Characterization of an Omnitrap-Orbitrap Platform Equipped with Infrared Multiphoton Dissociation, Ultraviolet Photodissociation, and Electron Capture Dissociation for the Analysis of Peptides and Proteins.

We describe an instrument configuration based on the Orbitrap Exploris 480 mass spectrometer that has been coupled to an Omnitrap platform. The Omnitrap possesses three distinct ion-activation regions that can be used to perform resonant-based collision-induced dissociation, several forms of electron-associated fragmentation, and ultraviolet photodissociation. Each section can also be combined with infrared multiphoton dissociation. In this work, we demonstrate all these modes of operation in a range of peptides and proteins. The results show that this instrument configuration produces similar data to previous implementations of each activation technique and at similar efficiency levels. We demonstrate that this unique instrument configuration is extremely versatile for the investigation of polypeptides.

Human Melanoma-Cell Metabolic Profiling: Identification of Novel Biomarkers Indicating Metastasis.

Melanoma is the most aggressive type of skin cancer, leading to metabolic rewiring and enhancement of metastatic transformation. Efforts to improve its early and accurate diagnosis are largely based on preclinical models and especially cell lines. Hence, we herein present a combinational Nuclear Magnetic Resonance (NMR)- and Ultra High Performance Liquid Chromatography-High-Resolution Tandem Mass Spectrometry (UHPLC-HRMS/MS)-mediated untargeted metabolomic profiling of melanoma cells, to landscape metabolic alterations likely controlling metastasis. The cell lines WM115 and WM2664, which belong to the same patient, were examined, with WM115 being derived from a primary, pre-metastatic, tumor and WM2664 clonally expanded from lymph-node metastases. Metabolite samples were analyzed using NMR and UHPLC-HRMS. Multivariate statistical analysis of high resolution NMR and MS (positive and negative ionization) results was performed by Principal Component Analysis (PCA), Partial Least Squares-Discriminant Analysis (PLS-DA) and Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA), while metastasis-related biomarkers were determined on the basis of VIP lists, S-plots and Student's t-tests. Receiver Operating Characteristic (ROC) curves of NMR and MS data revealed significantly differentiated metabolite profiles for each cell line, with WM115 being mainly characterized by upregulated levels of phosphocholine, choline, guanosine and inosine. Interestingly, WM2664 showed notably increased contents of hypoxanthine, myo-inositol, glutamic acid, organic acids, purines, pyrimidines, AMP, ADP, ATP and UDP(s), thus indicating the critical roles of purine, pyrimidine and amino acid metabolism during human melanoma metastasis.

Malignancy Grade-Dependent Mapping of Metabolic Landscapes in Human Urothelial Bladder Cancer: Identification of Novel, Diagnostic, and Druggable Biomarkers.

BACKGROUND: Urothelial bladder cancer (UBC) is one of the cancers with the highest mortality rate and prevalence worldwide; however, the clinical management of the disease remains challenging. Metabolomics has emerged as a powerful tool with beneficial applications in cancer biology and thus can provide new insights on the underlying mechanisms of UBC progression and/or reveal novel diagnostic and therapeutic schemes. METHODS: A collection of four human UBC cell lines that critically reflect the different malignancy grades of UBC was employed; RT4 (grade I), RT112 (grade II), T24 (grade III), and TCCSUP (grade IV). They were examined using Nuclear Magnetic Resonance, Mass Spectrometry, and advanced statistical approaches, with the goal of creating new metabolic profiles that are mechanistically associated with UBC progression toward metastasis. RESULTS: Distinct metabolic profiles were observed for each cell line group, with T24 (grade III) cells exhibiting the most abundant metabolite contents. AMP and creatine phosphate were highly increased in the T24 cell line compared to the RT4 (grade I) cell line, indicating the major energetic transformation to which UBC cells are being subjected during metastasis. Thymosin ß4 and ß10 were also profiled with grade-specific patterns of expression, strongly suggesting the importance of actin-cytoskeleton dynamics for UBC advancement to metastatic and drug-tolerant forms. CONCLUSIONS: The present study unveils a novel and putatively druggable metabolic signature that holds strong promise for early diagnosis and the successful chemotherapy of UBC disease.

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.

The Omnitrap Platform: A Versatile Segmented Linear Ion Trap for Multidimensional Multiple-Stage Tandem Mass Spectrometry.

Multidimensional multiple-stage tandem processing of ions is demonstrated successfully in a novel segmented linear ion trap. The enhanced performance is enabled by incorporating the entire range of ion activation methods into a single platform in a highly dynamic fashion. The ion activation network comprises external injection of reagent ions, radical neutral species, photons, electrons, and collisions with neutrals. Axial segmentation of the two-dimensional trapping field provides access to a unique functionality landscape through a system of purpose-designed regions for processing ions with maximum flexibility. Design aspects of the segmented linear ion trap, termed the Omnitrap platform, are highlighted, and motion of ions trapped by rectangular waveforms is investigated experimentally by mapping the stability diagram, tracing secular frequencies, and exploring different isolation techniques. All fragmentation methods incorporated in the Omnitrap platform involving radical chemistry are shown to provide complete sequence coverage for partially unfolded ubiquitin. Three-stage (MS3) tandem mass spectrometry experiments combining collision-induced dissociation of radical ions produced by electron meta-ionization and further involving two intermediate steps of ion isolation and accumulation are performed with high efficiency, producing information rich spectra with signal-to-noise levels comparable to those obtained in a two-stage (MS2) experiment. The advanced capabilities of the Omnitrap platform to provide in-depth top-down MSn characterization of proteins are portrayed. Performance is further enhanced by connecting the Omnitrap platform to an Orbitrap mass analyzer, while successful integration with time-of-flight analyzers has already been demonstrated.