Specific Fragmentation Research Articles

A Laboratory Rig and a Scale‐ and Energy‐Controlled Procedure for Tender Soil Fragmentation Test

A laboratory rig was developed for a scale‐ and energy‐controlled tender soil fragmentation test, which required multiple impacts and step‐by‐step decomposition of the soil structure on a macroscale. Soil sample size classes were scaled down by a factor of two, giving ranges of 128, 64, 32, 16, 8, 4, 2, 1, and 0.5 mm, respectively. These size classes were also used as control scales for respective fragmentation stages. Fragmentation tests were conducted to illustrate the possible influences from shape effects, falling weights, displacements, and fragmentation stages, and to investigate the relations among specific fragmentation energy, number of strokes per fragmentation stage, and fragmentation fractal dimension. Specific fragmentation energy was influenced by both the size and the shape of the soil samples. The cylindrical‐ and ball‐shaped soil samples behaved similarly. Thus ball samples were used for a benchmarking test on soil structural stabilities. Subsequent fragmentation on 60‐ and 80‐g ball samples with changed displacements from 5 to 45 cm yielded a decreased number of strokes from around 150 to 10. Whereas, their respective fragmentation fractal dimensions varied insignificantly, ranging from 1.52 to 1.64 for samples of 60‐g mass and 1.55 to 1.72 for 80 g, values much smaller than previously reported. Fragmentation fractal dimension appeared to be a nominal index for remolded soils under laboratory impacting actions. There was a slight increase in specific fragmentation energy as the number of impacts increased. But this increase was aggravated for a single or only few impacts. Thus 20 to 40 strokes was the appropriate stroke number to finish one fragmentation stage. A five‐stage fragmentation on a sample in the 32‐ to 64‐mm size range yielded fragmentation fractal dimensions of 1.29, 1.52, 1.45 and 1.61, for the first four stages, respectively, whereas their specific fragmentation energies increased linearly.

Precursor ion scanning and sequencing of arginine-ADP-ribosylated peptide by mass spectrometry

Arginine (Arg)-specific ADP-ribosylation is one of the posttranslational modifications of proteins and is thought to play an important role in reversibly regulating functions of the target proteins in eukaryotes. However, the physiological target protein has not been established. We examined the fragmentation pattern of both ADP-ribosyl-Arg (ADP-R-Arg) and Arg-ADP-ribosylated peptides by quadrupole tandem mass spectrometry and found a specific cleavage of ADP-R-Arg into N-(ADP-ribosyl)-carbodiimide (ADP-R-carbodiimide) and ornithine. Based on this specific fragmentation pattern, we successfully identified the modification site and sequence of Arg-ADP-ribosylated peptide using a two-step collision and showed that ADP-R-carbodiimide is an excellent marker ion for precursor ion scanning of Arg-ADP-ribosylated peptide. We propose that a combination of the precursor ion scanning with ADP-R-carbodiimide as a marker ion and two-step collision is useful in searching for physiological target proteins of Arg-ADP-ribosylation.

The N-glycome of human embryonic stem cells

BackgroundComplex carbohydrate structures, glycans, are essential components of glycoproteins, glycolipids, and proteoglycans. While individual glycan structures including the SSEA and Tra antigens are already used to define undifferentiated human embryonic stem cells (hESC), the whole spectrum of stem cell glycans has remained unknown. We undertook a global study of the asparagine-linked glycoprotein glycans (N-glycans) of hESC and their differentiated progeny using MALDI-TOF mass spectrometric and NMR spectroscopic profiling. Structural analyses were performed by specific glycosidase enzymes and mass spectrometric fragmentation analyses.ResultsThe data demonstrated that hESC have a characteristic N-glycome which consists of both a constant part and a variable part that changes during hESC differentiation. hESC-associated N-glycans were downregulated and new structures emerged in the differentiated cells. Previously mouse embryonic stem cells have been associated with complex fucosylation by use of SSEA-1 antibody. In the present study we found that complex fucosylation was the most characteristic glycosylation feature also in undifferentiated hESC. The most abundant complex fucosylated structures were Lex and H type 2 antennae in sialylated complex-type N-glycans.ConclusionThe N-glycan phenotype of hESC was shown to reflect their differentiation stage. During differentiation, hESC-associated N-glycan features were replaced by differentiated cell-associated structures. The results indicated that hESC differentiation stage can be determined by direct analysis of the N-glycan profile. These results provide the first overview of the N-glycan profile of hESC and form the basis for future strategies to target stem cell glycans.

P38 MITOGEN-ACTIVATED PROTEIN KINASE (MAPK) IS ACTIVATED BY NORADRENALINE AND SERVES A CARDIOPROTECTIVE ROLE, WHEREAS ADRENALINE INDUCES p38 MAPK DEPHOSPHORYLATION

1. The aim of the present study was to investigate the role of p38 mitogen-activated protein kinases (MAPK) in mediating the effect of noradrenaline (NA) on cardiomyocyte cell viability. 2. Cardiomyocytes from embryonic chick heart were treated with various concentrations of NA, phenylephrine or isoproterenol and p38 MAPK activation was determined by western blotting. Total cell death was assessed by the 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Apoptosis was determined by specific DNA fragmentation. 3. At 100 micromol/L, NA produced a significant increase in cell death that was associated with microscopic changes and DNA fragmentation indicative of apoptosis. The p38 MAPK inhibitor SB202190 (at 1 micromol/L beginning 1 h before NA), reduced NA-induced p38 MAPK activation and significantly accentuated NA-induced cell death. In contrast, the mitogen-activated protein kinase kinase ERK1/2 inhibitor PD98059 (at 1 micromol/L beginning 1 h before NA) did not significantly alter NA-induced cell death. These effects of NA were mediated, in part, through alpha-adrenoceptor because phenylephrine (100 micromol/L), like NA, also induced p38 MAPK activation. However, 100 micromol/L isoproterenol produced a sustained dephosphorylation of p38 MAPK. 4. These data show that NA-induced p38 MAPK activation, through alpha-adrenoceptor, has a protective role in cardiomyocytes to antagonize NA-induced cell death. In contrast, beta-adrenoceptor stimulation produces dephosphorylation of p38 MAPK.

Spectrometric Studies and Semi-Empirical Calculations of Some Thiomorpholides

Mass spectra of selected thiomorpholides have been analysed and specific fragmentation assignments have been done to characterised and weigh co-existing keto and enol tautomers. The predictive value of this methodology in regard to keto-enol equilibria occurring in gas phase is not only supported by the influence of substitution nature and size of these but also by the good correlation found between the selected fragments abundances ratio, semi-empirical calcula- tion (AM1) of the corresponding heats of tautomerization and nuclear magnetic resonance data. The results show that the thioketo-thioenol equilibrium can be studied by mass spectrometry where ionization in the ion source has a negligible ef- fect on the position of that equilibrium and by GC/MS where solvent separation in the analytical column seems to pre- clude shifts from the equilibrium reached at the injection port of the gas chromatograph.

Systematic LC-MS Analysis of Labile Post-Translational Modifications in Complex Mixtures

Most proteins are post-translationally modified and the characterization of modified peptides in complex mixtures generated by enzymatic digestion of multiple proteins remains a major analytical challenge. We describe an integrated LC-MS workflow implemented on a hybrid quadrupole time-of-flight (Q-ToF) instrument to detect modified peptides in a complex peptide sample and establish the nature of the modification. The method is based on the alternating acquisition of full mass spectra under different collision conditions inducing the cleavage of the substituents. Modified peptides are detected based on their specific fragmentation generating the nonmodified peptide backbone and reporter ions in the low mass region. The two mass analyzer stages of a Q-ToF instrument are used to eliminate the low mass chemical background in the quadrupole and thus facilitate the detection of low mass reporter ions in the ToF. Off-line data processing enables detection of one (or even multiple) modifications and the modified candidates are subsequently sequenced in a directed MS/MS mode. The technique was applied to the analysis of O-GlcNAc peptides, a very complex mixture of N-linked glycopeptides, and a phosphotyrosine peptide.

Fragmentation of adenine under energy control

We present results on the fragmentation of adenine dication as a function of the excitation energy. The adenine molecule is charged and excited in a single collision with Cl(+) ion at 3 keV and the excitation energy distribution is obtained for each fragmentation channel by measuring the kinetic energy loss of the projectile. This method named collision induced dissociation under energy control is based on the formation of a negative scattered projectile as a result of double electron capture from the target molecule. Comparison between the main dissociation channels of singly and doubly charged adenine shows that fragmentation patterns are very similar consisting mainly of the successive emission of neutral HCN or H(2)CN(+). The energy distributions of the parent adenine dication and the kinetic energy release of the fragments measured for the most abundant fragmentation channels confirms the assumption of successive emission dynamics. A specific fragmentation pathway of the adenine requiring less energy than the usual successive emission of neutral HCN could be identified. It consists of the emission of a charged H(2)CN(+) following on by the emission of a dimer of HCN (precisely HC(2)N(2)). This new channel, measured for a mean excitation energy of 8.4 eV for the adenine dication is very closed to the emission of HCN monomer measured at 7.9 eV. The implications of these results concern the formation of adenine in the sealed-tube reaction of HCN with liquid ammonia as well as the possible formation of the adenine molecule in the interstellar medium. This last point is briefly discussed in relation to astrobiology and exobiology interests.

Fragmentation Studies of Fulvic Acids Using Collision Induced Dissociation Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

The complex natural organic matter standard Suwannee river fulvic acid (SRFA) was analyzed by negative ion mode electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FTICR MS) using on-resonance collision induced dissociation (CID) of single ultrahigh resolved mass peaks in the ICR cell. Molecular formula assignment of precursor masses resulted in exactly one molecular formula for each of the peaks. Analyses of the corresponding fragment spectra and comparison to different standard substances revealed specific neutral losses and fragmentation patterns which result in structures consisting of a high degree of carboxyl- and fewer hydroxyl groups. The comparison of fragmented mass peaks within different pseudohomologous series (CH(2)-series, and CH(4) vs O exchange) suggested structurally based differences between these series. CID FTICR MS allowed isolating single mass peaks in a very complex natural organic matter spectrum. Subsequently, fragmentation gave structural insights into this material. Our results suggest that the structural diversity in complex humic substances is not as high as expected.

Study of active naphtodianthrone St John's Wort compounds by electrospray ionization Fourier transform ion cyclotron resonance and multi‐stage mass spectrometry in sustained off‐resonance irradiation collision‐induced dissociation and infrared multiphoton dissociation modes

Five well-known active naphtodianthrone constituents of Hypericum perforatum (St John's Wort) extracts have been investigated by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICRMS) and ESI-FTICRMSn. The studied compounds were hypericin, pseudohypericin, protohypericin, protopseudohypericin (biosynthetic precursors of the two former compounds, respectively) and isopseudohypericin (alkaline degradation product of pseudohypericin). Dissociation mass spectrometry measurements performed on the [M-H]- ion presented a variable efficiency as a function of the used activation mode. Sustained off-resonance irradiation collision-induced dissociation (SORI-CID) only led to a restricted number of fragment ions. In contrast, IRMPD ensured the detection of numerous product ions. Ions detected in ESI-FTICRMS and ESI-FTICRMSn experiments were measured with a very high mass accuracy (typically mass error is lower than 0.5 mDa at m/z close to 500) that allowed unambiguous formulae to be assigned to each signal observed in a mass spectrum. In spite of similar structures, specific fragmentation patterns were observed for the different compounds investigated. This study may be useful in the future to characterize in natural extracts these compounds (or derivatives of these compounds) by liquid chromatography/tandem mass spectrometry (LC/MS/MS) experiments by considering the MS/MS transitions highlighted in this paper.

Rapid and sensitive method for simultaneous determination of six carcinogenic aromatic amines in mainstream cigarette smoke by liquid chromatography/electrospray ionization tandem mass spectrometry

Aromatic amines are one of the sources of carcinogenicity in cigarette and tobacco smoke. Accurate quantification of these chemicals is needed to assess public health risk. A new validated rapid, sensitive and analyte specific liquid chromatography/electrospray ionization tandem mass spectrometric (LC/MS/MS) method has been developed for the simultaneous determination of six aromatic amines in mainstream cigarette smoke using research reference cigarette 2R4F. Three popular Indian brand cigarettes were also analyzed using the same procedure. The limit of detection of this method ranged from 0.04 to 0.59 ng/cig using an injection volume of 7 μl. The identification of each amine was established by chromatographic retention times, analyte specific fragmentation pattern and relative peak area ratios of two product/precursor ion pairs. The method showed excellent reproducibility and was also rapid, selective and robust for aromatic amine determination from cigarette smoke.

Group‐Based QSAR (G‐QSAR): Mitigating Interpretation Challenges in QSAR

AbstractSeveral approaches are widely being used as important tools for drug discovery. These approaches include Hansch method, Free‐Wilson method and conventional 2‐D/3‐D QSAR methods. The Hansch analysis assumes that substituents are independent of each other and does not include explicit interactions of groups. In the conventional QSAR method, the interpretation of model generated is rather difficult, as one does not a get clear direction about the site for improvement. A new Group‐Based QSAR (G‐QSAR) method is proposed which uses descriptors evaluated for the fragments of the molecules generated using specific fragmentation rules defined for a given dataset. Herein, we describe the application of G‐QSAR method on two different datasets belonging to a simple congeneric series and a complex noncongeneric series. This method provides models with predictive ability similar or better to conventional methods and in addition provides hints for sites of improvement in the molecules.

Identification and quantification of phenolic compounds from sunflower (Helianthus annuus L.) kernels and shells by HPLC-DAD/ESI-MSn

Abstract Phenolic compounds were extracted from defatted sunflower (Helianthus annuus L.) kernels and shells and characterised by HPLC with diode array and electrospray ionisation (ESI) mass spectrometric detection in the negative mode. Quantification of individual compounds was carried out by external calibration. Among the eleven compounds analysed 5-O-caffeoylquinic acid was predominant amounting up to 59.1 mg/100 g in the shells and 3050.5 mg/100 g in the kernels. The specific fragmentation patterns of mono- and dihydroxycinnamoylquinic acids allowed the unambiguous distinction of several stereoisomers which have not been described for sunflower seeds and seed shells so far. The total phenolic content of about 4200 mg/100 g on a dry matter basis revealed defatted sunflower meal to be a promising source of phenolic compounds that might be recovered and used as natural antioxidants. Furthermore, the press residues originating from sunflower oil extraction were shown to be still rich in phenolic antioxidants, thus, providing the opportunity to valorize these by-products in terms of sustainable agricultural production.

Specific Fragmentation of K-Shell Excited/Ionized Pyridine Derivatives Studied by Electron Impact: 2-Amino-3-methylpyridine and 3-Methylpyridine

Abstract Fragmentation of 2-amino-3-methylpyridine has been investigated by electron impact in the carbon and nitrogen K-shell excitation regions with a comparison to that of 3-methylpyridine. Fragment ions were analyzed with a quadrupole mass spectrometer. Yields of the NCNH2+ and C5H3+/C4HN+ fragment ions characteristically increase at the N 1s edge when compared to the C 1s excitation/ionization and valence ionization. This observation indicates that a valence electron forming the C–N bond in the pyridine ring is preferentially released through an Auger electron emission at the N 1s edge. The fragment ions produced via a hydrogen atom shift were observed as well.

Ion trap lC-MS/MS as a valid multi-method to determine trichothecenes and zearalenone in the food industry

In recent years the European Scientific Committee on Food has frequently expressed its opinion on Fusarium toxins, setting limits, regulations and guidelines in order to reduce their levels in raw materials and food commodities. Trichothecenes are known as DNA and mitochondrial metabolism inhibitors, representing the largest group (over 170 compounds) of Fusarium mycotoxins. Zearalenone (ZEA) is also produced by Fusarium species. It is found almost entirely in grains and has a mainly oestrogenic effect. Wheat-based products (such as bread and pasta) are the predominant source of deoxynivalenol (DON) and nivalenol (NIV) intake, while the highest amounts of T-2 and HT-2 toxins were observed mainly in oat-based products. Therefore, food companies are progressively being forced to set up analytical methods in their laboratories for determining these kinds of toxins in an accurate, sensitive and rapid way. Following this issue, we set up a liquid chromatography-electrospray ionisation tandem mass spectrometry (LC-ESI-IonTrap-MS/MS) method for the simultaneous determination of several types of A and B trichothecenes, together with ZEA, minimising the matrix effect by using specific fragmentation parameters in positive or negative ion modes and dedicated internal standards: quantification of trichothecenes was done with isotope substituted (13C15)-deoxynivalenol, while quantification of ZEA was performed by the contemporaneous use of Zearalanone (ZAN). Sample extraction was performed with acetonitrile/water mixtures, MycoSep® columns were used for fast and effective clean-up procedure and detection was carried out in the multiple reaction monitoring (MRM) mode. Spiking blank cereal samples, the method was validated in terms of detection limits (reaching µg/ kg levels), linearity, recovery, precision (RSD<5%) and accuracy. Method performances were finally tested on oat and durum wheat samples. In conclusion, ion trap instruments can currently provide high-throughput LC/MSn mycotoxin analysis where sensitivity, reliability and productivity coexist in an interesting and good compromise for food industry applications.

Assessment of missile hazards: Identification of reference fragmentation patterns

Industrial accidents involving fragment projection were investigated. The analysis of fracture mechanics fundamentals allowed the exploration of the relations between the fracture characteristics and the final event leading to equipment collapse. Reference fragmentation patterns were defined on the basis of the geometrical characteristics of the categories of process vessels that are more frequently involved in fragmentation accidents. Primary scenarios leading to fragment projection were correlated to specific fragmentation patterns. A database reporting a detailed analysis of more than 140 vessel fragmentation events provided the data needed to support and validate the approach. The available data also allowed the calculation of the expected probability of fragment projection following vessel fragmentation, and the probability of the alternative fragmentation patterns with respect to the different accidental scenarios, based on the observed frequencies over the available data set.

Specific fragmentation of [(CH 3) 2CO]Ar n heteroclusters induced by the Ar L 23- and O K-shell excitation

Ionic fragmentation following the inner-shell photoexcitation of [(CH 3) 2CO]Ar n heteroclusters was investigated in the Ar L 23- and O K-edge regions. A partial ion yield (PIY) measurement revealed that Ar n + and various acetone fragments ( H m + , CH m + , C 2 H m + , C 3 H m + , and CH m CO + ) were produced following the Ar L 23-shell excitation. In the O K-edge region, the PIY of O + exhibited a sharp resonance peak at the O 1s → π ∗(C O) resonance excitation. The selective O + formation was interpreted as the suppression of statistical fragmentation, which was dominant in isolated acetone, owing to the dissipation of excess energy into Ar clusters.

High‐throughput approaches towards the definitive identification of pharmaceutical drug metabolites. 1. Evidence for an ortho effect on the fragmentation of 4‐benzenesulfinyl‐3‐methylphenylamine using electrospray ionisation mass spectrometry

A 50 m/z unit loss from protonated 4-benzenesulfinyl-3-methylphenylamine has been observed and investigated using electrospray ionisation quadrupole ion trap mass spectrometry (ESI-QIT-MS). It was hypothesised that the specific fragmentation was affected by the presence of an ortho methyl group in relation to the sulfoxide functionality, i.e. an ortho effect influences the preferred dissociation pathway. This was because the des-methyl homologue did not display a 50 m/z unit loss. This fragmentation was shown to be a two-step process with sequential losses of a hydroxyl radical and a thiol radical. Molecular modelling calculations showed that the most favourable site of protonation for 4-benzenesulfinyl-3-methylphenylamine was the sulfoxide oxygen, which would facilitate the loss of a hydroxyl radical. Subsequent deuterium-exchange experiments confirmed that the loss was a hydroxyl radical and afforded definitive assignment of the site of protonation. Furthermore, the involvement of a single exchangeable hydrogen atom in the overall 50 m/z unit loss was demonstrated. Thus, supportive evidence was provided for the involvement of the ortho methyl group in the second stage of the fragmentation, leading to the loss of the thiol radical. Accurate mass measurements, performed using electrospray ionisation Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS), verified the elemental formulae of the individual losses. The ion structure following the 50 m/z unit loss was proposed to be a protonated aminofluorene and was supported by comparing the product ion spectrum of commercially available protonated 2-aminofluorene with the MS4 data of protonated 4-benzenesulfinyl-3-methylphenylamine. Fragmentation mechanisms are proposed. The relevance of the loss with regards to pharmaceutical drug metabolite identification is discussed.

Plant N-glycan profiling of minute amounts of material

Development of convenient strategies for identification of plant N-glycan profiles has been driven by the emergence of plants as an expression system for therapeutic proteins. In this article, we reinvestigated qualitative and quantitative aspects of plant N-glycan profiling. The extraction of plant proteins through a phenol/ammonium acetate procedure followed by deglycosylation with peptide N-glycosidase A (PNGase A) and coupling to 2-aminobenzamide provides an oligosaccharide preparation containing reduced amounts of contaminants from plant cell wall polysaccharides. Such a preparation was also suitable for accurate qualitative and quantitative evaluation of the N-glycan content by mass spectrometry. Combining these approaches allows the profiling to be carried out from as low as 500 mg of fresh leaf material. We also demonstrated that collision-induced dissociation (CID) mass spectrometry in negative mode of N-glycans harboring α(1,3)- or α(1,6)-fucose residue on the proximal GlcNAc leads to specific fragmentation patterns, thereby allowing the discrimination of plant N-glycans from those arising from mammalian contamination.

Is internucleosomal DNA fragmentation an indicator of programmed death in plant cells?

Specific DNA fragmentation into oligonucleosomal units occurs during programmed cell death (PCD) in both animal and plant cells, usually being regarded as an indicator of its apoptotic character. This internucleosomal DNA fragmentation is demonstrated in tobacco suspension and leaf cells, which were killed immediately by freezing in liquid nitrogen, and homogenization or treatment with Triton X-100. Although these cells could not activate and realize the respective enzymatic processes in a programmed manner, the character of DNA fragmentation was similar to that in the cells undergoing typical gradual PCD induced by 50 μM CdSO4. This internucleosomal DNA fragmentation was connected with the action of cysteine proteases and the loss of membrane, in particular tonoplast, integrity. The mechanisms of DNase activation in the rapidly killed cells, hypothetical biological relevance, and implications for the classification of cell death are discussed.

Profiling of Alk(en)ylresorcinols in cereals by HPLC-DAD-APcI-MS n

5-Alk(en)ylresorcinols in rye, wheat, spelt, and barley have been characterized by high-performance liquid chromatography coupled to atmospheric pressure chemical ionization multistage mass spectrometry (HPLC-APcI-MS(n)) for the first time. Among the 29 compounds analysed, several major and minor C(15), C(17), C(19), C(21), C(23), and C(25)-substituted resorcinols with saturated, monoenoic, dienoic, and/or oxygenated side-chains were characterized by their specific fragmentation patterns in collision-induced dissociation experiments. Additionally, a C(27:0) homologue, which has probably been overlooked in previous studies based on HPLC alone, was detected in all cereals analysed. Furthermore, we provide tentative evidence for the occurrence of alkylresorcinols with triolefinic side-chains, which have, to our knowledge, so far not been reported in any cereal species.