Top-down proteomics.

Proteoforms, which arise from post-translational modifications, genetic polymorphisms and RNA splice variants, play a pivotal role as drivers in biology. Understanding proteoforms is essential to unravel the intricacies of biological systems and bridge the gap between genotypes and phenotypes. By analysing whole proteins without digestion, top-down proteomics (TDP) provides a holistic view of the proteome and can decipher protein function, uncover disease mechanisms and advance precision medicine.

Top-down mass spectrometry of native proteoforms and their complexes: a community study.

The combination of native electrospray ionization with top-down fragmentation in mass spectrometry (MS) allows simultaneous determination of the stoichiometry of noncovalent complexes and identification of their component proteoforms and cofactors. Although this approach is powerful, both native MS and top-down MS are not yet well standardized, and only a limited number of laboratories regularly carry out this type of research. To address this challenge, the Consortium for Top-Down Proteomics initiated a study to develop and test protocols for native MS combined with top-down fragmentation of proteins and protein complexes across 11 instruments in nine laboratories.

Evaluating protein cross-linking as a therapeutic strategy to stabilize SOD1 variants in a mouse model of familial ALS.

Mutations in the gene encoding Cu-Zn superoxide dismutase 1 (SOD1) cause a subset of familial amyotrophic lateral sclerosis (fALS) cases. A shared effect of these mutations is that SOD1, which is normally a stable dimer, dissociates into toxic monomers that seed toxic aggregates. Considerable research effort has been devoted to developing compounds that stabilize the dimer of fALS SOD1 variants, but unfortunately, this has not yet resulted in a treatment.

Top-down mass spectrometry of native proteoforms and their complexes: A community study.

The combination of native electrospray ionisation with top-down fragmentation in mass spectrometry allows simultaneous determination of the stoichiometry of noncovalent complexes and identification of their component proteoforms and co-factors. While this approach is powerful, both native mass spectrometry and top-down mass spectrometry are not yet well standardised, and only a limited number of laboratories regularly carry out this type of research. To address this challenge, the Consortium for Top-Down Proteomics (CTDP) initiated a study to develop and test protocols for native mass spectrometry combined with top-down fragmentation of proteins and protein complexes across eleven instruments in nine laboratories.

Structure Relaxation Approximation (SRA) for Elucidation of Protein Structures from Ion Mobility Measurements (II). Protein Complexes.

Characterizing structures of protein complexes and their disease-related aberrations is essential to understanding molecular mechanisms of many biological processes. Electrospray ionization coupled with hybrid ion mobility/mass spectrometry (ESI-IM/MS) methods offer sufficient sensitivity, sample throughput, and dynamic range to enable systematic structural characterization of proteomes. However, because ESI-IM/MS characterizes ionized protein systems in the gas phase, it generally remains unclear to what extent the protein ions characterized by IM/MS have retained their solution structures.

massNet: integrated processing and classification of spatially resolved mass spectrometry data using deep learning for rapid tumor delineation.

Motivation: Mass spectrometry imaging (MSI) provides rich biochemical information in a label-free manner and therefore holds promise to substantially impact current practice in disease diagnosis. However, the complex nature of MSI data poses computational challenges in its analysis. The complexity of the data arises from its large size, high-dimensionality and spectral nonlinearity.

The Human Proteoform Project: Defining the human proteome.

Proteins are the primary effectors of function in biology, and thus, complete knowledge of their structure and properties is fundamental to deciphering function in basic and translational research. The chemical diversity of proteins is expressed in their many proteoforms, which result from combinations of genetic polymorphisms, RNA splice variants, and posttranslational modifications. This knowledge is foundational for the biological complexes and networks that control biology yet remains largely unknown.

Peak learning of mass spectrometry imaging data using artificial neural networks.

Mass spectrometry imaging (MSI) is an emerging technology that holds potential for improving, biomarker discovery, metabolomics research, pharmaceutical applications and clinical diagnosis. Despite many solutions being developed, the large data size and high dimensional nature of MSI, especially 3D datasets, still pose computational and memory complexities that hinder accurate identification of biologically relevant molecular patterns. Moreover, the subjectivity in the selection of parameters for conventional pre-processing approaches can lead to bias.

Multimodal platform for assessing drug distribution and response in clinical trials.

Background: Response to targeted therapy varies between patients for largely unknown reasons. Here, we developed and applied an integrative platform using mass spectrometry imaging (MSI), phosphoproteomics, and multiplexed tissue imaging for mapping drug distribution, target engagement, and adaptive response to gain insights into heterogeneous response to therapy.

Methods: Patient-derived xenograft (PDX) lines of glioblastoma were treated with adavosertib, a Wee1 inhibitor, and tissue drug distribution was measured with MALDI-MSI.

Increasing Top-Down Mass Spectrometry Sequence Coverage by an Order of Magnitude through Optimized Internal Fragment Generation and Assignment.

A major limitation of intact protein fragmentation is the lack of sequence coverage within proteins' interiors. We show that collisionally activated dissociation (CAD) produces extensive internal fragmentation within proteins' interiors that fill the existing gaps in sequence coverage, including disulfide loop regions that cannot be characterized using terminal fragments. A barrier to the adoption of internal fragments is the lack of methods for their generation and assignment.

Raf promotes dimerization of the Ras G-domain with increased allosteric connections.

Ras dimerization is critical for Raf activation. Here we show that the Ras binding domain of Raf (Raf-RBD) induces robust Ras dimerization at low surface densities on supported lipid bilayers and, to a lesser extent, in solution as observed by size exclusion chromatography and confirmed by SAXS. Community network analysis based on molecular dynamics simulations shows robust allosteric connections linking the two Raf-RBD D113 residues located in the Galectin scaffold protein binding site of each Raf-RBD molecule and 85 Å apart on opposite ends of the dimer complex.

Cyclic Thiosulfinates as a Novel Class of Disulfide Cleavable Cross-Linkers for Rapid Hydrogel Synthesis.

We recently reported that cyclic thiosulfinates are cysteine selective cross-linkers that avoid the "dead-end" modifications that contribute to other cross-linkers' toxicity. In this study, we generalize the chemistry of cyclic thiosulfinates to that of thiol selective cross-linking and apply them to the synthesis of hydrogels. Thiol-functionalized four-arm poly(ethylene glycol) and hyaluronic acid monomers were cross-linked with 1,2-dithiane-1-oxide to form disulfide cross-linked hydrogels within seconds.

Spatial Distribution of Transcytosis Relevant Phospholipids in Response to Omega-3 Dietary Deprivation.

The cell membrane of brain endothelial cells is enriched in omega-3 phospholipid species. Numerous omega-3 phospholipid species were recently proposed to be important for maintaining the low rate of transcytosis and, thus, could be important for regulating one of the mechanisms of the blood brain barrier (BBB). However, the spatial distribution of these phospholipid species within the brain was previously unknown.

Interlaboratory Study for Characterizing Monoclonal Antibodies by Top-Down and Middle-Down Mass Spectrometry.

The Consortium for Top-Down Proteomics (www.topdownproteomics.org) launched the present study to assess the current state of top-down mass spectrometry (TD MS) and middle-down mass spectrometry (MD MS) for characterizing monoclonal antibody (mAb) primary structures, including their modifications.

Loss of angiogenin function is related to earlier ALS onset and a paradoxical increase in ALS duration.

0.5-1% of ALS (Amyotrophic Lateral Sclerosis) and Parkinson's disease (PD) are associated with mutations in the angiogenin (ANG). These mutations are thought to cause disease through a loss of ANG function, but this hypothesis has not been evaluated statistically.

Localized Metabolomic Gradients in Patient-Derived Xenograft Models of Glioblastoma.

Glioblastoma (GBM) is increasingly recognized as a disease involving dysfunctional cellular metabolism. GBMs are known to be complex heterogeneous systems containing multiple distinct cell populations and are supported by an aberrant network of blood vessels. A better understanding of GBM metabolism, its variation with respect to the tumor microenvironment, and resulting regional changes in chemical composition is required.

Dysregulation of very-long-chain fatty acid metabolism causes membrane saturation and induction of the unfolded protein response.

The unfolded protein response (UPR) senses defects in the endoplasmic reticulum (ER) and orchestrates a complex program of adaptive cellular remodeling. Increasing evidence suggests an important relationship between lipid homeostasis and the UPR. Defects in the ER membrane induce the UPR, and the UPR in turn controls the expression of some lipid metabolic genes.

Unsupervised segmentation of mass spectrometric ion images characterizes morphology of tissues.

Motivation: Mass spectrometry imaging (MSI) characterizes the spatial distribution of ions in complex biological samples such as tissues. Since many tissues have complex morphology, treatments and conditions often affect the spatial distribution of the ions in morphology-specific ways. Evaluating the selectivity and the specificity of ion localization and regulation across morphology types is biologically important.

Best practices and benchmarks for intact protein analysis for top-down mass spectrometry.

One gene can give rise to many functionally distinct proteoforms, each of which has a characteristic molecular mass. Top-down mass spectrometry enables the analysis of intact proteins and proteoforms. Here members of the Consortium for Top-Down Proteomics provide a decision tree that guides researchers to robust protocols for mass analysis of intact proteins (antibodies, membrane proteins and others) from mixtures of varying complexity.

Automatic 3D Nonlinear Registration of Mass Spectrometry Imaging and Magnetic Resonance Imaging Data.

Multimodal integration between mass spectrometry imaging (MSI) and radiology-established modalities such as magnetic resonance imaging (MRI) would allow the investigations of key questions in complex biological systems such as the central nervous system. Such integration would provide complementary multiscale data to bridge the gap between molecular and anatomical phenotypes, potentially revealing new insights into molecular mechanisms underlying anatomical pathologies presented on MRI. Automatic coregistration between 3D MSI/MRI is a computationally challenging process due to dimensional complexity, MSI data sparsity, lack of direct spatial-correspondences, and nonlinear tissue deformation.

Genetically Encoded Fluorescent Proteins Enable High-Throughput Assignment of Cell Cohorts Directly from MALDI-MS Images.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) provides a unique in situ chemical profile that can include drugs, nucleic acids, metabolites, lipids, and proteins. MSI of individual cells (of a known cell type) affords a unique insight into normal and disease-related processes and is a prerequisite for combining the results of MSI and other single-cell modalities (e.g.

Molecular Characterization of Prostate Cancer with Associated Gleason Score Using Mass Spectrometry Imaging.

Diagnosis of prostate cancer is based on histologic evaluation of tumor architecture using a system known as the "Gleason score." This diagnostic paradigm, while the standard of care, is time-consuming, shows intraobserver variability, and provides no information about the altered metabolic pathways, which result in altered tissue architecture. Characterization of the molecular composition of prostate cancer and how it changes with respect to the Gleason score (GS) could enable a more objective and faster diagnosis.

Integrated mapping of pharmacokinetics and pharmacodynamics in a patient-derived xenograft model of glioblastoma.

Therapeutic options for the treatment of glioblastoma remain inadequate despite concerted research efforts in drug development. Therapeutic failure can result from poor permeability of the blood-brain barrier, heterogeneous drug distribution, and development of resistance. Elucidation of relationships among such parameters could enable the development of predictive models of drug response in patients and inform drug development.

Rapid discrimination of pediatric brain tumors by mass spectrometry imaging.

Purpose: Medulloblastoma, the most common primary pediatric malignant brain tumor, originates in the posterior fossa of the brain. Pineoblastoma, which originates within the pineal gland, is a rarer malignancy that also presents in the pediatric population. Medulloblastoma and pineoblastoma exhibit overlapping clinical features and have similar histopathological characteristics.