the quantitative determination of d-amphetamine in rat blood using MS³ scan mode on a hybrid triple quadrupole-linear ion trap mass spectrometer (LC–MS/MS/MS). Blood samples, following dilution with water, were prepared by fully automated protein precipitation with acetonitrile containing an internal standard. The chromatographic separation was achieved on a Waters XTerra C18 column (2.1 mm × 30 mm, 3.5 μm) using gradient elution at a flow rate of 1.0 mL/min over a 2 min run time. An Applied Biosystems API4000 QTRAP™ mass spectrometer equipped with turbo ion-spray ionization source was operated simultaneously in MS³ scan mode for the d-amphetamine and in multiple reaction monitoring (MRM) for the internal standard. The MS/MS/MS ion transition monitored was m/z 136.1 → 119.1 → 91.1 for the quantitation of d-amphetamine and for the internal standard (rolipram) the MS/MS ion transition monitored was m/z 276.1 → 208.2. The linear dynamic range was established over the concentration range 0.5–1000 ng/mL (r² = 0.9991). The method was rugged and sensitive with a lower limit of quantification (LLOQ) of 0.5 ng/mL. All the validation data, such as accuracy, precision, and inter-day repeatability, were within the required limits. This method was successfully applied to evaluate the pharmacokinetics of d-amphetamine in rat. On a more general extent, this work demonstrated that the selectivity of the fragmentation pathway (MS³) can be used as alternative approach to significantly improve detection capability in complex situation (e.g., small molecules in complex matrices) rather than increasing time for sample preparation and chromatographic separation.     

Rapid identification of vinca alkaloids by direct-injection electrospray ionisation tandem mass spectrometry and confirmation by high-performance liquid chromatography-mass spectrometry

A simple and rapid method for the identification of Vinca alkaloids from a crude extract of Catharanthus roseus G. Don (Apocynaceae) by direct-injection electrospray ionisation (ESI) and tandem mass spectrometry (MS/MS) has been developed. The alkaloids vindoline, vindolidine, vincristine and vinblastine were evaluated in a commercial extract of C. roseus using this method. Catharanthine and its isomers 19S-vindolinine and vindolinine were detected in the commercial product by direct injection ESI/MS/MS and confirmed by preparation and by HPLC-ESI/MS. For the characterisation of different fragment fingerprints, ESI/MS/MS is a sensitive, rapid and convenient technique by which to identify some constituents in complex and mixed plant extracts.     

Proteome analysis of an ectomycorrhizal fungus Boletus edulis under salt shock

Soil salinization has become a severe global problem and salinity is one of the most severe abiotic stresses inhibiting growth and survival of mycorrhizal fungi and their host plants. Salinity tolerance of ectomycorrhizal fungi and survival of ectomycorrhizal inocula is essential to reforestation and ecosystem restoration in saline areas. Proteomic changes of an ectomycorrhizal fungus, Boletus edulis, when exposed to salt stress conditions (4 % NaCl, w/v) were determined using two-dimensional electrophoresis (2DE) and mass spectrometry (MS) techniques. Twenty-two protein spots, 14 upregulated and 8 downregulated, were found changed under salt stress conditions. Sixteen changed protein spots were identified by nanospray ESI Q-TOF MS/MS and liquid chromatography MS/MS. These proteins were involved in biosynthesis of methionine and S-adenosylmethionine, glycolysis, DNA repair, cell cycle control, and general stress tolerance, and their possible functions in salinity adaptation ofBoletus edulis were discussed.