Dissociation Tandem Mass Spectrometry Research Articles

Energy-Resolved Mass Spectrometry and Mid-Infrared Spectroscopy for Purity Assessment of a Synthetic Peptide Cyclised by Intramolecular Huisgen Click Chemistry

Cyclic peptides have higher stability and better properties as therapeutic agents than their linear peptide analogues. Consequently, intramolecular click chemistry is becoming an increasingly popular method for the synthesis of cyclic peptides from their isomeric linear peptides. However, assessing the purity of these cyclic peptides by mass spectrometry is a significant challenge, as the linear and cyclic peptides have identical masses. In this paper, we have evaluated the analytical capabilities of energy-resolved mass spectrometry (ER MS) and mid-infrared microscopy (IR) to address this challenge. On the one hand, mixtures of both peptides were subjected to collision-induced dissociation tandem mass spectrometry (CID MS/MS) experiments in an ion trap mass spectrometer at several excitation energies. Two different calibration models were used: a univariate model (at a single excitation voltage) and a multivariate model (using multiple excitation voltages). The multivariate model demonstrated slightly enhanced analytical performance, which can be attributed to more effective signal averaging when multiple excitation voltages are considered. On the other hand, IR microscopy was used for the quantification of the relative amount of linear peptide. This was achieved through univariate calibration, based on the absorbance of an alkyne band specific to the linear peptide, and through Partial Least Squares (PLS) multivariate calibration. The PLS calibration model demonstrated superior performance in comparison to univariate calibration, indicating that consideration of the full IR spectrum is preferable to focusing on the specific peak of the linear peptide. The advantage of IR microscopy is that it is linear across the entire working interval, from linear peptide molar ratios of 0 (equivalent to pure cyclic peptide) up to 1 (pure linear peptide). In contrast, the ER MS calibration models exhibited linearity only up to 0.3 linear peptide molar ratio. However, ER MS showed better performances in terms of the limit of detection, intermediate precision and the root-mean-square-error of calibration. Therefore, ER MS is the optimal choice for the detection and quantification of the lowest relative amounts of linear peptides.

Unravelling the Structure of One-Electron Oxidation Products of Model Peptide Backbones Containing Methionine by IRMPD Spectroscopy: the Effect of the Neighbouring Groups.

A series of Methionine (Met) derivatives, where either the amino group and/or the carboxylic acid group is blocked by acetyl and/or methyl ester functionalities, has been investigated by Collision Induced Dissociation-tandem mass spectrometry (CID-MS2) and Infrared Multiple Photon Dissociation (IRMPD) spectroscopy. The CID-MS2 experiments were performed using a Fourier-transform ion-cyclotron-resonance (FT-ICR) mass spectrometer equipped with an electrospray ionization (ESI) source. The IRMPD spectra were recorded employing a Paul type ion-trap coupled with the free-electron laser (FEL) FELIX in the fingerprint region from 600 to 2000 cm-1. We show that the oxidation of the methionine residue with protected terminal groups induces the formation of a sulfoxide function. However, compared to the IRMPD spectrum of protonated methionine and methionine sulfoxide, significant spectral differences are observed in the spectra of model protected peptide backbones containing methionine. DFT calculations show that protonation occurs on the sulfoxide group in the gas phase for these model peptide backbones containing methionine, shifting the diagnostic signature of the sulfoxide group from 1000 to 862 cm-1.

Structural elucidation of 14-membered ring macrolide antibiotics using electrospray ionization tandem mass spectrometry and density functional theory calculations.

Macrolides are critical antibiotics featuring a macrocyclic lactone core with deoxy sugars. Understanding their gas-phase fragmentation is challenging but essential for improving structural elucidation in mass spectrometry, which has implications for drug discovery and development. We used electrospray ionization collision-induced dissociation tandem mass spectrometry (ESI-CID-MS) combined with quantum chemical calculations to investigate the fragmentation pathways of erythromycin A and roxithromycin. This approach helps elucidate the preferred fragmentation routes influenced by protonation sites. Macrolides showed similar fragmentation patterns, including sequential losses of saccharide or amino sugar units and dehydration from the macrocycle core. Multiple competitive pathways were observed, influenced by protonation sites. Computational studies confirmed the most favorable protonation sites and their impact on fragmentation, providing insights into key diagnostic product ions. Subsequent fragments involved rearrangement pathways such as alkene formation and cleavages via remote hydrogen transfers and pericyclic reactions. Our integrated approach offers a comprehensive understanding of macrolide fragmentation, enhancing structural elucidation and potential applications in drug development. This study advances mass spectrometry analysis of macrolides, contributing to pharmaceutical research by integrating orthogonal annotation methods and fragmentation studies.

Structural characterization and bioactivity profiling of the fungal peptaibiotic tolypin reveal protective effects against influenza viruses.

In a bioprospection for new antivirals, we tested nonribosomally biosynthesized polypeptide antibiotics in MDCK II cells for their actions on influenza A and B viruses (IAV/IBV). Only tolypin, a mixture of closely related 16-residue peptaibiotics from the fungus Tolypocladium inflatum IE 1897, showed promising activity. It was selected for further investigation and structural characterization byultrahigh performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HR-MS/MS) and ultrahigh performance liquid chromatography coupled to in-source collision-induced dissociation tandem mass spectrometry (UHPLC-isCID-HR-MS/MS), revealing 12 partially co-eluting individual peptides that were fully sequenced. Since tolypin-related efrapeptins are potent inhibitors of F1/Fo-ATPase, we screened tolypin for its toxicity against MDCK II cells and larvae of the greater wax moth Galleria mellonella. We found that a nontoxic concentration of tolypin (1 µg/mL) reduced the titer of two IBV strains by 4-5 log values, and that of an H3N2 strain by 1-2 log values, but the H1N1pdm strain was not affected. The higher concentrations of tolypin were cytostatic to MDCK II cells, shifted their metabolism from oxidative phosphorylation to glycolysis, and induced paralysis in G. mellonella, supporting the inhibition of F1/Fo-ATPase as the mode of action. Our results lay the foundations for future work to investigate the interplay between viral replication and cellular energy metabolism, as well as the development of drugs that target host factors.

Structural and quantitative characterization of membrane N-glycans from MIN6 mouse pancreatic beta cells using liquid chromatography-quadrupole-Orbitrap tandem mass spectrometry

MIN6, a mouse pancreatic beta cell line, is used in diabetes research, and the cellular N-glycoproteins in membrane are important in regulating the metabolism of insulin secretion. However, the identities of N-glycans in MIN6 cells are yet to be fully elucidated. In this study, the structures of N-glycans were analyzed using liquid chromatography-electrospray ionization-higher energy collisional dissociation-tandem mass spectrometry. The abundances (%) of each N-glycan relative to the total N-glycans (100 %) were also obtained. Fifty N-glycans (with relative abundance of each > 0.5 %) were obtained, revealing 22 bisecting N-acetylglucosamine (GlcNAc; associated with cell adhesion and growth; sum of relative abundance of each: 27.1 %), 21 core-fucosylated (associated with glucose sensing and insulin secretion regulation; 28.3 %), and 16 sialylated (N-acetylneuraminic acid; related to the expression of glucose transporters and diabetes;15.5 %) N-glycans. Membranes contained higher bisecting GlcNAc and core-fucosylation, similar sialylation, but less high-mannosylation than the lysate (the cellular contents). Notably, all bisecting GlcNAc N-glycans were categorized into structures with (16.6 %) or without (10.5 %) core-fucosylation and with (6.9 %) or without (20.2 %) sialylation. The bisecting GlcNAc structures were not found in human islets; moreover, sialylation levels were 6.9 times higher than for human islets. These structural characteristics of N-glycans affect their cell adhesion and distribution through homologous interactions between beta cells, leading to increased insulin secretion efficiency. This study is the first to identify the structures and quantities of 50 N-glycans in MIN6 cell membranes that may play an important role in regulating the functions of pancreatic beta cells.

Comprehensive identification strategy for rapid profiling of chemical constituents using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry with Rhubarb as an example

In this study, the collision induced dissociation tandem mass spectrometry (CID-MS/MS) fragmentation pathway of chemical components in rhubarb was wholly explored using 34 standards by UHPLC-QTOF-MS/MS in negative ion mode. In consequently, the diagnostic product ions for speedy screening and categorization of chemical components in rhubarb were ascertained based on their MS/MS splitting decomposition patterns and intensity analysis. According to these findings, a fresh two-step data mining strategy had set up. The initial key step involves the use of characteristic product ions and neutral loss to screen for different types of substituents and basic skeletons of compounds. The subsequent key step is to screen and classify different types of compounds based on their characteristic product ions. This method can be utilized for rapid research, classification, and identification of compounds in rhubarb. A total of 356 compounds were rapidly identified or tentatively characterized in three rhubarb species extracts, including 150 acylglucoside, 125 anthraquinone, 65 flavanols and 15 other compounds. This study manifests that the analytical strategy is feasible for the analysis of complex natural products in rhubarb.

Investigating the Mechanism of Microwave-Assisted Enzymolysis Synergized with Magnetic Bead Adsorption for Reducing Ovalbumin Allergenicity through Biomass Spectrometry Analysis.

This study found that, after microwave treatment at 560 W for 30 s, alkaline protease enzymolysis significantly reduced the allergenicity of ovalbumin (OVA). Furthermore, specific adsorption of allergenic anti-enzyme hydrolyzed peptides in the enzymatic products by immunoglobulin G (IgG) bound to magnetic bead further decreased the allergenicity of OVA. The results indicated that microwave treatment disrupts the structure of OVA, increasing the accessibility of OVA to the alkaline protease. A comparison between 17 IgG-binding epitopes identified through high-performance liquid chromatography-higher energy collisional dissociation-tandem mass spectrometry and previously reported immunoglobulin E (IgE)-binding epitopes revealed a complete overlap in binding epitopes at amino acids (AA)125-135, AA151-158, AA357-366, and AA373-381. Additionally, partial overlap was observed at positions AA41-59, AA243-252, and AA320-340. Consequently, these binding epitopes were likely pivotal in eliciting the allergic reaction to OVA, warranting specific attention in future studies. In conclusion, microwave-assisted enzymolysis synergized with magnetic bead adsorption provides an effective method to reduce the allergenicity of OVA.

Characterization of PEGylation sites in Neulasta and a biosimilar candidate with a combined fragmentation strategy in mass spectrometry analysis.

In the development of biosimilar products to Neulasta, it is essential to determine the intact molecular mass and confirm precise PEGylation sites. In this study, we applied a combination of techniques, including post-column addition of triethylamine in reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) to determine the intact molecular mass, and in-source fragmentation (ISF) and higher-energy collision dissociation-tandem mass spectrometry (HCD-MS/MS) to identify the PEGylation site. Our results show that both the pegfilgrastim biosimilar candidate and Neulasta lots are mono-PEGylated at the N-terminal end. Furthermore, we show that the combined ISF and HCD-MS/MS method can be used for identifying the PEGylation sites in the diPEGylated variant of pegfilgrastim. The diPEGylated variant has modification sites at the N-terminal end and a lysine at position 35 in the protein sequence.

A spectroscopic test suggests that fragment ion structure annotations in MS/MS libraries are frequently incorrect

Modern untargeted mass spectrometry (MS) analyses quickly detect and resolve thousands of molecular compounds. Although features are readily annotated with a molecular formula in high-resolution small-molecule MS applications, the large majority of them remains unidentified in terms of their full molecular structure. Collision-induced dissociation tandem mass spectrometry (CID-MS2) provides a diagnostic molecular fingerprint to resolve the molecular structure through a library search. However, for de novo identifications, one must often rely on in silico generated MS2 spectra as reference. The ability of different in silico algorithms to correctly predict MS2 spectra and thus to retrieve correct molecular structures is a topic of lively debate, for instance in the CASMI contest. Underlying the predicted MS2 spectra are the in silico generated product ion structures, which are normally not used in de novo identification, but which can serve to critically assess the fragmentation algorithms. Here we evaluate in silico generated MSn product ion structures by comparison with structures established experimentally by infrared ion spectroscopy (IRIS). For a set of three dozen product ion structures from five precursor molecules, we find that virtually all fragment ion structure annotations in three major in silico MS2 libraries (HMDB, METLIN, mzCloud) are incorrect and caution the reader against their use for structure annotation of MS/MS ions.

Characterizing the D-Amino Acid Position in Peptide Epimers by Using Higher-Energy Collisional Dissociation Tandem Mass Spectrometry: A Case Study of Liraglutide.

D-amino acid-containing peptides (DAACPs) occur in biological and artificial environments. Since the importance of DAACPs has been recognized, various mass spectrometry-based analytical approaches have been developed. However, the capability of higher-energy collisional dissociation (HCD) fragmentation to characterize DAACP sites has not been evaluated. In this study, we compared the normalized spectra intensity under different conditions of HCD and used liraglutide along with its DAACPs as examples. Our results indicated that the difference in the intensity of y ions between DAACPs and all-L liraglutide could not only distinguish them but also localize the sites of D-amino acids in the DAACPs. Our data demonstrate the potential of using HCD for the site characterization of DAACPs, which may have great impact in biological studies and peptide drug development.

Direct and Detailed Site-Specific Glycopeptide Characterization by Higher-Energy Electron-Activated Dissociation Tandem Mass Spectrometry.

Glycosylation is widely recognized as the most complex post-translational modification due to the widespread presence of macro- and microheterogeneities, wherein its biological consequence is closely related to both the glycosylation sites and the glycan fine structures. Yet, efficient site-specific detailed glycan characterization remains a significant analytical challenge. Here, utilizing an Orbitrap-Omnitrap platform, higher-energy electron-activated dissociation (heExD) tandem mass spectrometry (MS/MS) revealed extraordinary efficacy for the structural characterization of intact glycopeptides. HeExD produced extensive fragmentation within both the glycan and the peptide, including A-/B-/C-/Y-/Z-/X-ions from the glycan motif and a-/b-/c-/x-/y-/z-type peptide fragments (with or without the glycan). The intensity of cross-ring cleavage and backbone fragments retaining the intact glycan was highly dependent on the electron energy. Among the four electron energy levels investigated, electronic excitation dissociation (EED) provided the most comprehensive structural information, yielding a complete series of glycosidic fragments for accurate glycan topology determination, a wealth of cross-ring fragments for linkage definition, and the most extensive peptide backbone fragments for accurate peptide sequencing and glycosylation site localization. The glycan fragments observed in the EED spectrum correlated well with the fragmentation patterns observed in EED MS/MS of the released glycans. The advantages of EED over higher-energy collisional dissociation (HCD), stepped collision energy HCD (sceHCD), and electron-transfer/higher-energy collisional dissociation (EThcD) were demonstrated for the characterization of a glycopeptide bearing a biantennary disialylated glycan. EED can produce a complete peptide backbone and glycan sequence coverage even for doubly protonated precursors. The exceptional performance of heExD MS/MS, particularly EED MS/MS, in site-specific detailed glycan characterization on an Orbitrap-Omnitrap hybrid instrument presents a novel option for in-depth glycosylation analysis.

Detection and analysis of triacylglycerol regioisomers via electron activated dissociation (EAD) tandem mass spectrometry

Triacylglycerols (TGs) are important components of human diet. The positional distribution of fatty acids (FAs) on the glycerol backbone affects the chemistry and physical properties of fats. Especially for infants, the structure of TGs plays an important role in the growth and development. However, limited by detecting technology, accurately identifying regioisomers of ABA/AAB and BAC/ABC/ACB type TGs is a significant challenge for human milk utilization and the development of infant formula. For this, we exploit a novel method for identifying the regioisomers of ABA/AAB and BAC/ABC/ACB type TGs within complex lipid mixtures, via used electron activated dissociation (EAD) tandem mass spectrometry. The distribution information of acyl chains at the sn-2 and sn-1/3 positions of glycerol backbone and double bonds in unsaturated FAs can be easily obtained by fragmenting TG ions with energetic electrons (15 eV). Then, the standard curve was established by correlating the peak area intensity of sn-2 characteristic product ion with the content of TG regioisomers standard. These analytical methods successfully enabled the identification and quantification of TG regioisomers in human milk, cow milk, infant formula, palm oil, and sunflower oil. Additionally, the distribution of the double-bond positions of unsaturated FAs in these samples was also identified. Compared to traditional methods, this approach eliminates the need for complex processing and analysis procedures, enabling rapid structural characterization of ABA/AAB and BAC/ABC/ACB type TGs within 17 min. Hence, we provide a rapid and convenient methodology for detecting and analyzing ABA/AAB and BAC/ABC/ACB type TG regioisomers, thereby offering valuable assistance in the development of specialized formulations and facilitating effective process control for ensuring the quality of edible oils and fats.

Isomeric Separation of α2,3/α2,6-Linked 2-Aminobenzamide (2AB)-Labeled Sialoglycopeptides by C18-LC-MS/MS.

Determination of the relative expression levels of the α2,3/α2,6-sialic acid linkage isomers on glycoproteins is critical to the analysis of various human diseases such as cancer, inflammation, and viral infection. However, it remains a challenge to separate and differentiate site-specific linkage isomers at the glycopeptide level. Some derivatization methods on the carboxyl group of sialic acid have been developed to generate mass differences between linkage isomers. In this study, we utilized chemical derivatization that occurred on the vicinal diol of sialic acid to separate linkage isomers on a reverse-phase column using a relatively short time. 2-Aminobenzamide (2AB) labeling derivatization, including periodate oxidation and reductive amination, took only ∼3 h and achieved high labeling efficiency (>90%). Within a 66 min gradient, the sialic acid linkage isomers of 2AB-labeled glycopeptides from model glycoproteins can be efficiently resolved compared to native glycopeptides. Two different methods, neuraminidase digestion and higher-energy collision dissociation tandem mass spectrometry (HCD-MS2) fragmentation, were utilized to differentiate those isomeric peaks. By calculating the diagnostic oxonium ion ratio of Gal2ABNeuAc and 2ABNeuAc fragments, significant differences in chromatographic retention times and in mass spectral peak abundances were observed between linkage isomers. Their corresponding MS2 PCA plots also helped to elucidate the linkage information. This method was successfully applied to human blood serum. A total of 514 2AB-labeled glycopeptide structures, including 152 sets of isomers, were identified, proving the applicability of this method in linkage-specific structural characterization and relative quantification of sialic acid isomers.

Determination and gas-phase stability evaluation of metal complexes by nanoelectrospray ionization and collision-induced dissociation tandem mass spectrometry.

The structures of metal complexes determine their stable functioning in product performance. Electrospray ionization mass spectrometry (ESI-MS) is used in studying metal complexes despite exhibiting limitations in analyzing labile complexes. Therefore, identifying a method for detecting unstable complexes and evaluating their stabilities is necessary, providing a theoretical basis for material selection and performance evaluation. The standard complexes Zn(BTZ)2 , Fe(acac)3 , and Sn(Oct)2 were analyzed using nanoESI quadrupole orbitrap MS (nanoESI-MS) and compared with ESI-MS for two temperature modes. The three complexes and alkylamine-Ag+ complexes were analyzed using nanoESI and collision-induced dissociation MS/MS (CID-MS/MS). Breakdown plots of the survival yield against collision energies expressed in terms of the center-of-mass were constructed according to the obtained product ion spectra. Quantum chemical calculations based on density functional theory were performed to calculate the binding energies between the alkylamines and Ag+ . The three standard complexes were detected in the native structures using nanoESI-MS, confirming the advantage of nanoESI over ESI for detecting unstable complexes. The gas-phase stabilities of the amine-Ag+ complexes, estimated using the breakdown plots constructed by plotting the data obtained via nanoESI and CID-MS/MS, were consistent with the established theories, previous studies, and binding energies calculated using computational methods. NanoESI-MS is suitable for detecting labile complexes and enables the structural analyses of unknown complex additives. A novel approach based on nanoESI and CID-MS/MS was developed to determine the gas-phase stabilities of complexes, enabling their quantification and comparison and providing a technical basis for product improvement, which is essential in developing industrial materials.

Sequencing of Morpholino Antisense Oligonucleotides Using Electron Capture Dissociation Mass Spectrometry.

We report the first sequencing of morpholino antisense oligonucleotides (phosphorodiamidate morpholino oligomers, PMOs) using electron capture dissociation (ECD) mass spectrometry. In this research, we found dissociation of the backbone of 18- to 25-mer PMOs to produce d and z ions as the major ions, and 100% cleavage coverage (sequence coverage) was obtained with these ions. This is a critical contrast with beam-type collision-induced dissociation, which dominantly induces base loss, so it is difficult to obtain sequence information. The results showed that an electron beam energy (typically 15 eV) can be used universally for PMOs with different sequences, lengths, and charge states so that no detailed optimization is required for multiprecursor targeting liquid chromatography coupled with tandem mass spectrometry measurements. We also confirmed that the ECD reaction speed was compatible with the high-performance liquid chromatography time scale. Finally, we demonstrated a liquid chromatography electron capture dissociation tandem mass spectrometry workflow to survey the modification sites of the emulated PMO impurities.

Pseudomonassin, a New Bioactive Ribosomally Synthesised and Post-Translationally Modified Peptide from Pseudomonas sp. SST3.

Genome mining and metabolomics have become valuable tools in natural products research to evaluate and identify potential new chemistry from bacteria. In the search for new compounds from the deep-sea organism, Pseudomonas sp. SST3, from the South Shetland Trough, Antarctica, a co-cultivation with a second deep-sea Pseudomonas zhaodongensis SST2, was undertaken to isolate pseudomonassin, a ribosomally synthesised and post-translationally modified peptide (RiPP) that belongs to a class of RiPP called lasso peptides. Pseudomonassin was identified using a genome-mining approach and isolated by means of mass spectrometric guided isolation. Extensive metabolomics analysis of the co-cultivation of Pseudomonas sp. SST3 and P. zhaodongensis SST2, Pseudomonas sp. SST3 and Escherichia coli, and P. zhaodongensis SST2 and E. coli were performed using principal component analysis (PCA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA), which revealed potential new metabolites in the outlier regions of the co-cultivation, with other metabolites identified previously from other species of Pseudomonas. The sequence of pseudomonassin was completely deduced using high collision dissociation tandem mass spectrometry (HCD-MS/MS). Preliminary studies on its activity against the pathogenic P. aeruginosa and its biofilm formation have been assessed and produced a minimum inhibitory concentration (MIC) of 63 μg/mL and 28 μg/mL, respectively.

OH radical-induced oxidation in nucleosides and nucleotides unraveled by tandem mass spectrometry and IRMPD spectroscopy.

OH●-induced oxidation products of DNA nucleosides and nucleotides have been structurally characterized by collision-induced dissociation tandem mass spectrometry (CID-MS2) and Infrared Multiple Photon Dissociation (IRMPD) spectroscopy. CID-MS2 results have shown that the addition of one oxygen atom occurs on the nucleobase moiety. The gas-phase geometries of +16 mass increment products of 2'-deoxyadenosine (dA(O)H+), 2'-deoxyadenosine 5'-monophosphate (dAMP(O)H+), 2'-deoxycytidine (dC(O)H+), and 2'-deoxycytidine 5'-monophosphate (dCMP(O)H+) are extensively investigated by IRMPD spectroscopy and quantum-chemical calculations. We show that a carbonyl group is formed at the C8 position after oxidation of 2'-deoxyadenosine and its monophosphate derivative. For 2'-deoxycytidine and its monophosphate derivative, the oxygen atom is added to the C5 position to form a C-OH group. IRMPD spectroscopy has been employed for the first time to provide direct structural information on oxidative lesions in DNA model systems.

In-Depth Characterization of Sphingoid Bases via Radical-Directed Dissociation Tandem Mass Spectrometry.

Sphingoid base (SPH) is a basic structural unit of all classes of sphingolipids. A sphingoid base typically consists of an aliphatic chain that may be desaturated between C4 and C5, an amine group at C2, and a variable number of OH groups located at C1, C3, and C4. Variations in the chain length and the occurrence of chemical modifications, such as methyl branching, desaturation, and hydroxylation, lead to a large structural diversity and distinct functional properties of sphingoid bases. However, conventional tandem mass spectrometry (MS/MS) via collision-induced dissociation (CID) faces challenges in characterizing these modifications. Herein, we developed an MS/MS method based on CID-triggered radical-directed dissociation (RDD) for in-depth characterization of sphingoid bases. The method involves derivatizing the sphingoid amine with 3-(2,2,6,6-tetramethylpiperidin-1-yloxymethyl)-picolinic acid 2,5-dioxopyrrolidin-1-yl ester (TPN), followed by MS2 CID to unleash the pyridine methyl radical moiety for subsequent RDD. This MS/MS method was integrated on a reversed-phase liquid chromatography-mass spectrometry workflow and further applied for in-depth profiling of total sphingoid bases in bovine heart and Caenorhabditis elegans. Notably, we identified and relatively quantified a series of unusual sphingoid bases, including SPH id17:2 (4,13) and SPH it19:0 in C. elegans, revealing that the metabolic pathways of sphingolipids are more diverse than previously known.

Application of liquid chromatography quadrupole time-of-flight mass spectrometry for the elucidation of fenamiphos photodegradation by-products

The degradation of the insecticide fenamiphos was undertaken by using a homogeneous photocatalysis employing iron (III) aqua complex, [Fe(H2O)5(OH)]2+. The irradiation was studied by excitation at 365 nm and the generated primary by-products were elucidated by Liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry in the positive ion mode, that leads to the formation of the protonated forms of the studied compounds, [M + H+]. Collision-induced dissociation tandem mass spectrometry (CID-MS/MS) of the protonated molecules was carried out, and the effect of the collision energy as well as the elemental compositions of the product ions were used in order to propose the chemical structures. The primary processes were then i) hydroxylation on the aromatic ring; ii) the loss of propene from the aminoisopropyl chain and iii) the loss of the methylene at the ethoxy moiety.

Identification, quantification, and structural role of N-glycans in two highly purified isoforms of sheep testicular hyaluronidase

Animal-derived hyaluronidase, which hydrolyzes the polysaccharide hyaluronic acid, has been used in medical applications despite its limited purity. Additionally, the N-glycan characterization of sheep testicular hyaluronidase (STH) and its structural role remain poorly understood. In this study, STH was purified from the commercially available STH preparation (containing at least 14 impurity proteins) using heparin-affinity chromatography followed by size exclusion chromatography. The structure and quantity of N-glycans of STH were investigated using liquid chromatography-electrospray ionization-high energy collision dissociation-tandem mass spectrometry. Two isoforms, H3S1 and H3S2, of STH were obtained (purity >98 %) with a yield of 3.4 % and 5.1 %, respectively. Fourteen N-glycans, including nine core-fucosylated N-glycans (important for the stability and function of glycoproteins), were identified in both H3S1 and H3S2, with similar quantities of each N-glycan. The amino acid sequences of the proteolytic peptides of H3S1 and H3S2 were compared with those reported in STH. The hyaluronic acid-degrading activity of deglycosylated H3S1 and H3S2 was reduced to 70.8 % and 71.1 % compared to that (100 %) of H3S1 and H3S2, respectively. This is the first report of N-glycan characterization of two highly purified isoforms of STH. These H3S1 and H3S2 will be useful for medical use without unwanted effects of partially purified STH.