SciCombinator

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Concept: Tandem mass spectrometry

171

The primary structural information of proteins employed as biotherapeutics is essential if one wishes to understand their structure-function relationship, as well as in the rational design of new therapeutics and for quality control. Given both the large size (around 150 kDa) and the structural complexity of intact immunoglobulin G (IgG), which includes a variable number of disulfide bridges, its extensive fragmentation and subsequent sequence determination by means of tandem mass spectrometry (MS) are challenging. Here, we applied electron transfer dissociation (ETD), implemented on a hybrid Orbitrap Fourier transform mass spectrometer (FTMS), to analyze a commercial recombinant IgG in a liquid chromatography (LC)-tandem mass spectrometry (MS/MS) top-down experiment. The lack of sensitivity typically observed during the top-down MS of large proteins was addressed by averaging time-domain transients recorded in different LC-MS/MS experiments before performing Fourier transform signal processing. The results demonstrate that an improved signal-to-noise ratio, along with the higher resolution and mass accuracy provided by Orbitrap FTMS (relative to previous applications of top-down ETD-based proteomics on IgG), is essential for comprehensive analysis. Specifically, ETD on Orbitrap FTMS produced about 33% sequence coverage of an intact IgG, signifying an almost 2-fold increase in IgG sequence coverage relative to prior ETD-based analysis of intact monoclonal antibodies of a similar subclass. These results suggest the potential application of the developed methodology to other classes of large proteins and biomolecules.

Concepts: Immune system, Protein, Mass spectrometry, Fourier transform, Tandem mass spectrometry, Fourier transform ion cyclotron resonance, Collision-induced dissociation, Electron transfer dissociation

168

In this study, for the first time, both neuropeptides isotocin (IT) and arginine vasotocin (AVT) have been identified and measured in urophysis, the neurohaemal organ of the caudal neurosecretory system of teleost fish. So far, AVT, but not IT, was quantified by radioimmunoassay (RIA) in urophysis of several fish species. We have used high-performance liquid chromatographic assay with fluorescence detection (HPLC-FL) preceded by solid-phase extraction (SPE) and liquid chromatography-electrospray ionization triple-quadrupole tandem mass spectrometry (LC-ESI MS/MS) technique to determine both neuropeptides in urophysis of three fish species. The efficiency of peptide’s SPE extraction was 79-85 %. In HPLC-FL method, the limits of detection (LOD) and quantification (LOQ) were estimated as 1.0 and 3.4 pmol/mL for IT and 0.25 and 2.20 pmol/mL for AVT. In LC-MS/MS method, LOD and LOQ were estimated as 0.4 and 1.2 pmol/mL for IT and 0.06 and 0.2 pmol/mL for AVT. The chromatographic methods are good alternative for RIA, because enable to measure both nonapeptides simultaneously in one sample. In round goby (Neogobius melanostomus), three-spined stickleback (Gasterosteus aculeatus) and sea bream (Sparus aurata), urophysial IT concentrations ranged between 0.056 and 0.678 pmol/mg tissue and AVT concentrations ranged between 0.0008 (or even below detection threshold) and 0.084 pmol/mg tissue.

Concepts: Mass spectrometry, Measurement, Analytical chemistry, Actinopterygii, Tandem mass spectrometry, Three-spined stickleback, Gasterosteiformes, Gilt-head bream

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The World Anti-Doping Agency (WADA) has recently added desmopressin, a synthetic analogue of the endogenous peptide hormone arginine vasopressin, to the Prohibited List, owing to the potential masking effects of this drug on hematic parameters useful to detect blood doping. A qualitative method for detection of desmopressin in human urine by high-performance liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS/MS) has been developed and validated. Desmopressin purification from urine was achieved by means of delipidation with a 60:40 di-isopropyl ether/n-butanol and solid-phase extraction with WCX cartridges. The lower limit of detection was 25 pg/mL. Extraction recovery was determined as 59.3% (SD 29.4), and signal reduction owing to ion suppression was estimated to be 42.7% (SD 12.9). The applicability of the method was proven by the analysis of real urine samples obtained after intravenous, oral and intranasal administration of desmopressin, achieving unambiguous detection of the peptide in all the cases. Copyright © 2012 John Wiley & Sons, Ltd.

Concepts: Mass spectrometry, Hormone, Oxytocin, Vasopressin, Diabetes insipidus, Tandem mass spectrometry, Desmopressin, World Anti-Doping Agency

28

High-throughput identification of proteins with the latest generation of hybrid high-resolution mass spectrometers is opening new perspectives in microbiology. I present, here, an overview of tandem mass spectrometry technology and bioinformatics for shotgun proteomics that make 2D-PAGE approaches obsolete. Non-labelling quantitative approaches have become more popular than labelling techniques on most proteomic platforms because they are easier to carry out while their quantitative outcome is rather robust. Parameters for recording mass spectrometry data, however, need to be chosen carefully and statistics to assess the confidence of the results should not be neglected. Interestingly, next-generation sequencing methodologies make any microbial model quickly amenable to proteomics, leading to the documentation of a wide range of organisms from diverse environments. Some recent discoveries made using microbial proteomics have challenged some biological dogma, such as: (i) initiation of the translation does not occur predominantly from ATG codons in some microorganisms, (ii) non-canonical initiation codons are used to regulate the production of specific but important proteins and (iii) a gene may code for multiple polypeptide species, heterogeneous in terms of sequences. Microbial diversity and microbial physiology can now be revisited by means of exhaustive comparative proteomic surveys where thousands of proteins are detected and quantified. Proteogenomics, consisting of better annotating of genomes with the help of proteomic evidence, is paving the way for integrated multi-omic approaches in microbiology. Finally, meta-proteomic tools and approaches are emerging for tackling the high complexity of the microbial world as a whole, opening new perspectives for assessing how microbial communities function.

Concepts: Protein, Gene, Bioinformatics, Mass spectrometry, Organism, Tandem mass spectrometry, Top-down proteomics, Shotgun proteomics

28

Limonene, considered a green solvent, was successfully used to extract simvastatin, lovastatin, and their hydroxy-acid metabolites from human plasma samples. The extraction process was followed by the direct injection of a large volume aliquot (100 μL) from the limonene layer into a Zorbax SB-C(18) Rapid Resolution chromatographic column (50 mm length × 4.6 mm i.d. × 1.8 µm d.p.), operated under gradient elution reversed-phase separation mechanism. Tandem mass spectrometry operated under the multiple reaction monitoring mode was used for detection, providing low quantitation limits in the 0.25-0.5 ng/mL concentration interval. This method was validated and used for quantitation of simvastatin and its hydroxy acid metabolite in incurred plasma samples obtained from two volunteers participating in a bioequivalence study, using lovastatin and its hydroxy analog as internal standards. The results were statistically compared with those produced by means of an alternative RPLC-tandem MS using protein precipitation with acetonitrile. The quality attributes of the two methods are comparatively discussed. The agreement between the quality characteristics of the two methods and the experimental results obtained on real samples may be considered as a consistent basis for the simultaneous use of limonene as extraction medium and injection diluent for hydrophobic compounds in bioanalytical approaches. Copyright © 2012 John Wiley & Sons, Ltd.

Concepts: Mass spectrometry, Statin, Chromatography, Analytical chemistry, CYP3A4, Tandem mass spectrometry, Statins, Liquid-liquid extraction

28

The goal of this study was to monitor the anti-proliferative activity of Rosmarinus officinalis and Salvia officinalis extracts against cancer cells and to correlate this activity with their phytochemical profiles using liquid chromatography/diode array detection/electrospray ion trap tandem mass spectrometry (LC/DAD/ESI-MS(n)). For the quantitative estimation of triterpenic acids in the crude extracts an NMR based methodology was used and compared with the HPLC measurements, both applied for the first time, for the case of betulinic acid. Both extracts exerted cytotoxic activity through dose-dependent impairment of viability and mitochondrial activity of rat insulinoma m5F (RINm5F) cells. Decrease of RINm5F viability was mediated by nitric oxide (NO)-induced apoptosis. Importantly, these extracts potentiated NO and TNF-α release from macrophages therefore enhancing their cytocidal action. The rosemary extract developed more pronounced antioxidant, cytotoxic and immunomodifying activities, probably due to the presence of betulinic acid and a higher concentration of carnosic acid in its phytochemical profile.

Concepts: Mass spectrometry, PH, Lamiaceae, Medicinal plants, Tandem mass spectrometry, Fourier transform ion cyclotron resonance, Betulinic acid, Rosemary

28

The phenolic compounds were extracted from green and yellow leaves, stalks, and seeds of garlic ( Allium ursinum L.). The extracts were analyzed by liquid chromatography-photodiode array detector-electrospray ionization-tandem mass spectrometry (LC-PDA-ESI-MS/MS). In total, 21 compounds were detected. The flavonol derivatives were identified on the basis of their ultraviolet (UV) spectra and fragmentation patterns in collision-induced dissociation experiments. On the basis of accurate MS and MS/MS data, six compounds were newly identified in bear’s garlic, mainly the kaempferol derivatives. As far as the investigated parts of garlic are concerned, the kaempferol derivatives were found to be predominant in yellow leaves [2362.96 mg/100 g of dry matter (dm)], followed by green leaves (1856.31 mg/100 g of dm). Seeds contained the minimal phenolic compounds, less than stalks. The yellow leaves of A. ursinum possessed a much larger content of compounds acylated with p-coumaric acid than green leaves (1299.97 versus 855.67 mg/100 g of dm, respectively). The stalks and seeds contained much more non-acetylated than acetylated flavonoid glycosides with p-coumaric acid compounds (162.4 versus 62.82 mg/100 g of dm and 105.49 versus 24.18 mg/100 g of dm, respectively).

Concepts: Mass spectrometry, Garlic, Allium, Quercetin, Flavonoid, Phenols, Tandem mass spectrometry, Myricetin

28

Isobaric labeling strategies (e.g. iTRAQ or TMT) are commonly applied in tandem mass spectrometric (MS/MS) level quantitative proteomics. However, we frequently observed missing isotope reporter ion signals in a large-scale liquid chromatography/matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometric (LC/MALDI-TOF/TOF) quantitative proteomics experiment. To understand this issue, we systematically investigated the processing of MS/MS spectra into peak lists prior to peptide identification and quantification.

Concepts: Scientific method, Mass spectrometry, Research, Proteomics, Tandem mass spectrometry, Tandem mass tags, Quantitative proteomics, SILAC

28

We have synthesized cis- and trans-dihydroanatoxin-a and cis- and trans-dihydrohomoanatoxin-a using a short synthetic route. The relative configuration of N-tert-butoxycarbonyl-cis-dihydroanatoxin-a was determined by X-ray crystallography, while that of N-tert-butoxycarbonyl-trans-dihydroanatoxin-a was confirmed by epimerization leading to the cis-diastereoisomer. The relative configuration of N-tert-butoxycarbonyl-trans- and cis-dihydrohomoanatoxin-a was inferred from their NMR spectra. Using an optimized LC-MS/MS analytical method and pure standards we have simultaneously determined anatoxin-a, homoanatoxin-a and their dihydroderivatives in axenic strains of cyanobacteria and in environmental samples from the Tarn River, France. However, in these analytical conditions, the cis- and trans-dihydroanatoxin-a and cis- and trans-dihydrohomoanatoxin-a could not be separated. In axenic strains, the dihydroderivatives represented less than 3% of the total toxin content, while in field samples dihydroanatoxin-a represented from 17% to 90% of the total toxin content. Thus, the reduction of anatoxin-a to dihydroanatoxin-a is predominant in the environment. The ratio of anatoxin-a concentration over that of homoanatoxin-a in axenic strains was variable, and among the eight strains studied we found three exclusive anatoxin-a producers and five producers of homoanatoxin-a and anatoxin-a, the latter representing from 0.5% to 2.0% of the total. In the strains studied, we have amplified by PCR, and sequenced the region of anaG coding for the methylation domain proposed to be responsible for the formation of homoanatoxin-a. The sequences showed at least 88% identity and we could not relate the toxin profile of the strains to the sequence of the methylation domain.

Concepts: DNA, Cyanobacteria, Bacteria, Mass spectrometry, Ratio, Sequence, Tandem mass spectrometry, Cyanotoxin

28

Protein toxins, such as botulinum neurotoxins (BoNTs), Clostridium perfringens epsilon toxin (ETX), staphylococcal enterotoxin B (SEB), shiga toxin (STX), and plant toxin ricin, are involved in a number of diseases and are considered as potential agents for bioterrorism and warfare. From a bioterrorism and warfare perspective, these agents are likely to cause maximum damage to a civilian or military population through an inhalational route of exposure and aerosol is considered the envisaged mode of delivery. Unambiguous detection of toxin from aerosol is of paramount importance, both for bringing mitigation protocols into operation and for implementation of effective medical countermeasures, in case a “biological cloud” is seen over a population. A multiplex, unambiguous, and qualitative detection of protein toxins is reported here using tandem mass spectrometry with MALDI-TOF-TOF. The methodology involving simple sample processing steps was demonstrated to identify toxins (ETX, Clostridium perfringes phospholipase C, and SEB) from blind spiked samples. The novel directed search approach using a list of unique peptides was used to identify toxins from a complex protein mixture. The bioinformatic analysis of seven protein toxins for elucidation of unique peptides with conservation status across all known sequences provides a high confidence for detecting toxins originating from any geographical location and source organism. Use of tandem MS data with peptide sequence information increases the specificity of the method. A prototype for generation of aerosol using a nebulizer and collection using a cyclone collector was used to provide a proof of concept for unambiguous detection of toxin from aerosol using precursor directed tandem mass spectrometry combined with protein database searching. ETX prototoxin could be detected from aerosol at 0.2 ppb concentration in aerosol.

Concepts: Protein, Mass spectrometry, Peptide, Exotoxin, Toxin, Tandem mass spectrometry, Clostridium perfringens, Microbial toxins