Conotoxin Prediction: New Features to Increase Prediction Accuracy.

Conotoxins are toxic, disulfide-bond-rich peptides from cone snail venom that target a wide range of receptors and ion channels with multiple pathophysiological effects. Conotoxins have extraordinary potential for medical therapeutics that include cancer, microbial infections, epilepsy, autoimmune diseases, neurological conditions, and cardiovascular disorders. Despite the potential for these compounds in novel therapeutic treatment development, the process of identifying and characterizing the toxicities of conotoxins is difficult, costly, and time-consuming. This challenge requires a series of diverse, complex, and labor-intensive biological, toxicological, and analytical techniques for effective characterization. While recent attempts, using machine learning based solely on primary amino acid sequences to predict biological toxins (e.g., conotoxins and animal venoms), have improved toxin identification, these methods are limited due to peptide conformational flexibility and the high frequency of cysteines present in toxin sequences. This results in an enumerable set of disulfide-bridged foldamers with different conformations of the same primary amino acid sequence that affect function and toxicity levels. Consequently, a given peptide may be toxic when its cysteine residues form a particular disulfide-bond pattern, while alternative bonding patterns (isoforms) or its reduced form (free cysteines with no disulfide bridges) may have little or no toxicological effects. Similarly, the same disulfide-bond pattern may be possible for other peptide sequences and result in different conformations that all exhibit varying toxicities to the same receptor or to different receptors. We present here new features, when combined with primary sequence features to train machine learning algorithms to predict conotoxins, that significantly increase prediction accuracy.

Glycoproteomic Analyses of Influenza A Viruses Using timsTOF Pro MS.

N-Linked glycosylation in hemagglutinin and neuraminidase glycoproteins of influenza viruses affects antigenic and receptor binding properties, and precise analyses of site-specific glycoforms in these proteins are critical in understanding the antigenic and immunogenic properties of influenza viruses. In this study, we developed a glycoproteomic approach by using a timsTOF Pro mass spectrometer (MS) to determine the abundance and heterogeneity of site-specific glycosylation for influenza glycoproteins. Compared with a Q Exactive HF MS, the timsTOF Pro MS method without the hydrophilic interaction liquid chromatography column enrichment achieved similar glycopeptide coverage and quantities but was more effective in identifying low-abundance glycopeptides. We quantified the distributions of intact site-specific glycopeptides in hemagglutinin of A/chicken/Wuxi/0405005/2013 (H7N9) and A/mute swan/Rhode Island/A00325125/2008 (H7N3). Results showed that hemagglutinin for both viruses had complex N-glycans at N22, N38, N240, and N483 but only high-mannose glycans at N411 and, however, that the type and quantities of glycans were distinct between these viruses. Collisional cross section (CCS) provided by the ion mobility spectrometry from the timsTOF Pro MS data differentiated sialylation linkages of the glycopeptides. In summary, timsTOF Pro MS method can quantify intact site-specific glycans for influenza glycoproteins without enrichment and thus facilitate influenza vaccine development and production.

Nanohydrophobic Interaction Chromatography Coupled to Ultraviolet Photodissociation Mass Spectrometry for the Analysis of Intact Proteins in Low Charge States.

The direct correlation between proteoforms and biological phenotype necessitates the exploration of mass spectrometry (MS)-based methods more suitable for proteoform detection and characterization. Here, we couple nano-hydrophobic interaction chromatography (nano-HIC) to ultraviolet photodissociation MS (UVPD-MS) for separation and characterization of intact proteins and proteoforms. High linearity, sensitivity, and sequence coverage are obtained with this method for a variety of proteins. Investigation of collisional cross sections of intact proteins during nano-HIC indicates semifolded conformations in low charge states, enabling a different dimension of separation in comparison to traditional, fully denaturing reversed-phase separations. This method is demonstrated for a mixture of intact proteins from Escherichia coli ribosomes; high sequence coverage is obtained for a variety of modified and unmodified proteoforms.

Evaluating the Effects of Metal Adduction and Charge Isomerism on Ion-Mobility Measurements using m-Xylene Macrocycles as Models.

Gas-phase ion-mobility spectrometry provides a unique platform to study the effect of mobile charge(s) or charge location on collisional cross section and ion separation. Here, we evaluate the effects of cation/anion adduction in a series of xylene and pyridyl macrocycles that contain ureas and thioureas. We explore how zinc binding led to unexpected deprotonation of the thiourea macrocyclic host in positive polarity ionization and subsequently how charge isomerism due to cation (zinc metal) and anion (chloride counterion) adduction or proton competition among acceptors can affect the measured collisional cross sections in helium and nitrogen buffer gases. Our approach uses synthetic chemistry to design macrocycle targets and a combination of ion-mobility spectrometry mass spectrometry experiments and quantum mechanics calculations to characterize their structural properties. We demonstrate that charge isomerism significantly improves ion-mobility resolution and allows for determination of the metal binding mechanism in metal-inclusion macrocyclic complexes. Additionally, charge isomers can be populated in molecules where individual protons are shared between acceptors. In these cases, interactions via drift gas collisions magnify the conformational differences. Finally, for the macrocyclic systems we report here, charge isomers are observed in both helium and nitrogen drift gases with similar resolution. The separation factor does not simply increase with increasing drift gas polarizability. Our study sheds light on important properties of charge isomerism and offers strategies to take advantage of this phenomenon in analytical separations.

Lipid analysis by ion mobility spectrometry combined with mass spectrometry: A brief update with a perspective on applications in the clinical laboratory.

Ion mobility spectrometry (IMS) is an analytical technique where ions are separated in the gas phase based on their mobility through a buffer gas in the presence of an electric field. An ion passing through an IMS device has a characteristic collisional cross section (CCS) value that depends on the buffer gas used. IMS can be coupled with mass spectrometry (MS), which characterizes an ion based on a mass-to-charge ratio (m/z), to increase analytical specificity and provide further physicochemical information. In particular, IMS-MS is of ever-increasing interest for the analysis of lipids, which can be problematic to accurately identify and quantify in bodily fluids by liquid chromatography (LC) with MS alone due to the presence of isomers, isobars, and structurally similar analogs. IMS provides an additional layer of separation when combined with front-end LC approaches, thereby, enhancing peak capacity and analytical specificity. CCS (and also ion mobility drift time) can be plotted against m/z ion intensity and/or LC retention time in order to generate in-depth molecular profiles of a sample. Utilization of IMS-MS for routine clinical laboratory testing remains relatively unexplored, but areas do exist for potential implementation. A brief update is provided here on lipid analysis using IMS-MS with a perspective on some applications in the clinical laboratory.

Trapped Ion Mobility Spectrometry and Parallel Accumulation-Serial Fragmentation in Proteomics.

Recent advances in efficiency and ease of implementation have rekindled interest in ion mobility spectrometry, a technique that separates gas phase ions by their size and shape and that can be hybridized with conventional LC and MS. Here, we review the recent development of trapped ion mobility spectrometry (TIMS) coupled to TOF mass analysis. In particular, the parallel accumulation-serial fragmentation (PASEF) operation mode offers unique advantages in terms of sequencing speed and sensitivity. Its defining feature is that it synchronizes the release of ions from the TIMS device with the downstream selection of precursors for fragmentation in a TIMS quadrupole TOF configuration. As ions are compressed into narrow ion mobility peaks, the number of peptide fragment ion spectra obtained in data-dependent or targeted analyses can be increased by an order of magnitude without compromising sensitivity. Taking advantage of the correlation between ion mobility and mass, the PASEF principle also multiplies the efficiency of data-independent acquisition. This makes the technology well suited for rapid proteome profiling, an increasingly important attribute in clinical proteomics, as well as for ultrasensitive measurements down to single cells. The speed and accuracy of TIMS and PASEF also enable precise measurements of collisional cross section values at the scale of more than a million data points and the development of neural networks capable of predicting them based only on peptide sequences. Peptide collisional cross section values can differ for isobaric sequences or positional isomers of post-translational modifications. This additional information may be leveraged in real time to direct data acquisition or in postprocessing to increase confidence in peptide identifications. These developments make TIMS quadrupole TOF PASEF a powerful and expandable platform for proteomics and beyond.

Optical Microscopy-Guided Laser Ablation Electrospray Ionization Ion Mobility Mass Spectrometry: Ambient Single Cell Metabolomics with Increased Confidence in Molecular Identification.

Single cell analysis is a field of increasing interest as new tools are continually being developed to understand intercellular differences within large cell populations. Laser-ablation electrospray ionization mass spectrometry (LAESI-MS) is an emerging technique for single cell metabolomics. Over the years, it has been validated that this ionization technique is advantageous for probing the molecular content of individual cells in situ. Here, we report the integration of a microscope into the optical train of the LAESI source to allow for visually informed ambient in situ single cell analysis. Additionally, we have coupled this 'LAESI microscope' to a drift-tube ion mobility mass spectrometer to enable separation of isobaric species and allow for the determination of ion collision cross sections in conjunction with accurate mass measurements. This combined information helps provide higher confidence for structural assignment of molecules ablated from single cells. Here, we show that this system enables the analysis of the metabolite content of Allium cepa epidermal cells with high confidence structural identification together with their spatial locations within a tissue.

Online Parallel Accumulation-Serial Fragmentation (PASEF) with a Novel Trapped Ion Mobility Mass Spectrometer.

In bottom-up proteomics, peptides are separated by liquid chromatography with elution peak widths in the range of seconds, whereas mass spectra are acquired in about 100 microseconds with time-of-flight (TOF) instruments. This allows adding ion mobility as a third dimension of separation. Among several formats, trapped ion mobility spectrometry (TIMS) is attractive because of its small size, low voltage requirements and high efficiency of ion utilization. We have recently demonstrated a scan mode termed parallel accumulation - serial fragmentation (PASEF), which multiplies the sequencing speed without any loss in sensitivity (Meier et al., PMID: 26538118). Here we introduce the timsTOF Pro instrument, which optimally implements online PASEF. It features an orthogonal ion path into the ion mobility device, limiting the amount of debris entering the instrument and making it very robust in daily operation. We investigate different precursor selection schemes for shotgun proteomics to optimally allocate in excess of 100 fragmentation events per second. More than 600,000 fragmentation spectra in standard 120 min LC runs are achievable, which can be used for near exhaustive precursor selection in complex mixtures or accumulating the signal of weak precursors. In 120 min single runs of HeLa digest, MaxQuant identified more than 6,000 proteins without matching to a library and with high quantitative reproducibility (R > 0.97). Online PASEF achieves a remarkable sensitivity with more than 2,500 proteins identified in 30 min runs of only 10 ng HeLa digest. We also show that highly reproducible collisional cross sections can be acquired on a large scale (R > 0.99). PASEF on the timsTOF Pro is a valuable addition to the technological toolbox in proteomics, with a number of unique operating modes that are only beginning to be explored.

Collidoscope: An Improved Tool for Computing Collisional Cross-Sections with the Trajectory Method.

Ion mobility-mass spectrometry (IM-MS) can be a powerful tool for determining structural information about ions in the gas phase, from small covalent analytes to large, native-like or denatured proteins and complexes. For large biomolecular ions, which may have a wide variety of possible gas-phase conformations and multiple charge sites, quantitative, physically explicit modeling of collisional cross sections (CCSs) for comparison to IMS data can be challenging and time-consuming. We present a "trajectory method" (TM) based CCS calculator, named "Collidoscope," which utilizes parallel processing and optimized trajectory sampling, and implements both He and N2 as collision gas options. Also included is a charge-placement algorithm for determining probable charge site configurations for protonated protein ions given an input geometry in pdb file format. Results from Collidoscope are compared with those from the current state-of-the-art CCS simulation suite, IMoS. Collidoscope CCSs are within 4% of IMoS values for ions with masses from ~18 Da to ~800 kDa. Collidoscope CCSs using X-ray crystal geometries are typically within a few percent of IM-MS experimental values for ions with mass up to ~3.5 kDa (melittin), and discrepancies for larger ions up to ~800 kDa (GroEL) are attributed in large part to changes in ion structure during and after the electrospray process. Due to its physically explicit modeling of scattering, computational efficiency, and accuracy, Collidoscope can be a valuable tool for IM-MS research, especially for large biomolecular ions. Graphical Abstract ᅟ.

Factors influencing compact-extended structure equilibrium in oligomers of aß1-40 peptide--an ion mobility mass spectrometry study.

Oligomers formed by amyloid ß (Aß) peptide are widely believed to be the main neurotoxic agent in Alzheimer's disease. Studies discovered a broad variety of oligomeric forms, which display different levels of toxicity. Some of these forms may further assemble into mature fibrils, while other might be off-pathway from conversion to fibrils and assemble into alternative forms. To better understand a relationship between the structure and toxicity of Aß oligomers, we require systematic characterization and classification of all possible forms, facilitating rational design of the beneficial modifiers of their activity. In previous ion mobility analysis of Aß1-40 oligomers, we have detected the coexistence of two alternative structural forms (compact and extended) in a pool of low-order Aß1-40 oligomers. These forms may represent two pathways of the oligomer evolution, leading either to fibrils or to off-pathway oligomers, which are potential candidates for the neurotoxic species. Here, we have analyzed the impact of incubation time, the presence of selected metal ions and the effect of a series of point mutations on mutual population of alternative forms. We have shown that a salt bridge D23K28 provides stabilization of the compact form whereas G25 is required for the existence of the extended form. We have found that binding of metal ions also stabilizes the compact form. These results improve our understanding of the possible molecular mechanism of the bifurcation of structural evolution of non-monomeric Aß species into an off-fibril pathway, ultimately leading to the formation of potentially neurotoxic species.

Transferrin as a drug carrier: Cytotoxicity, cellular uptake and transport kinetics of doxorubicin transferrin conjugate in the human leukemia cells.

Leukemias are one of most common malignancies worldwide. There is a substantial need for new chemotherapeutic drugs effective against this cancer. Doxorubicin (DOX), used for treatment of leukemias and solid tumors, is poorly efficacious when it is administered systemically at conventional doses. Therefore, several strategies have been developed to reduce the side effects of this anthracycline treatment. In this study we compared the effect of DOX and doxorubicin-transferrin conjugate (DOX-TRF) on human leukemia cell lines: chronic erythromyeloblastoid leukemia (K562), sensitive and resistant (K562/DOX) to doxorubicin, and acute lymphoblastic leukemia (CCRF-CEM). Experiments were also carried out on normal cells, peripheral blood mononuclear cells (PBMC). We analyzed the chemical structure of DOX-TRF conjugate by using mass spectroscopy. The in vitro growth-inhibition assay XTT, indicated that DOX-TRF is more cytotoxic for leukemia cells sensitive and resistant to doxorubicin and significantly less sensitive to normal cells compared to DOX alone. During the assessment of intracellular DOX-TRF accumulation it was confirmed that the tested malignant cells were able to retain the examined conjugate for longer periods of time than normal lymphocytes. Comparison of kinetic parameters showed that the rate of DOX-TRF efflux was also slower in the tested cells than free DOX. The results presented here should contribute to the understanding of the differences in antitumor activities of the DOX-TRF conjugate and free drug.