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.

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 β4 and β10 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.

The configuration of the first prototype timsOmni instrument, which integrates an Omnitrap linear ion trap into a timsTOF platform, is presented and applied to antibody analysis. A modified electrode design for the electron-based fragmentation (ExD) section of the Omnitrap platform is introduced, enhancing both the robustness and performance of the method. Optimal characterization of antibodies requires characterizing light and heavy chains as pairs in addition to sequencing their variable domains and identifying any modifications. This is best addressed using protein centric proteomics as heterogeneity information such as the specific clonal origin of each identified fragment can be retained. Furthermore, by acting on intact proteins that retain part of their structure such as disulfide bonds, it is possible to target selected regions for fragmentation such as some of the hypervariable complementarity determining regions (CDRs) that are unique for each clone and necessary for target recognition. Using electron-based fragmentation methods, we employed the prototype timsOmni mass spectrometer to obtain full CDR3 sequences for paired heavy and light chains with high confidence. Optimal results were obtained by performing Electron Induced Dissociation (EID) at ~35 eV electron energy, on native-like fragment antigen-binding (Fab) precursor ions. This approach yields both (a, x) as well as (c, z) fragment ion pairs with the potential to enhance both sequence coverage and annotation confidence. Overall, the timsOmni mass spectrometer presented here serves as an advanced and versatile platform for protein-centric proteomics, demonstrating exceptional sequencing power for comprehensive protein characterization.

Deep characterization of intact proteoforms remains an analytical challenge in functional proteomics, particularly for heterogenous multi-site post-translational modifications at distinct amino acid residues. Histones are among the most dynamically and diversely post-translationally modified proteins in eukaryote cells, carrying multiple, co-occurring and reversible modifications that can give rise to isomeric proteoform species. Tandem mass spectrometry with multimodal fragmentation capabilities is a promising approach for deep characterization of intact proteoforms, such as modified histones. We applied the novel timsOmni mass spectrometer, which incorporates the Omnitrap platform enabling multimodal MSⁿ workflows, for residue-level mapping of histone modifications, including acetylation and methylation. Recombinant histones H3.1 and H4 were in vitro acetylated by enzymes GCN5, PCAF and p300 to generate mono- and multi-acetylated proteoforms. Complementary MS ² electron- and collision-based dissociation (ECD, EID, _R CID and ECciD), together with MS ³ strategies, produced complete or near-complete backbone fragmentation of intact protein ions (>92% amino acid sequence coverage). For monoacetylated species generated by the more site-selective lysine acetyltransferases, the dominant proteoform matched the known catalytic preferences of the enzymes (H3.1K14ac for GCN5 and PCAF, and H4K8ac for PCAF), while minor positional isomers were also identified and their relative abundance estimated. In contrast, the broader substrate specificity of p300 produced a wide distribution of H4 proteoforms bearing up to seven acetylated lysine residues. Species carrying six and seven acetylations were characterized by multimodal MS ² /MS ³ experiments, enabling localization of individual acetylation sites and discrimination of positional isomers. Finally, endogenous histone proteoforms from liver extracts were analyzed, yielding sequence coverages of 92–93% for the most abundant species and enabling confident localization of multiple PTMs (acetylation and methylation). These results illustrate that multimodal MS ⁿ fragmentation of intact proteins supports residue-level assignment of combinatorial histone marks and coexisting positional isomers. Graphical Highlights: Multimodal MS²/MS³ maps histone PTMs on intact proteins. ECD, EID, _R CID, and ECciD provide complete or near-complete sequence coverage. MS³ localizes acetylation sites, distinguishes positional isomers. Endogenous H4 proteoforms are assigned with site-specific PTM mapping.