Linkage Position Research Articles

Efficient and fully non-halogen solution processed Cs2AgBiBr6 perovskite solar cell

For traditional hole transport material (HTM) 2,2′,7,7′-tetrakis(N,N′-di-pmethoxyphenylamino)9,9′-spirbiuorene(Spiro-OMeTAD) used in Cs2AgBiBr6 perovskite solar cell (PSC), the strong dependence of its performance on the hygroscopic and volatility additives, together with the mismatched energy level with Cs2AgBiBr6 and gold electrodes, are considered to be the two key factors restricting the improvement of performance. Considering this, we reported a series of dopant-free D-π-D type HTMs comprising of FNP as donor unit and Cz-Mp dimer linked benzene as π-bridge, termed o-PCz, m-PCz, and p-PCz by adjusting the linkage position of π-bridge moieties. HTM p-PCz with planar rigid structure, exhibits higher hole mobility and better film morphology compared with o-PCz and m-PCz. Notably, o-PCz, m-PCz, and p-PCz all show good dissolvability in non-halogenated solvent tetrahydrofuran (THF), making it possible to fabricate Cs2AgBiBr6 PSCs with full non-halogenated solution process. As a result, the dopant-free p-PCz based Cs2AgBiBr6 PSC device achieved an efficiency of 2.44 % with enhanced ambient and thermal stability. Hence, the successful development of dopant-free p-PCz would make a great contribution for efficient and stable lead-free Cs2AgBiBr6 PSCs with full non-halogenated solution process.

Radical-directed dissociation mass spectrometry for differentiation and relative quantitation of isomeric ether-linked phosphatidylcholines

BackgroundEther-linked phosphatidylcholines (PCs) include both plasmanyl and plasmenyl PCs, which contain an ether or a vinyl ether bond at the sn-1 linkage position, respectively. Profiling and quantifying ether PCs with accurate structural information is challenging because of the common presence of isomeric and isobaric species in a lipidome. ResultsIn the present study, radical directed dissociation (RDD) from collision-induced dissociation (CID) of the bicarbonate anion adduct of ether PCs has been investigated to differentiate and relatively quantify ether PCs. Alkyl- and alkenyl- PCs give diagnostic characteristic fragment patterns that enable their confident identification and isomer differentiation. Additionally, the sn-position specific product ions have proven effective for relative quantitation among isomers in ether PCs and their isobaric PC species. Using this methodology, we successfully identified a total of 30 PC-O species, 21 PC-P species at the chain composition level, and 22 species of isobaric PC at the sn-position level in the human plasma lipid extract. The quantitative analysis revealed that ether PCs with a 20:4 fatty acyl chain are relatively more abundant in human plasma. Finally, the profile of ether PCs in type 2 diabetic (T2D) groups compared to normal control groups revealed a significant decrease in PC-O 18:1/20:5. We also found it is the PC species containing a 17-carbon fatty acyl chain, rather than their isobaric ether PCs, that shows a decreasing trend in the T2D groups. Significanceether-linked PCs are firstly investigated by RDD mass spectrometry.

Development of a cyclic ion mobility spectrometry-mass spectrometry-based collision cross-section database of permethylated human milk oligosaccharides.

Human milk oligosaccharides (HMOs) are an important class of biomolecules responsible for the healthy development of the brain-gut axis of infants. Unfortunately, their accurate characterization is largely precluded due to a variety of reasons - there are over 200 possible HMO structures whereas only 10s of these are available as authentic analytical standards. Furthermore, their isomeric heterogeneity stemming from their many possible glycosidic linkage positions and corresponding α/β anomericities further complicates their analyses. While liquid chromatography coupled to tandem mass spectrometry remains the gold standard for HMO analyses, it often times cannot resolve all possible isomeric species and thus warrants the development of other orthogonal approaches. High-resolution ion mobility spectrometry coupled to mass spectrometry has emerged as a rapid alternative to condensed-phase separations but largely has remained limited to qualitative information related to the resolution of isomers. In this work, we have assessed the use of permethylation to improve both the resolution and sensitivity of HMO analyses with cyclic ion mobility separations coupled with mass spectrometry. In addition to this, we have developed the first-ever high-resolution collision cross-section database for permethylated HMOs using our previously established calibration protocol. We envision that this internal reference database generated from high-resolution cyclic ion mobility spectrometry-mass spectrometry will greatly aid in the accurate characterization of HMOs and provide a valuable, orthogonal, approach to existing liquid chromatography-tandem mass spectrometry-based methods.

Robust Covalent Organic Framework Photocatalysts for H2O2 Production: Linkage Position Matters.

Covalent organic frameworks (COFs) are promising photocatalysts for hydrogen peroxide (H2O2) synthesis. However, the nature of organic polymers makes the balance between high activity and stability challenging. We demonstrate that the linkage position matters in the design of robust COF photocatalysts with durable high activity without sacrificial reagents. COFs with ortho- and para-linkages (o-COFs and p-COFs) were constructed by 1,3,5-triformylphloroglucinol with benzene-, pyridine-, pyrazine-orthodiamines and paradiamines. The pyrzaine-containing o-COFs with two pyridinic nitrogen atoms exhibited a H2O2 production rate of 4396 μmol g-1 h-1 together with long-time continuous H2O2 photosynthesis performance in pure water (48 h), superior to the corresponding p-COFs. A four-step reaction mechanism is proposed by density function calculations. Moreover, the active sites and origin of stability enhancement for o-COFs are clarified. This work provides a simple and effective molecular design strategy in the design of robust COF photocatalysts for artificial H2O2 photosynthesis.

Intriguing Role of Weak Electron Donor in Dictating the Luminescent Characteristics of DS‐A‐DW Materials

AbstractThe short wavelength (SW) emission region of the materials employing strong donor (DS) phenothiazine (PTZ) is generally correlated to its quasi‐axial (QAPTZ) conformer arrangement. This propels to investigate the specific role of weak donors (DW) such as carbazole (CZ) in determining the emission characteristics of molecular heredity (MH) type structural design encompassing dual donors of varying strength. For this purpose, a common DS(PTZ)–Acceptor unit is linked to the DW(CZ) unit via different linkage positions to form M1, M2, and M3, which not only alters the absorption and dual emission characteristics but also influences the delayed fluorescence phenomenon. The SW emission originates by minimizing the aggregation factor rather than by literature envisaged QAPTZ conformer. Incorporating M1 as a single molecular white light emitter (SMWLE) in near UV light emitting diode (LED) devices results in cold white light emission with CRI of 68 and CIE of (0.24, 0.33), while its corresponding solution‐processed organic light‐emitting diode devices delivers warm white electroluminescence with an EQE of 5.8%. Overall, this work demonstrates the significant contribution of the DW in attaining the white light emission characteristics of MH molecular design not only at the molecular level but also in practical light‐emitting devices.

Robust Covalent Organic Framework Photocatalysts for H2O2 Production: Linkage Position Matters

AbstractCovalent organic frameworks (COFs) are promising photocatalysts for hydrogen peroxide (H2O2) synthesis. However, the nature of organic polymers makes the balance between high activity and stability challenging. We demonstrate that the linkage position matters in the design of robust COF photocatalysts with durable high activity without sacrificial reagents. COFs with ortho‐ and para‐linkages (o‐COFs and p‐COFs) were constructed by 1,3,5‐triformylphloroglucinol with benzene‐, pyridine‐, pyrazine‐orthodiamines and paradiamines. The pyrzaine‐containing o‐COFs with two pyridinic nitrogen atoms exhibited a H2O2 production rate of 4396 μmol g−1 h−1 together with long‐time continuous H2O2 photosynthesis performance in pure water (48 h), superior to the corresponding p‐COFs. A four‐step reaction mechanism is proposed by density function calculations. Moreover, the active sites and origin of stability enhancement for o‐COFs are clarified. This work provides a simple and effective molecular design strategy in the design of robust COF photocatalysts for artificial H2O2 photosynthesis.

Ulvans are not equal - Linkage and substitution patterns in ulvan polysaccharides differ with Ulva morphology

Ulva are hardy green seaweeds that contain the sulfated polysaccharide ulvan and grow in two distinct morphologies: foliose and tubular. The authors hypothesise that ulvan from tubular species are more structurally complex than ulvans from foliose species. Herein, using standardised methods, the glycosyl linkage positions and sulfate ester substitutions of constituent monosaccharides of ulvan isolated from foliose (U. lacinulata and U. stenophylloides) and tubular (U. prolifera and U. ralfsii) species of Ulva were investigated. Comparison of native ulvans with 80 and 100 °C desulfated counterparts indicated that 4-linked rhamnose is predominantly 3-O-sulfated in all four ulvans. Ulvans from the foliose species predominantly contained →3,4)-Rhap-(1→, →4)-GlcAp-(1→ and →4)-IdoAp-(1→, collectively accounting for 67 to 81 mol% of the total linkages. In contrast, these same linkages in ulvans from the tubular species only collectively accounted for 29 to 36 mol%. Instead, ulvan from tubular species contained a combination of →2,3,4)-Rhap-(1→, terminal Rhap-(1→, →4)-GlcAp-(1→, →4)-Xylp-(1→, and/or →4)-Galp-(1→ in high proportions; some of the latter three residues were also likely O-2 sulfated. The results presented here suggest that ulvan from foliose species are predominantly unbranched polysaccharides composed of repeat disaccharides while ulvans from tubular species contain a greater diversity of branch and sulfate substitution locations.

Optimization of culture conditions to express AA9 Polysaccharide monooxygenases AN3860 in Escherichia coli

Lignocellulose biomass is a copious source for second generation biomaterial production. The participant of Polysaccharide monooxygenases enzyme (PMO) in the reactions which convert lignocellulose biomass into monosaccharides enhances the activity and improve the efficiency of hydrolysis of hydrolase enzymes on lignocellulose substrate. Enzyme AN3860, obtained from Aspergillus nidulans strain belonging to AA9 PMO, is expected to catalyze flexibly at C1 and C4 carbon positions of β-glycosidic linkage. As an enzyme with high potential of improving cellulose crystals hydrolysis capacity, AN3860 was successfully cloned into the expression system of E. coli BL21 (DE3) strain. In this study, the culture process of recombinant strain with AN3680 gene is optimized to increase the target proteins yield, thus ensure the outcome of purification process, and save production cost. The results demonstrate that the E. coli recombinant strains grow sufficiently in TB (Terrific Broth) culture media and the highest yield of AN3680 protein achieved when the concentration of Isopropyl β-D-1-thiogalactopyranoside (IPTG) is 0.05 mM and the temperature of the reaction is 30 0C at 150 rpm. After 6 hours of induction, the biomass reaches 500 mg/L and the yield of AN3860 account for (7-10) % total protein generated. The recombinant AN3860 protein is later harvested on larger scale and purified by Ni-NTA column chromatography method for analysis of bioactivities on lignocellulose substrates in the future. ® 2022 Journal of Science and Technology - NTTU

Structural determination of fructooligosaccharides and raffinose family oligosaccharides using logically derived sequence tandem mass spectrometry.

Fructooligosaccharides (FOS) and raffinose family oligosaccharides (RFOs) are two highly abundant water-soluble carbohydrates in plants. The typical procedures for the FOS and RFO structural determination using mass spectrometry involve permethylation, followed by the hydrolysis of the permethylated oligosaccharides into monosaccharides, and then the identification of linkage positions using GC mass spectrometry. However, the determination of linkage position sequence is not straightforward, thus this method is limited to small oligosaccharides or oligosaccharides with simple linkages. In this study, we employed a new mass spectrometry method, logically derived sequence tandem mass spectrometry, to determine the structures of FOS and RFOs. We first showed that the monosaccharide and disaccharide CID spectra of aldohexose and ketohexose can be rationalized using dissociation mechanisms. Then we demonstrated that the linkage positions of FOS and RFOs can be identified, the sequence of the linkages can be determined, and the ketohexose and aldohexose in FOS and RFOs can be differentiated, suggesting this new method is useful for structural determination of FOS and RFOs.

In-Source Decay MALDI and High-Energy Collision-Induced Dissociation Mass Spectrometry of Alkali Metal-Adducted Underivatized Oligosaccharides.

In-source decay (ISD) and high-energy collision-induced dissociation (HE-CID) were explored to provide structural information on alkali metal-adducted linear and stacked oligosaccharides (oligosaccharides with increased flexibility due to linkage type). These oligosaccharides include isomeric tetrasaccharides, maltoheptaose, and several human milk oligosaccharides (HMOs). Matrix-assisted laser desorption ionization (MALDI) ion production efficiency, as well as the product ion intensities, and the number of product ions formed in ISD and HE-CID of these oligosaccharides were influenced by the matrix, the ionic radius of the metal ion used for adduction, and the affinity of metal ions for specific functional groups in the oligosaccharides. 2,4,6-Trihydroxyacetophenone (THAP) was the best matrix for HE-CID of oligosaccharides, 4-dimethylaminobenzaldehyde (DMABA) worked best for ISD of tetrasaccharides and pentasaccharides, while 2,5-dihydroxybenzoic acid (DHB) was the best matrix for ISD and HE-CID of long chain oligosaccharides. In general, the number of product ions formed followed the trend Li+ > Na+ > K+ > Rb+ > Cs+, except for HMOs where Na+ ≥ Li+ > K+ > Rb+ > Cs+ occurred. The type of product ions formed and their intensities varied based on the position of the glycosidic bond linkage and the content of the monosaccharide. ISD and HE-CID produced diagnostic ions that could structurally differentiate isomers. Overall, HE-CID of alkali-metal adducted oligosaccharides produces intense glycosidic bond cleavages and low intensity cross-ring and internal cleavages. In contrast, ISD generates mainly cross-ring cleavages and internal cleavages at intensities higher than in HE-CID. In addition, ISD produced unique product ions that complement results from HE-CID.

Tuning the Photophysical Properties of Acceptor-Donor-Acceptor Di-2-(2-oxindolin-3-ylidene) Malononitrile Materials via Extended π-Conjugation: A Joint Experimental and Theoretical Study.

Many optoelectronic applications require organic semiconductor (OSC) materials with high electron affinity. In this work, a series of novel acceptor-donor-acceptor (A-D-A) materials with low-lying LUMO energy levels were designed and characterized. In this strategy, two acceptor dyes, bis-isatin and di-2-(2-oxindolin-3-ylidene) malononitrile, were connected by various π-bridges (benzene ring, benzo[c][1,2,5]thiadiazole, monothiophene, trithiophene). We varied the length of the π-conjugation of the central core and the linkage position of the acceptor core (4- vs. 6-position of the phenyl ring) to investigate the effect on the optical and electrochemical properties of the materials. We performed density functional theory (DFT) and time-dependent DFT (TD-DFT) studies to gain insight into the dyes' electronic properties by determining the energy levels. Our findings demonstrate that with increasing acceptor strength and π-conjugation length of the core, the wavelength of the longest absorption maximum as well as their respective extinction coefficients are enhanced, which results in band-gap reduction either by lowering the LUMO and/or raising the HOMO energy level of the molecules. The potential practical utility of these materials as electron-transport materials for perovskite solar cells (PSCs) has been demonstrated.

Thermal annealing promoted room temperature phosphorescence: motion models and internal mechanism.

Thermal annealing has been proven to be an efficient method to optimize the device performance of organic and polymeric opto-electronic materials. However, no detailed information of aggregate structures was obtained for a deeper understanding of what happens during thermal annealing. Herein, through modulation of molecular configurations by tunable linkage positions, and the amplified amplitudes of molecular motions by incorporation of additional methylene units, accurate changes of aggregated structures upon thermal annealing have been achieved, accompanying with the 'turn-on' room temperature phosphorescence (RTP) response by about 4800- and 177-fold increase of lifetimes. The stretching and swing motion models have been proposed, which afforded an efficient way to investigate the science of dynamic aggregation in depth.

Characterization of Unbranched Ubiquitin Tetramers by Combining Ultraviolet Photodissociation with Proton Transfer Charge Reduction Reactions.

Polyubiquitination is an important post-translational modification (PTM) that regulates various biological functions. The linkage sites and topologies of polyubiquitination chains are important factors in determining the fate of polyubiquitinated proteins. Characterization of polyubiquitin chains is the first step in understanding the biological functions of protein ubiquitination, but it is challenging owing to the repeating nature of the ubiquitin chains and the difficulty in deciphering linkage positions. Here, we combine ultraviolet photodissociation (UVPD) mass spectrometry and gas-phase proton transfer charge reduction (PTCR) to facilitate the assignment of product ions generated from Lys6-, Lys11-, Lys29-, Lys33-, Lys48-, and Lys63-linked ubiquitin tetramers. UVPD results in extensive fragmentation of intact proteins in a manner that allows the localization of PTMs. However, UVPD mass spectra of large proteins (>30 kDa) are often congested due to the overlapping isotopic distribution of highly charged fragment ions. UVPD + PTCR improved the identification of PTM-containing fragment ions, allowing the localization of linkage sites in all six tetramers analyzed. UVPD + PTCR also increased the sequence coverage obtained from the PTM-containing fragment ions in each of the four chains of each tetramer by 7 to 44% when compared to UVPD alone.

Permethylation and Metal Adduction: A Toolbox for the Improved Characterization of Glycolipids with Cyclic Ion Mobility Separations Coupled to Mass Spectrometry.

Lipids are an important class of molecules involved in various biological functions but remain difficult to characterize through mass-spectrometry-based methods because of their many possible isomers. Glycolipids, specifically, play important roles in cell signaling but display an even greater level of isomeric heterogeneity as compared to other lipid classes stemming from the introduction of a carbohydrate and its corresponding linkage position and α/β anomericity at the headgroup. While liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) remains the gold standard technique in lipidomics, it is still unable to characterize all isomeric species, thus presenting the need for new, orthogonal, methodologies. Ion mobility spectrometry-mass spectrometry (IMS-MS) can provide an additional dimension of information that supplements LC-MS/MS workflows, but has seen little use for glycolipid analyses. Herein, we present an analytical toolbox that enables the characterization of various glycolipid isomer sets using high-resolution cyclic ion mobility separations coupled with mass spectrometry (cIMS-MS). Specifically, we utilized a combination of both permethylation and metal adduction to fully resolve isomeric sphingolipids and ceramides with our cIMS-MS platform. We also introduce a new metric that can enable comparing peak-to-peak resolution across varying cIMS-MS pathlengths. Overall, we envision that our presented methodologies are highly amenable to existing LC-MS/MS-based workflows and can also have broad utility toward other omics-based analyses.

Characterization of antisense oligonucleotide and guide ribonucleic acid diastereomers by hydrophilic interaction liquid chromatography coupled to mass spectrometry

Oligonucleotides have become an essential modality for a variety of therapeutic approaches, including cell and gene therapies. Rapid progress in the field has attracted significant research in designing novel oligonucleotide chemistries and structures. Beyond their polar nature, the length of large RNAs and presence of numerous diastereomers for phosphorothioate (PS)-modified RNAs pose heightened challenges for their characterization. In this study, the stereochemistry of a fully-modified antisense oligonucleotide (ASO) and partially-modified guide RNAs (gRNAs) was investigated using HILIC and orthogonal techniques. The profiles of three lots of a fully-modified ASO with PS linkages were compared using ion-pairing RPLC (IPRP) and HILIC. Interestingly, three isomer peaks were partially resolved by HILIC for two lots while only one peak was observed on the IPRP profile. Model oligonucleotides having the same sequence of the five nucleotides incorporated to the 3′-end of the gRNA but differing in their number and position of PS linkages were investigated by HILIC, IPRP, ion mobility spectrometry–mass spectrometry (IM-MS) and nuclear magnetic resonance (NMR). An strategy was ultimately designed to aid in the characterization of gRNA stereochemistry. Ribonuclease (RNase) T1 digestion enabled the characterization of gRNA diastereomers by reducing their number from 32 at the gRNA intact level to 4 or 8 at the fragment level. To our knowledge, this is the first time that HILIC has successfully been utilized for the profiling of diastereomers for various oligonucleotide formats and chemical modifications.

Rotor Speed and Position Estimation Analysis of Interior PMSM Machines in Low and Medium-High Speed Regions Adopting an Improved Flux Observer for Electric Vehicle Applications

This paper proposes a nonlinear flux linkage observer for the PMSM speed controls without motion sensors, introducing the deviation among the real stator flux linkage and an estimated stator flux linkage to suppress feedback and integral flux drift. In the position detection of an interior PMSM without a speed sensor, the traditional back EMF integration method uses a pure integrator, or LPF, to estimate the stator flux. Its inherent defects inevitably lead to inaccurate flux estimation, which directly affects the estimation of the motor mover position, resulting in the decline in motor control operation and the distortion of phase current. This paper uses an improved integrator with adaptive compensation. The projected value of the stator flux linkage has been derived from the estimated value of the rotor permanent magnetic flux linkage position angle and the algebraic model (m-model) of the stator flux linkage, along with a synchronous coordinate system. The IPMSM stator coil flux linkage obtained from the stator coil current and integral voltage models in the static coordinate system is compared to form a feedback closed-loop to suppress the integral drift, and using the cross-product approach of the actual and estimated flux linkage yields the projected value of the IPMSM rotor speed and position through a PLL. Compared with the existing motion-sensorless observers, the methodology proposed in this article is simple and exhibits better dynamic and static estimation performance. Extensive and comprehensive MATLAB computer simulation and experimental findings validate the proposed motion-sensorless control mechanism.

Bionic Design of a Miniature Jumping Robot

In response to the problem of low energy storage density in the structure of existing miniature jumping robots, this study designed a parallel single-degree-of-freedom double six-link jumping robot by imitating the physiological structure and jumping mechanism of wax cicadas. The designed six-link mechanism was first mathematically modeled, and to accommodate the jumping structure of this robot, a six-link mechanism with a smaller cam pushrod stroke was obtained by optimizing the linkage size and position parameters in the model. The dynamics of the robot’s jumping process were then analyzed utilizing the second type of Lagrange equation to determine the joint angles of the robot’s jumping phase. The results were compared with an ADAMS-based jumping simulation to verify the validity of the analysis of the dynamics. The feasibility of the structural design was then validated using ADAMS simulations. Finally, a physical prototype of the jumping robot was produced and tested; the findings revealed that the robot had good jumping performance, was stable in the air, fully discharged 600.2 mJ of energy, and was able to overcome obstacles measuring 220 mm in height and 330 mm in distance. The design of the jumping robot provides a novel approach to improving energy storage density and serves as a foundation for future research on footed jumping robots.

Dibenzo [a, g] corannulene-phenoxazine dyads: Facile synthesis and optoelectronic properties

Corannulene has a peculiar topology, while phenoxazine (PXZ) features extraordinary optoelectronic properties. In this study, two dibenzo [a, g] corannulene-phenoxazine dyads, BBC-C-PXZ and BBC-N-PXZ, connected via C–C and C–N bonds, respectively, were successfully prepared using PXZ as an electron donor and dibenzo [a, g] corannulene (BBC) as an electron acceptor. Photoelectrical properties of the compounds were studied using UV–vis absorption, luminescence spectroscopy, electrochemical tests and theoretical calculations. Both dyads showed Curve-Twist intramolecular charge transfer (C-TICT) in solution and aggregated state, which could be explained by the BBC-C-PXZ concave-convex crystal packing mode. They further exhibited moderate to good singlet oxygen quantum yields, the BBC-N-PXZ was 42.3% in toluene and BBC-C-PXZ was only 14.3% in n-hexane. The BBC-N-PXZ (connected by the C–N bond) exhibited contrasting reversible and anti-fatigue “turn-on” mechanochromic behavior, which displayed a blue shift under external stimulation, while the BBC-C-PXZ (linked by the C–C bond) did not exhibit this phenomenon. Analyses of the D-A (donor-acceptor) dyad systems revealed that the introduction of curved polycyclic aromatic hydrocarbons and diversified linkage positions result in unique optical and electrical properties of materials, and it provides new advances and breakthroughs in the design of organic electronic materials.

The collision-induced dissociation mechanism of sodiated Hex-HexNAc disaccharides.

Determining carbohydrate structures, such as their compositions, linkage positions, and in particular the anomers and stereoisomers, is a great challenge. Isomers of different anomers or stereoisomers have the same sequences of chemical bonds, but have different orientations of some chemical bonds which are difficult to be distinguished by mass spectrometry. Collision-induced dissociation (CID) tandem mass spectroscopy (MS/MS) is a widely used technique for characterizing carbohydrate structures. Understanding the carbohydrate dissociation mechanism is important for obtaining the structural information from MS/MS. In this work, we studied the CID mechanism of galactose-N-acetylgalactosamine (Gal-GalNAc) and glucose-N-acetylglucosamine (Glc-GlcNAc) disaccharides with 1→3 and 1→4 linkages. For Gal-GalNAc disaccharides, the CID mass spectra of sodium ion adducts show significant difference between the α- and β-anomers of GalNAc at the reducing end, while no difference in the CID mass spectra between two anomers of Glc-GlcNAc disaccharides was found. Quantum chemistry calculations show that for Gal-GalNAc disaccharides, the difference of the dissociation barriers between dehydration and glycosidic bond cleavage is significantly small in the β-anomer compared to that in the α-anomer; while these differences are similar between the α- and β-anomers of Glc-GlcNAc disaccharides. These differences can be attributed to the different orientations of hydroxyl and N-acetyl groups located at GalNAc and GlcNAc. The calculation results are consistent with the CID spectra of isotope labelled disaccharides. Our study provides an insight into the CID of 1→3 and 1→4 linked Gal-GalNAc and Glc-GlcNAc disaccharides. This information is useful for determining the anomeric configurations of GalNAc in oligosaccharides.

De novo glycan sequencing by electronic excitation dissociation MS2-guided MS3 analysis on an Omnitrap-Orbitrap hybrid instrument.

Comprehensive de novo glycan sequencing remains an elusive goal due to the structural diversity and complexity of glycans. Present strategies employing collision-induced dissociation (CID) and higher energy collisional dissociation (HCD)-based multi-stage tandem mass spectrometry (MSn) or MS/MS combined with sequential exoglycosidase digestions are inherently low-throughput and difficult to automate. Compared to CID and HCD, electron transfer dissociation (ETD) and electron capture dissociation (ECD) each generate more cross-ring cleavages informative about linkage positions, but electronic excitation dissociation (EED) exceeds the information content of all other methods and is also applicable to analysis of singly charged precursors. Although EED can provide extensive glycan structural information in a single stage of MS/MS, its performance has largely been limited to FTICR MS, and thus it has not been widely adopted by the glycoscience research community. Here, the effective performance of EED MS/MS was demonstrated on a hybrid Orbitrap-Omnitrap QE-HF instrument, with high sensitivity, fragmentation efficiency, and analysis speed. In addition, a novel EED MS2-guided MS3 approach was developed for detailed glycan structural analysis. Automated topology reconstruction from MS2 and MS3 spectra could be achieved with a modified GlycoDeNovo software. We showed that the topology and linkage configurations of the Man9GlcNAc2 glycan can be accurately determined from first principles based on one EED MS2 and two CID-EED MS3 analyses, without reliance on biological knowledge, a structure database or a spectral library. The presented approach holds great promise for autonomous, comprehensive and de novo glycan sequencing.