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Substantial efforts are underway to deepen our understanding of human brain morphology, structure, and function using high‐resolution imaging as well as high‐content molecular profiling technologies. The current work adds to these approaches by providing a comprehensive and quantitative protein expression map of 13 anatomically distinct brain regions covering more than 11,000 proteins. This was enabled by the optimization, characterization, and implementation of a high‐sensitivity and high‐throughput microflow liquid chromatography timsTOF tandem mass spectrometry system (LC–MS/MS) capable of analyzing more than 2,000 consecutive samples prepared from formalin‐fixed paraffin embedded (FFPE) material. Analysis of this proteomic resource highlighted brain region‐enriched protein expression patterns and functional protein classes, protein localization differences between brain regions and individual markers for specific areas. To facilitate access to and ease further mining of the data by the scientific community, all data can be explored online in a purpose‐built R Shiny app (https://brain‐region‐atlas.proteomics.ls.tum.de). Synopsis: This resource provides a comprehensive and quantitative protein expression atlas of thirteen anatomically distinct brain regions covering more than 11,000 proteins. This was enabled by the optimization and implementation of an advanced high‐sensitivity and high‐throughput micro‐flow liquid chromatography timsTOF tandem mass spectrometry system (LC‐MS/MS). Optimization of a fast, sensitive and robust micro‐flow liquid chromatography timsTOF tandem mass spectrometry protocol.Validation of high‐throughput applicability of the timsTOF pipeline by analyzing > 2,000 consecutive formalin‐fixed brain tissue samples.Generation of the most comprehensive map of a human brain proteome to date covering more than 11,000 proteins.The 13 brain regions show distinct proteomic signatures including new marker proteins. [ABSTRACT FROM AUTHOR]

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Fucose is a signaling carbohydrate that is attached at the end of glycan processing. It is involved in a range of processes, such as the selectin‐dependent leukocyte adhesion or pathogen‐receptor interactions. Mass‐spectrometric techniques, which are commonly used to determine the structure of glycans, frequently show fucose‐containing chimeric fragments that obfuscate the analysis. The rearrangement leading to these fragments—often referred to as fucose migration—has been known for more than 25 years, but the chemical identity of the rearrangement product remains unclear. In this work, we combine ion‐mobility spectrometry, radical‐directed dissociation mass spectrometry, cryogenic IR spectroscopy of ions, and density‐functional theory calculations to deduce the product of the rearrangement in the model trisaccharides Lewis x and blood group H2. The structural search yields the fucose moiety attached to the galactose with an α(1→6) glycosidic bond as the most likely product. [ABSTRACT FROM AUTHOR]

Fluorination is a potent method to modulate chemical properties of glycans. Here, we study how C3‐ and C6‐fluorination of glucosyl building blocks influence the structure of the intermediate of the glycosylation reaction, the glycosyl cation. Using a combination of gas‐phase infrared spectroscopy and first‐principles theory, glycosyl cations generated from fluorinated and non‐fluorinated monosaccharides are structurally characterized. The results indicate that neighboring group participation of the C2‐benzoyl protecting group is the dominant structural motif for all building blocks, correlating with the β‐selectivity observed in glycosylation reactions. The infrared signatures indicate that participation of the benzoyl group in enhanced by resonance effects. Participation of remote acyl groups such as Fmoc or benzyl on the other hand is unfavored. The introduction of the less bulky fluorine leads to a change in the conformation of the ring pucker, whereas the structure of the active dioxolenium site remains unchanged. [ABSTRACT FROM AUTHOR]

A vast number of experiments have now shown that helium nanodroplets are an exemplary cryogenicmatrix for spectroscopic investigations. The experimental techniques are well established and involve in most cases the pickup of evaporated neutral species by helium droplets. These techniques have been extended within our research group to enable nanodroplet pickup of anions or cations stored in an ion trap. By using electrospray ionization (ESI) in combination with modern mass spectrometric methods to supply ions to the trap, an immense variety of mass-to-charge selected species can be doped into the droplets and spectroscopically investigated. We have combined this droplet doping methodology with IR action spectroscopy to investigate anions and cations ranging in size from a few atoms to proteins that consist of thousands of atoms. Herein, we show examples of small complexes of fluoride anions (F-) with CO2 and H2O and carbohydrate molecules. In the case of the small complexes, novel compounds could be identified, and quantum chemistry can in some instances quantitatively explain the results. For biologically relevant complex carbohydrate molecules, the IR spectra are highly diagnostic and allow the differentiation of species that would be difficult or impossible to identify by more conventional methods. [ABSTRACT FROM AUTHOR]

Glycolipids are complex glycoconjugates composed of a glycan headgroup and a lipid moiety. Their modular biosynthesis creates a vast amount of diverse and often isomeric structures, which fulfill highly specific biological functions. To date, no gold-standard analytical technique can provide a comprehensive structural elucidation of complex glycolipids, and insufficient tools for isomer distinction can lead to wrong assignments. Herein we use cryogenic gas-phase infrared spectroscopy to systematically investigate different kinds of isomerism in immunologically relevant glycolipids. We show that all structural features, including isomeric glycan headgroups, anomeric configurations and different lipid moieties, can be unambiguously resolved by diagnostic spectroscopic fingerprints in a narrow spectral range. The results allow for the characterization of isomeric glycolipid mixtures and biological applications. Glycolipids are glycoconjugates with important biological functions, but techniques for their analysis are deficient. Here, the authors report the use of cryogenic gas-phase infrared spectroscopy to investigate isomerism in a set of immunologically relevant glycolipids, and show that their structural features can be accurately resolved based on a narrow spectral fingerprint region. [ABSTRACT FROM AUTHOR]

Isolation of biomolecules in vacuum facilitates characterization of the intramolecular interactions that determine three-dimensional structure, but experimental quantification of conformer thermochemistry remains challenging. Infrared spectroscopy of molecules trapped in helium nanodroplets is a promising methodology for the measurement of thermochemical parameters. When molecules are captured in a helium nanodroplet, the rate of cooling to an equilibrium temperature of ca. 0.4 K is generally faster than the rate of isomerization, resulting in "shock-freezing" that kinetically traps molecules in local conformational minima. This unique property enables the study of temperature-dependent conformational equilibria via infrared spectroscopy at 0.4 K, thereby avoiding the deleterious effects of spectral broadening at higher temperatures. Herein, we demonstrate the first application of this approach to ionic species by coupling electrospray ionization mass spectrometry (ESI-MS) with helium nanodroplet infrared action spectroscopy to probe the structure and thermochemistry of deprotonated DNA dinucleotides. Dinucleotide anions were generated by ESI, confined in an ion trap at temperatures between 90 and 350 K, and entrained in traversing helium nanodroplets. The infrared action spectra of the entrained ions show a strong dependence on pre-pickup ion temperature, consistent with the preservation of conformer population upon cooling to 0.4 K. Non-negative matrix factorization was utilized to identify component conformer infrared spectra and determine temperature-dependent conformer populations. Relative enthalpies and entropies of conformers were subsequently obtained from a van't Hoff analysis. IR spectra and conformer thermochemistry are compared to results from ion mobility spectrometry (IMS) and electronic structure methods. The implementation of ESI-MS as a source of dopant molecules expands the diversity of molecules accessible for thermochemical measurements, enabling the study of larger, non-volatile species. [ABSTRACT FROM AUTHOR]

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1‐Deoxysphingolipids are a recently described class of sphingolipids that have been shown to be associated with several disease states including diabetic and hereditary neuropathy. The identification and characterization of 1‐deoxysphingolipids and their metabolites is therefore highly important. However, exact structure determination requires a combination of sophisticated analytical techniques due to the presence of various isomers, such as ketone/alkenol isomers, carbon–carbon double‐bond (C=C) isomers and hydroxylation regioisomers. Here we demonstrate that cryogenic gas‐phase infrared (IR) spectroscopy of ionized 1‐deoxysphingolipids enables the identification and differentiation of isomers by their unique spectroscopic fingerprints. In particular, C=C bond positions and stereochemical configurations can be distinguished by specific interactions between the charged amine and the double bond. The results demonstrate the power of gas‐phase IR spectroscopy to overcome the challenge of isomer resolution in conventional mass spectrometry and pave the way for deeper analysis of the lipidome. [ABSTRACT FROM AUTHOR]

Copyright of Angewandte Chemie is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The stereoselective formation of 1,2‐cis‐glycosidic bonds is challenging. However, 1,2‐cis‐selectivity can be induced by remote participation of C4 or C6 ester groups. Reactions involving remote participation are believed to proceed via a key ionic intermediate, the glycosyl cation. Although mechanistic pathways were postulated many years ago, the structure of the reaction intermediates remained elusive owing to their short‐lived nature. Herein, we unravel the structure of glycosyl cations involved in remote participation reactions via cryogenic vibrational spectroscopy and first principles theory. Acetyl groups at C4 ensure α‐selective galactosylations by forming a covalent bond to the anomeric carbon in dioxolenium‐type ions. Unexpectedly, also benzyl ether protecting groups can engage in remote participation and promote the stereoselective formation of 1,2‐cis‐glycosidic bonds. [ABSTRACT FROM AUTHOR]

Glycosaminoglycans (GAGs) are a physio- and pharmacologically highly relevant class of complex saccharides, possessing a linear sequence and strongly acidic character. Their repetitive linear core makes them seem structurally simple at first glance, yet differences in sulfation and epimerization lead to an enormous structural diversity with only a few GAGs having been successfully characterized to date. Recent infrared action spectroscopic experiments on sulfated mono- and disaccharide ions show great promise. Here, we assess the potential of two types of gas-phase action spectroscopy approaches in the range from 1000 to 1800 cm−1 for the structural analysis of complex GAG oligosaccharides. Synthetic tetra- and pentasaccharides were chosen as model compounds for this benchmark study. Utilizing infrared multiple photon dissociation action spectroscopy at room temperature, diagnostic bands are largely unresolved. In contrast, cryogenic infrared action spectroscopy of ions trapped in helium nanodroplets yields resolved infrared spectra with diagnostic features for monosaccharide composition and sulfation pattern. The analysis of GAGs could therefore significantly benefit from expanding the conventional MS-based toolkit with gas-phase cryogenic IR spectroscopy. [ABSTRACT FROM AUTHOR]

Fucose migration reactions represent a substantial challenge in the analysis of fucosylated glycan structures by mass spectrometry. In addition to the well-established observation of transposed fucose residues in glycan-dissociation product ions, recent experiments show that the rearrangement can also occur in intact glycan ions. These results suggest a low-energy barrier for migration of the fucose residue and broaden the relevance of fucose migration to include other types of mass spectrometry experiments, including ion mobility-mass spectrometry and ion spectroscopy. In this work, we utilize cold-ion infrared spectroscopy to provide further insight into glycan scrambling in intact glycan ions. Our results show that the mobility of the proton is a prerequisite for the migration reaction. For the prototypical fucosylated glycans Lewis x and blood group antigen H-2, the formation of adduct ions or the addition of functional groups with variable proton affinity yields significant differences in the infrared spectra. These changes correlate well with the promotion or inhibition of fucose migration through the presence or absence of a mobile proton. [ABSTRACT FROM AUTHOR]

Abstract: The proton‐bound dicarboxylate motif, RCOO−⋅H+⋅−OOCR, is a prevalent chemical configuration found in many condensed‐phase systems. The proton‐bound formate dimer HCOO−⋅H+⋅−OOCH was studied utilizing cold‐ion IR action spectroscopy in the range 400–1800 cm−1. The spectrum obtained at ca. 0.4 K of ions captured in He nanodroplets was compared to that measured at ca. 10 K by photodissociation of Ar‐ion complexes. Similar band patterns are obtained by the two techniques that are consistent with calculations for a C2 symmetry structure with a proton shared equally between the two formate moieties. Isotopic substitution experiments point to the nominal parallel stretch of the bridging proton appearing as a sharp, dominant feature near 600 cm−1. Multidimensional anharmonic calculations reveal that the bridging proton motion is strongly coupled to the flanking −COO− framework, an effect that is in line with the expected change in −C=O bond rehybridization upon protonation. [ABSTRACT FROM AUTHOR]

Abstract: The proton‐bound dicarboxylate motif, RCOO−⋅H+⋅−OOCR, is a prevalent chemical configuration found in many condensed‐phase systems. The proton‐bound formate dimer HCOO−⋅H+⋅−OOCH was studied utilizing cold‐ion IR action spectroscopy in the range 400–1800 cm−1. The spectrum obtained at ca. 0.4 K of ions captured in He nanodroplets was compared to that measured at ca. 10 K by photodissociation of Ar‐ion complexes. Similar band patterns are obtained by the two techniques that are consistent with calculations for a C2 symmetry structure with a proton shared equally between the two formate moieties. Isotopic substitution experiments point to the nominal parallel stretch of the bridging proton appearing as a sharp, dominant feature near 600 cm−1. Multidimensional anharmonic calculations reveal that the bridging proton motion is strongly coupled to the flanking −COO− framework, an effect that is in line with the expected change in −C=O bond rehybridization upon protonation. [ABSTRACT FROM AUTHOR]

Abstract: Fucose is an essential deoxysugar that is found in a wide range of biologically relevant glycans and glycoconjugates. A recurring problem in mass spectrometric analyses of fucosylated glycans is the intramolecular migration of fucose units, which can lead to erroneous sequence assignments. This migration reaction is typically assigned to activation during collision‐induced dissociation (CID) in tandem mass spectrometry (MS). In this work, we utilized cold‐ion spectroscopy and show for the first time that fucose migration is not limited to fragments obtained in tandem MS and can also be observed in intact glycan ions. This observation suggests a possible low‐energy barrier for this transfer reaction and generalizes fucose migration to an issue that may universally occur in any type of mass spectrometry experiment. [ABSTRACT FROM AUTHOR]

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The diversity of stereochemical isomers present in glycans and glycoconjugates poses a formidable challenge for comprehensive structural analysis. Typically, sophisticated mass spectrometry (MS)-based techniques are used in combination with chromatography or ion-mobility separation. However, coexisting structurally similar isomers often render an unambiguous identification impossible. Other powerful techniques such as gas-phase infrared (IR) spectroscopy have been limited to smaller glycans, since conformational flexibility and thermal activation during the measurement result in poor spectral resolution. This limitation can be overcome by using cold-ion spectroscopy. The vibrational fingerprints of cold oligosaccharide ions exhibit a wealth of well-resolved absorption features that are diagnostic for minute structural variations. The unprecedented resolution of cold-ion spectroscopy coupled with tandem MS may render this the key technology to unravel complex glycomes. [ABSTRACT FROM AUTHOR]

Copyright of Angewandte Chemie is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Here we present a combined experimental and theoretical study on the secondary structure of isolated proteins as a function of charge state. In infrared spectra of the proteins ubiquitin and cytochrome c, amide I (C=O stretch) and amide II (N-H bend) bands can be found at positions that are typical for condensed-phase proteins. For high charge states a new band appears, substantially red-shifted from the amide II band observed at lower charge states. The observations are interpreted in terms of Coulomb-driven transitions in secondary structures from mostly helical to extended C5-type hydrogen-bonded structures. Support for this interpretation comes from simple energy considerations as well as from quantum chemical calculations on model peptides. This transition in secondary structure is most likely universal for isolated proteins that occur in mass spectrometric experiments. [ABSTRACT FROM AUTHOR]

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An approach to mono- and divalent C-aminoglycosides starting from a new enantiopure 1,2-oxazine derivative is described. The introduction of a vinyl group into the 1,3-dioxolanyl substituent of a 1,2-oxazine allowed the Lewis acid promoted preparation of a vinyl-substituted bicyclic 1,2-oxazinone. After reduction of the carbonyl group, exhaustive hydrogenolysis provided branched C-aminoglycosides either with β-D-talose or β-D-idose configuration. The vinyl group of the protected rearrangement product 8 also allowed a self-metathesis with Grubbs II catalyst providing a 'dimeric' compound as an E/Z mixture. Its hydrogenolysis furnished the divalent C-aminoglycoside in good overall yield. [ABSTRACT FROM AUTHOR]

The original version of this Article contained an error in Fig. 1, in which an oxygen atom was missing from the ‘Acetoxonium type’ structure. This has been corrected in both the PDF and HTML versions of the Article. [ABSTRACT FROM AUTHOR]

Glycosyl cations are the key intermediates during the glycosylation reaction that covalently links building blocks during the synthetic assembly of carbohydrates. The exact structure of these ions remained elusive due to their transient and short-lived nature. Structural insights into the intermediate would improve our understanding of the reaction mechanism of glycosidic bond formation. Here, we report an in-depth structural analysis of glycosyl cations using a combination of cold-ion infrared spectroscopy and first-principles theory. Participating C2 protective groups form indeed a covalent bond with the anomeric carbon that leads to C1-bridged acetoxonium-type structures. The resulting bicyclic structure strongly distorts the ring, which leads to a unique conformation for each individual monosaccharide. This gain in mechanistic understanding fundamentally impacts glycosynthesis and will allow to tailor building blocks and reaction conditions in the future. Glycosyl cations are key intermediates in glycosylation reactions, but their structure has remained elusive due to their transient nature. Here, the authors perform an in-depth structural analysis and report that C2-participating protective groups induce acetoxonium cations with distinct ring conformations. [ABSTRACT FROM AUTHOR]

Charge‐induced unzipping of isolated proteins leads to a defined secondary structure characterized by intramolecular C5 hydrogen bonds. The cover picture shows how Coulomb repulsion can pull apart helical elements to form an extended linear conformation similar to a single‐strand β sheet. As G. von Helden and co‐workers report in their Communication on page 3295 ff., this conformation is likely a universal form of secondary structure in highly charged protein ions. [ABSTRACT FROM AUTHOR]

Das ladungsinduzierte Entwinden von isolierten Proteinen führt zu einer definierten Sekundärstruktur, die durch intramolekulare C5 ‐ Wasserstoffbrücken charakterisiert ist. Das Titelbild zeigt, wie die Coulomb ‐ Abstoßung helikale Elemente auseinanderzwingt und die Bildung einer linearen Konformation, ähnlich einem β ‐ Faltblatt ‐ Einzelstrang, bewirkt. Wie G. von Helden und Mitarbeiter in ihrer Zuschrift auf S. 3356 ff. feststellen, ist diese Konformation vermutlich eine universelle Sekundärstruktur für hoch geladene Proteinionen. [ABSTRACT FROM AUTHOR]