Despite significant advancements in sample preparation, instrumentation and data analysis, single-cell proteomics is currently limited by proteomic depth and quantitative performance. Here we demonstrate highly improved depth of proteome coverage as well as accuracy and precision for quantification of ultra-low input amounts. Using a tailored library, we identify up to 7,400 protein groups from as little as 250 pg of HeLa cell peptides at a throughput of 50 samples per day.
View Article and Find Full Text PDFThe field of crosslinking mass spectrometry has seen substantial advancements over the past decades, enabling the structural analysis of proteins and protein complexes and serving as a powerful tool in protein-protein interaction studies. However, data analysis of large non-cleavable crosslink studies is still a mostly unsolved problem due to its n-squared complexity. We here introduce an algorithm for the identification of non-cleavable crosslinks implemented in our crosslinking search engine MS Annika that is based on sparse matrix multiplication and allows for proteome-wide searches on commodity hardware.
View Article and Find Full Text PDFComprehensive proteomic analysis is essential to elucidate molecular pathways and protein functions. Despite tremendous progress in proteomics, current studies still suffer from limited proteomic coverage and dynamic range. Here, we utilize micropillar array columns (µPACs) together with wide-window acquisition and the AI-based CHIMERYS search engine to achieve excellent proteomic comprehensiveness for bulk proteomics, affinity purification mass spectrometry and single cell proteomics.
View Article and Find Full Text PDFThis work demonstrates the utility of high-throughput nanoLC-MS and label-free quantification (LFQ) for sample-limited bottom-up proteomics analysis, including single-cell proteomics (SCP). Conditions were optimized on a 50 μm internal diameter (I.D.
View Article and Find Full Text PDFPurpose: Prevention of fractures is an unmet need in glucocorticoid (GC)-treated Duchenne muscular dystrophy. This study explored factors associated with incident vertebral fractures (VFs) to inform future fracture prevention efforts.
Methods: VFs were evaluated prospectively at study baseline and 12 months on lateral spine radiographs in participants aged 4 to 25 years with Duchenne muscular dystrophy.
Cross-linking mass spectrometry has become a powerful tool for the identification of protein-protein interactions and for gaining insight into the structures of proteins. We previously published MS Annika, a cross-linking search engine which can accurately identify cross-linked peptides in MS2 spectra from a variety of different MS-cleavable cross-linkers. In this publication, we present MS Annika 2.
View Article and Find Full Text PDFOsteonecrosis (ON) is a serious complication of childhood acute lymphoblastic leukemia. We determined the prevalence of osteonecrotic lesions in our patient population by a one-time multisite magnetic resonance imaging (MRI) more than 1 year following leukemia therapy. MRI findings were evaluated in relationship to clinical factors (including longitudinal changes in bone mineral density [BMD]).
View Article and Find Full Text PDFThe analysis of ultralow input samples or even individual cells is essential to answering a multitude of biomedical questions, but current proteomic workflows are limited in their sensitivity and reproducibility. Here, we report a comprehensive workflow that includes improved strategies for all steps, from cell lysis to data analysis. Thanks to convenient-to-handle 1 μL sample volume and standardized 384-well plates, the workflow is easy for even novice users to implement.
View Article and Find Full Text PDFDigital subtraction angiography provides excellent spatial and temporal resolution; however, it lacks the capability to depict the nonvascular anatomy of the brain and spinal cord.A review of the institutional database identified five patients in whom a new integrated fusion workflow of cross-sectional imaging and 3D rotational angiography (3DRA) provided important diagnostic information and assisted in treatment planning. These included two acutely ruptured brain arteriovenous malformations (AVM), a small superficial brainstem AVM after radiosurgery, a thalamic microaneurysm, and a spine AVM, and fusion was crucial for diagnosis and influenced further treatment.
View Article and Find Full Text PDFRibosomes are produced in large quantities during oogenesis and are stored in the egg. However, the egg and early embryo are translationally repressed. Here, using mass spectrometry and cryo-electron microscopy analyses of ribosomes isolated from zebrafish (Danio rerio) and Xenopus laevis eggs and embryos, we provide molecular evidence that ribosomes transition from a dormant state to an active state during the first hours of embryogenesis.
View Article and Find Full Text PDFIn the field of liquid chromatography-mass spectrometry (LC-MS)-based proteomics, increases in the sampling depth and proteome coverage have mainly been accomplished by rapid advances in mass spectrometer technology. The comprehensiveness and quality of the data that can be generated do, however, also depend on the performance provided by nano-liquid chromatography (nanoLC) separations. Proper selection of reversed-phase separation columns can be important to provide the MS instrument with peptides at the highest possible concentration and separated at the highest possible resolution.
View Article and Find Full Text PDFCross-linking mass spectrometry has matured to a frequently used tool for the investigation of protein structures as well as interactome studies up to a system-wide level. The growing community generated a broad spectrum of applications, linker types, acquisition strategies and specialized data analysis tools, which makes it challenging to decide for an appropriate analysis workflow. Here, we report a large and flexible synthetic peptide library as reliable instrument to benchmark crosslink workflows.
View Article and Find Full Text PDFAlthough bone fragility may already be present at diagnosis of pediatric acute lymphoblastic leukemia (ALL), routine performance of dual-energy X-ray absorptiometry (DXA) in every child is not universally feasible. The aim of this study was to develop and validate a risk prediction model for low lumbar spine bone mineral density (LS BMD Z-score ≤ -2.0) at diagnosis, as an important indicator for fracture risk and further treatment-related BMD aggravation.
View Article and Find Full Text PDFContext: Osteoporotic fractures are an important cause of morbidity in children with glucocorticoid-treated rheumatic disorders.
Objective: This work aims to evaluate the incidence and predictors of osteoporotic fractures and potential for recovery over six years following glucocorticoid (GC) initiation in children with rheumatic disorders.
Methods: Children with GC-treated rheumatic disorders were evaluated through a prospective inception cohort study led by the Canadian STeroid-induced Osteoporosis in the Pediatric Population (STOPP) Consortium.
Cross-linking mass spectrometry (XL-MS) has become a powerful technique that enables insights into protein structures and protein interactions. The development of cleavable cross-linkers has further promoted XL-MS through search space reduction, thereby allowing for proteome-wide studies. These new analysis possibilities foster the development of new cross-linkers, which not every search engine can deal with out of the box.
View Article and Find Full Text PDFVertebral fractures are clinically important sequelae of a wide array of pediatric diseases. In this study, we examined the accuracy of case-finding strategies for detecting incident vertebral fractures (IVF) over 2 years in glucocorticoid-treated children (n = 343) with leukemia, rheumatic disorders, or nephrotic syndrome. Two clinical situations were addressed: the prevalent vertebral fracture (PVF) scenario (when baseline PVF status was known), which assessed the utility of PVF and low lumbar spine bone mineral density (LS BMD; Z-score <-1.
View Article and Find Full Text PDFCross-linking mass spectrometry (XL-MS) has matured into a potent tool to identify protein-protein interactions or to uncover protein structures in living cells, tissues, or organelles. The unique ability to investigate the interplay of proteins within their native environment delivers valuable complementary information to other advanced structural biology techniques. This Review gives a comprehensive overview of the current possible applications as well as the remaining limitations of the technique, focusing on cross-linking in highly complex biological systems like cells, organelles, or tissues.
View Article and Find Full Text PDF5'-AMP-activated protein kinase (AMPK) and the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) are main players in the cellular adaptive response to metabolic and oxidative/xenobiotic stress, respectively. AMPK does not only balance the rate of fuel catabolism versus anabolism but also emerges as regulator of gene expression. We here examined the influence of AMPK on Nrf2-dependent gene transcription and the potential interplay of the two cellular stress hubs.
View Article and Find Full Text PDFCross-linking mass spectrometry is an increasingly used, powerful technique to study protein-protein interactions or to provide structural information. Due to substochiometric reaction efficiencies, cross-linked peptides are usually low abundance. This results in challenging data evaluation and the need for an effective enrichment.
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