Biology subjects

Reber, V.

Publications and source records attributed to Reber, V..

4 recordsLinked to original sources

bioRxiv2026

In-source fragmentation in mass spectrometry-based proteomics: prevalence, impact, and strategies for mitigation

Peptide-level analyses are becoming increasingly popular in mass spectrometry-based proteomics and are being applied, for example, in immunopeptidomics, structural proteomics, and analyses of post-translational modifications. In such analyses, peptides that are not biologically meaningful but instead arise as artifacts prior to mass spectrometry analysis pose the risk of data misinterpretation. Here, we describe an approach based on retention time analysis and precise chromatographic peak matching to identify peptides generated by in-source fragmentation (ISF), which occurs between chromatographic separation of peptide mixtures and the first mass filter of a tandem mass spectrometer (MS). To understand the prevalence and properties of ISF, we generated 13 proteomics datasets and analyzed them along with additional 25 previously published datasets spanning a broad range of sample types, MS, and proteomics approaches including classical bottom-up proteomics, immunopeptidomics, structural proteomics, and phosphoproteomics. We found that, in typical trypsin-digested samples on average 1 % of fully-tryptic peptides and 22 % of semi-tryptic peptides originated from ISF. However, we observed large variations between datasets, and in-source fragments exceeded, in some cases, a third of the total peptide identifications. The extent of ISF was dependent on the peptide sequence, the instrument, method parameters, and sample complexity. Although ISF did not impair relative quantification across samples, it generated peptides that could be misinterpreted qualitatively, inflated peptide identifications, and comprised up to 37 percent of peptides shorter than 9 amino acids in immunopeptidomics datasets. We propose that, for peptide-centric applications, our open-source ISF detection approach be used to re-annotate peptides generated by ISF and remove them to avoid misinterpretation of data. ISF is an increasing concern with improving mass spectrometers, as they enable detection of an ever-increasing number of m/z features, including low abundance features like ISF products. Our work thus addresses a growing issue in proteomics and presents solutions to mitigate the impact of in-source fragment peptides. In the future, improved feature detection algorithms may enable elucidation of new ISF patterns affecting side chains that have been missed so far, which could contribute to explaining the vast space of as-yet unannotated proteomics data.

biochemistry↗

bioRxiv2025

Allosteric Gain-of-Function and Off-Mechanism Effects of ATP-Competitive Kinase Inhibitors Revealed by Multimodal Proteomics

ATP competitive kinase inhibitors represent one of the largest classes of targeted anti-cancer drugs. While their primary mechanism is to block catalytic activity, they can also trigger paradoxical phenotypic effects that cannot be explained by catalytic inhibition alone. These observations point to a hidden layer of drug action that modulates non-catalytic kinase functions via changes in kinase conformation and protein-protein interactions (PPIs). Here we developed a multimodal proteomics approach combining limited proteolysis coupled mass spectrometry on affinity purified samples (AP-LiP-MS), AP-MS, and proximity labeling-MS to map inhibitor-induced conformation and PPI changes. We show that inhibitor binding causes structural rearrangements in the AIDs of all tested kinases, consistent with a transition to an open, active-like kinase conformation. These structural shifts drive distinct kinase-protein interaction changes that control non-catalytic functions: sequestration of AMPK by inhibited CAMKK2 blocks phosphorylation by other kinases, CHEK1 inhibition causes dissociation from the mitochondrial protein CLPB and leads to mitochondrial fragmentation, and structural changes in inhibited PRKCA trigger rapid relocalization to cell junctions. Thus we identify the ATP-binding site as a major organizing center of kinase conformation and interaction. Our work suggests that these on-target, off-mechanism effects are highly prevalent and provides the analytical framework to systematically characterize a frequently overlooked phenomenon highly relevant for understanding drug side effects to guide the development of novel therapeutics.

molecular biology↗

bioRxiv2025

Structure-function relationship of alpha-synuclein fibrillar polymorphs derived from distinct synucleinopathies

The aggregation of the protein alpha-synuclein (Syn) is a common feature of multiple neurodegenerative diseases collectively called synucleinopathies, for which the pathobiology is not well understood. The different phenotypic characteristics of the synucleinopathies Parkinsons disease (PD), Dementia with Lewy Bodies (DLB) and Multiple System Atrophy (MSA) have been proposed to originate from the distinct structures adopted by Syn in its amyloid forms. Here, using covalent labeling and limited proteolysis coupled to mass spectrometry (LiP-MS) in vitro and in situ within neuronal cells and directly in native patient brain homogenates, we show that pathogenic Syn from distinct synucleinopathies (PD, DLB and MSA) are structurally different. Further, we found that fibrillar structural differences are associated with different fibril interactomes and neuronal responses. We discovered disease-specific ubiquitination patterns and turnover profiles for pathogenic Syn species, detected molecular pathways responding specifically to the uptake of different Syn fibrillar polymorphs, and identified a subset of the involved proteins as candidate direct interactors of Syn. In particular, components of the Ubiquitin-proteasomal System (UPS), including E3 ubiquitin ligases, chaperones, and Deubiquitinating proteins, showed disease/polymorph-specific interaction patterns, possibly accounting for different resistance of patient-derived Syn fibrils to degradation. Genetic modulation with CRISPR-based tools showed that members of the UPS degradation pathway (three E3 ligases: UBE3A, TRIM25, HUWE1 and the AAA+ ATPase VCP) reduced Syn inclusions, in a strain-specific manner. LiP-MS also identified sets of proteins with altered protease susceptibility in postmortem brain homogenates of PD, DLB, and MSA patients. These sets were largely disease-specific and included proteins altered in cells treated with fibrils derived from patients with the matching disease. Our findings provide insight into cellular processes involved in the accumulation and turnover of Syn pathogenic aggregates in PD, DLB and MSA in a disease specific manner and constitutes a resource of potential novel drug targets in these synucleinopathies.

systems biology↗

bioRxiv2025

Alpha-synuclein fibrils induce budding of mitochondrial-derived vesicles

-synuclein (-syn) aggregation is a hallmark of synucleinopathies, a class of neurodegenerative disorders such as Parkinsons disease (PD). Several lines of evidence indicate the involvement of mitochondria in the disease pathology. Despite extensive study, the link between -syn aggregation and mechanisms of mitochondrial toxicity remains not fully understood. Using high-resolution imaging with electron microscopy, we examined cells exposed to -syn fibrils vs control cells with a focus on mitochondria. We found that upon exposure to -syn fibrils, mitochondria increase in size, cristae structure gets defects, and mitochondria enhance the budding of mitochondrial-derived vesicles (MDVs). MDV formation reflects an evolutionarily conserved mechanism reminiscent of bacterial outer membrane vesicle (OMV) biogenesis. Structural proteomics analysis by mass spectrometry corroborates this microscopy observation by identifying changes in multiple proteins that regulate cristae structure, MDV formation, and trafficking. Our results provide a new link between -syn and mitochondria and identify novel pathways responding to -syn aggregates, particularly that -synuclein directly triggers MDV generation. The processes we detected could be of interest for diagnostics and potential therapeutic interventions.

neuroscience↗