Kevin Pagel

Identification of carbohydrate anomers using ion mobility–mass spectrometry

Carbohydrates are ubiquitous biological polymers that are important in a broad range of biological processes. However, owing to their branched structures and the presence of stereogenic centres at each glycosidic linkage between monomers, carbohydrates are harder to characterize than are peptides and oligonucleotides. Methods such as nuclear magnetic resonance spectroscopy can be used to characterize glycosidic linkages, but this technique requires milligram amounts of material and cannot detect small amounts of coexisting isomers. Mass spectrometry, on the other hand, can provide information on carbohydrate composition and connectivity for even small amounts of sample, but it cannot be used to distinguish between stereoisomers. Here, we demonstrate that ion mobility–mass spectrometry—a method that separates molecules according to their mass, charge, size, and shape—can unambiguously identify carbohydrate linkage-isomers and stereoisomers. We analysed six synthetic carbohydrate isomers that differ in composition, connectivity, or configuration. Our data show that coexisting carbohydrate isomers can be identified, and relative concentrations of the minor isomer as low as 0.1 per cent can be detected. In addition, the analysis is rapid, and requires no derivatization and only small amounts of sample. These results indicate that ion mobility–mass spectrometry is an effective tool for the analysis of complex carbohydrates. This method could have an impact on the field of carbohydrate synthesis similar to that of the advent of high-performance liquid chromatography on the field of peptide assembly in the late 1970s.

Alternate dissociation pathways identified in charge-reduced protein complex ions

Tandem mass spectrometry (MS) of large protein complexes has proven to be capable of assessing the stoichiometry, connectivity, and structural details of multiprotein assemblies. While the utility of tandem MS is without question, a deeper understanding of the mechanism of protein complex dissociation will undoubtedly drive the technology into new areas of enhanced utility and information content. We present here the systematic analysis of the charge state dependent decay of the noncovalently associated complex of human transthyretin, generated by collision-induced dissociation (CID). A crown ether based charge reduction approach was applied to generate intact transthyretin tetramers with charge states ranging from 15+ to 7+. These nine charge states were subsequently analyzed by means of tandem MS and ion mobility spectrometry. Three different charge-dependent mechanistic regimes were identified: (1) common asymmetric dissociation involving ejection of unfolded monomers, (2) expulsion of folded monomers from the intact tetramer, and (3) release of C-terminal peptide fragments from the intact complex. Taken together, the results presented highlight the potential of charge state modulation as a method for directing the course of gas-phase dissociation and unfolding of protein complexes.

Ion Mobility–Mass Spectrometry of Complex Carbohydrates: Collision Cross Sections of Sodiated N-linked Glycans

Currently, the vast majority of complex carbohydrates are characterized using mass spectrometry (MS)-based techniques. Measuring the molecular mass of a sugar, however, immediately poses a fundamental problem: entire classes of the constituting monosaccharide building blocks exhibit an identical atomic composition and, consequently, also an identical mass. Therefore, carbohydrate MS data can be highly ambiguous and often it is simply not possible to clearly assign a particular molecular structure. A promising approach to overcome the above-mentioned limitation is to implement an additional gas-phase separation dimension using ion mobility spectrometry (IMS), which is a method in which molecules of identical mass and structure but different structure can be separated according to their shape and collision cross section (CCS). With the emergence of commercially available hybrid ion mobility-mass spectrometry (IM-MS) instruments in 2006, IMS technology became readily available. Because of the nonhomogeneous, traveling wave (TW) field utilized in these instruments, however, CCS values currently cannot be determined directly from the drift times measured. Instead, an external calibration using compounds of known CCS and similar molecular identity is required. Here, we report a calibration protocol for TW IMS instruments using a series of sodiated N-glycans that were released from commercially available glycoproteins using an easy-to-follow protocol. The underlying CCS values were determined using a modified Synapt HDMS instrument with a linear drift tube, which was described in detail previously. Our data indicate that, under in-source fragmentation conditions, only a few glycans are required to obtain a TW IMS calibration of sufficient quality. In this context, however, the type of glycan was shown to be of tremendous importance. Furthermore, our data clearly demonstrate that carbohydrate isomers with identical mass but different conformation can be distinguished based on their CCS when all the associated errors are taken into account.

Protomers of Benzocaine: Solvent and Permittivity Dependence

The immediate environment of a molecule can have a profound influence on its properties. Benzocaine, the ethyl ester of para-aminobenzoic acid that finds an application as a local anesthetic, is found to adopt in its protonated form at least two populations of distinct structures in the gas phase, and their relative intensities strongly depend on the properties of the solvent used in the electrospray ionization process. Here, we combine IR-vibrational spectroscopy with ion mobility-mass spectrometry to yield gas-phase IR spectra of simultaneously m/z and drift-time-resolved species of benzocaine. The results allow for an unambiguous identification of two protomeric species: the N- and O-protonated forms. Density functional theory calculations link these structures to the most stable solution and gas-phase structures, respectively, with the electric properties of the surrounding medium being the main determinant for the preferred protonation site. The fact that the N-protonated form of benzocaine can be found in the gas phase is owed to kinetic trapping of the solution-phase structure during transfer into the experimental setup. These observations confirm earlier studies on similar molecules where N- and O-protonation have been suggested.

Secondary Structure of Ac-Alan-LysH+ Polyalanine Peptides (n = 5,10,15) in Vacuo: Helical or Not?

The polyalanine-based peptide series Ac-Alan-LysH+ (n = 5−20) is a prime example that a secondary structure motif that is well-known from the solution phase (here: helices) can be formed in vacuo. Here we revisit the series members n = 5,10,15, using density functional theory (van der Waals corrected generalized gradient approximation) for structure predictions, which are then corroborated by room temperature gas-phase infrared vibrational spectroscopy. We employ a quantitative comparison based on Pendry’s reliability factor (popular in surface crystallography). In particular, including anharmonic effects into calculated spectra by way of ab initio molecular dynamics produces remarkably good experiment−theory agreement. We find the longer molecules (n = 10,15) to be firmly α-helical in character. For n = 5, calculated free-energy differences show different H-bond networks to still compete closely. Vibrational spectroscopy indicates a predominance of α-helical motifs at 300 K, but the lowest-energy conform...

Intrinsically Disordered p53 and Its Complexes Populate Compact Conformations in the Gas Phase

Spontaneous shrinking: the intrinsically disordered tumor suppressor protein p53 was analyzed by using a combination of ion mobility mass spectrometry and molecular dynamics simulations. Structured p53 subdomains retain their overall topology upon transfer into the gas phase. When intrinsically disordered segments are introduced into the protein sequence, however, the complex spontaneously collapses in the gas phase to a compact conformation.

Photodissociation of Conformer-Selected Ubiquitin Ions Reveals Site-Specific Cis/Trans Isomerization of Proline Peptide Bonds

Ultraviolet photodissociation (UVPD) of gas-phase proteins has attracted increased attention in recent years. This growing interest is largely based on the fact that, in contrast to slow heating techniques such as collision induced dissociation (CID), the cleavage propensity after absorption of UV light is distributed over the entire protein sequence, which can lead to a very high sequence coverage as required in typical top-down proteomics applications. However, in the gas phase, proteins can adopt a multitude of distinct and sometimes coexisting conformations, and it is not clear how this three-dimensional structure affects the UVPD fragmentation behavior. Using ion mobility-UVPD-mass spectrometry in conjunction with molecular dynamics simulations, we provide the first experimental evidence that UVPD is sensitive to the higher order structure of gas-phase proteins. Distinct UVPD spectra were obtained for different extended conformations of 11(+) ubiquitin ions. Assignment of the fragments showed that the majority of differences arise from cis/trans isomerization of one particular proline peptide bond. Seen from a broader perspective, these data highlight the potential of UVPD to be used for the structural analysis of proteins in the gas phase.

Amide-I and -II Vibrations of the Cyclic β-Sheet Model Peptide Gramicidin S in the Gas Phase

In the condensed phase, the peptide gramicidin S is often considered as a model system for a beta-sheet structure. Here, we investigate gramicidin S free of any influences of the environment by measuring the mid-IR spectra of doubly protonated (deuterated) gramicidin S in the gas phase. In the amide I (i.e., C=O stretch) region, the spectra show a broad split peak between 1580 and 1720 cm(-1). To deduce structural information, the conformational space has been searched using molecular dynamics methods and several structural candidates have been further investigated at the density functional level. The calculations show the importance of the interactions of the charged side-chains with the backbone, which is responsible for the lower frequency part of the amide I peak. When this interaction is inhibited via complexation with two 18-crown-6 molecules, the amide I peak narrows and shows two maxima at 1653 and 1680 cm(-1). A comparison to calculations shows that for this complexed ion, four C=O groups are in an antiparallel beta-sheet arrangement. Surprisingly, an analysis of the calculated spectra shows that these beta-sheet C=O groups give rise to the vibrations near 1680 cm(-1). This is in sharp contrast to expectations based on values for the condensed phase, where resonances of beta-sheet sections are thought to occur near 1630 cm(-1). The difference between those values might be caused by interactions with the environment, as the condensed phase value is mostly deduced for beta-sheet sections that are embedded in larger proteins, that interact strongly with solvent or that are part of partially aggregated species.

How Metal Ions Affect Amyloid Formation: Cu2+‐ and Zn2+‐Sensitive Peptides

The common feature of proteins involved in many neurodegenerative diseases is their ability to adopt at least two different stable conformations. The conformational transition that shifts the equilibrium from the functional, mostly partially α‐helical structure, to the β‐sheet rich amyloid can be triggered by numerous factors, such as mutations in the primary structure or changes in the environment. We present a set of model peptides that, without changes in their primary structure, react in a predictable fashion in the presence of transition metal ions by adopting different conformations and aggregate morphologies. These de novo designed peptides strictly follow the characteristic heptad repeat of the α‐helical coiled‐coil structural motif. Furthermore, domains that favor β‐sheet formation have been incorporated to make the system prone to amyloid formation. As a third feature, histidine residues create sensitivity towards the presence of transition metal ions. CD spectroscopy, ThT fluorescence experiments, and transmission electron microscopy were used to characterize peptide conformation and aggregate morphology in the presence of Cu2+ and Zn2+. Furthermore, the binding geometry within peptide–Cu2+ complexes was characterized by electron paramagnetic resonance spectroscopy.

Estimating collision cross sections of negatively charged N-glycans using traveling wave ion mobility-mass spectrometry.

Glycosylation is one of the most common post-translational modifications occurring in proteins. A detailed structural characterization of the involved carbohydrates, however, is still one of the greatest challenges in modern glycoproteomics, since multiple regio- and stereoisomers with an identical monosaccharide composition may exist. Recently, ion mobility-mass spectrometry (IM-MS), a technique in which ions are separated according to their mass, charge, and shape, has evolved as a promising technique for the separation and structural analysis of complex carbohydrates. This growing interest is based on the fact that the measured drift times can be converted into collision cross sections (CCSs), which can be compared, implemented into databases, and used as additional search criteria for structural identification. However, most of the currently used commercial IM-MS instruments utilize a nonuniform traveling wave field to propel the ions through the IM cell. As a result, CCS measurements cannot be performed directly and require calibration. Here, we present a calibration data set consisting of over 500 reference CCSs for negatively charged N-glycans and their fragments. Moreover, we show that dextran, already widely used as a calibrant in high performance liquid chromatography, is also a suitable calibrant for CCS estimations. Our data also indicate that a considerably increased error has to be taken into account when reference CCSs acquired in a different drift gas are used for calibration.