A computational tool to infer enzyme activity using post-translational modification profiling data.

Enzymes play a pivotal role in orchestrating complex cellular responses to external stimuli and environmental changes through signal transduction pathways. Despite their crucial roles, measuring enzyme activities is typically indirect and performed on a smaller scale, unlike protein abundance measured by high-throughput proteomics. Moreover, it is challenging to derive the activity of enzymes from proteome-wide post-translational modification (PTM) profiling data.

Insight into distribution and composition of nonhuman N-Glycans in mammalian organs via MALDI-TOF and MALDI-MSI.

The major hurdle of xenotransplantation is the immune response triggered by human natural antibodies interacting with carbohydrate antigens on the transplanted animal organ. Specifically, terminal glycoprotein motifs such as galactose-α1,3-galactose (α-Gal) and N-glycolylneuraminic acid (Neu5Gc) are significant obstacles. Little is known about the abundance and compositions of asparagine-linked complex carbohydrates (N-glycans) carrying these motifs in mammalian organs.

Acquisition of Fc-afucosylation of PfEMP1-specific IgG is age-dependent and associated with clinical protection against malaria.

Protective immunity to malaria depends on acquisition of parasite-specific antibodies, with Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) being one of the most important target antigens. The effector functions of PfEMP1-specific IgG include inhibition of infected erythrocyte (IE) sequestration and opsonization of IEs for cell-mediated destruction. IgG glycosylation modulates antibody functionality, with increased affinity to FcγRIIIa for IgG lacking fucose in the Fc region (Fc-afucosylation).

Parasitic infections during pregnancy in Gabon affect glycosylation patterns of maternal and child antibodies.

Antibody glycosylation patterns can affect antibody functionality and thereby contribute to protection against invading pathogens. During pregnancy, maternal antibodies can be transferred through the placenta and contribute to modulating both the mother's and her child's immune responses. Although several studies of IgG glycosylation during pregnancy have been carried out, very few cohorts studied were from sub-Saharan Africa, where exposure to microorganisms and parasites is high.

Shotgun Proteomics Protocol for Insects.

Shotgun proteomics can be applied to identify and study insect species in diverse research areas such as agriculture, forensics, biodiversity conservation, and food safety. In this chapter, we have provided a detailed protocol for shotgun proteomics analytical methods involving enzymatic digestion of insect proteins using trypsin, separation using high-performance liquid chromatography, and detection of separated peptides using high-resolution mass spectrometry. The protocol also covers the utilization of bioinformatics software for protein identification and spectral library building, proposing both proteomic database-dependent and independent methods.

JUMPlib: Integrative Search Tool Combining Fragment Ion Indexing with Comprehensive TMT Spectral Libraries.

The identification of peptides is a cornerstone of mass spectrometry-based proteomics. Spectral library-based algorithms are well-established methods to enhance the identification efficiency of peptides during database searches in proteomics. However, these algorithms are not specifically tailored for tandem mass tag (TMT)-based proteomics due to the lack of high-quality TMT spectral libraries.

Development of a High-Throughput Platform for Quantitation of Histone Modifications on a New QTOF Instrument.

Histone post-translational modifications (PTMs) regulate gene expression patterns through epigenetic mechanisms. The five histone proteins (H1, H2A, H2B, H3, and H4) are extensively modified, with over 75 distinct modification types spanning more than 200 sites. Despite strong advances in mass spectrometry (MS)-based approaches, identification and quantification of modified histone peptides remains challenging because of factors, such as isobaric peptides, pseudo-isobaric PTMs, and low stoichiometry of certain marks.

Addressing Sample Mix-Ups: Tools and Approaches for Large-Scale Multi-Omics Studies.

Advances in high-throughput omics technologies have enabled system-wide characterization of biological samples across multiple molecular levels, such as the genome, transcriptome, and proteome. However, as sample sizes rapidly increase in large-scale multi-omics studies, sample mix-ups have become a prevalent issue, compromising data integrity and leading to erroneous conclusions. The interconnected nature of multi-omics data presents an opportunity to identify and correct these errors.

Challenges in the identification and quantification of an unknown impurity in chenodeoxycholic acid drug substance.

In 2018 the Amsterdam University Medical Centre decided to prepare chenodeoxycholic acid (CDCA) capsules (also known as pharmacy compounding) for patients with the genetic metabolic disease cerebrotendinous xanthomatosis (CTX) when the product with a marketing authorization was commercially unavailable for patients. However, after reanalysis, unknown impurities were identified in the CDCA active pharmaceutical ingredient (API) using thin-layer chromatography from the European Pharmacopoeia (Ph.Eur.

ASGR1 deficiency improves atherosclerosis but alters liver metabolism in ApoE mice.

The asialoglycoprotein receptor 1 (ASGR1), a multivalent carbohydrate-binding receptor that primarily is responsible for recognizing and eliminating circulating glycoproteins with exposed galactose (Gal) or N-acetylgalactosamine (GalNAc) as terminal glycan residues, has been implicated in modulating the lipid metabolism and reducing cardiovascular disease burden. In this study, we investigated the impact of ASGR1 deficiency (ASGR1 on atherosclerosis by evaluating its effects on plaque formation, lipid metabolism, circulating immunoinflammatory response, and circulating N-glycome under the hypercholesterolemic condition in ApoE-deficient mice. After 16 weeks of a western-type diet, ApoE/ASGR1 mice presented lower plasma cholesterol and triglyceride levels compared to ApoE.

Fc-Afucosylation of VAR2CSA-Specific Immunoglobulin G and Clinical Immunity to Placental Plasmodium falciparum Malaria.

Background: Acquired immunity to Plasmodium falciparum malaria is mainly mediated by immunoglobulin G (IgG) targeting erythrocyte membrane protein 1 (PfEMP1). These adhesins mediate infected erythrocyte (IE) sequestration, protecting IEs from splenic destruction. PfEMP1-specific IgG is therefore thought to protect mainly by inhibiting IE sequestration.

Spatial Lipidomics Reveals Myelin Defects and Protumor Macrophage Infiltration in Malignant Peripheral Nerve Sheath Tumor Adjacent Nerves.

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive sarcomas arising from peripheral nerves, accounting for 3% to 5% of soft tissue sarcomas. MPNSTs often recur locally, leading to poor survival. Achieving tumor-free surgical margins is essential to prevent recurrence, but current methods for determining tumor margins are limited, highlighting the need for improved biomarkers.

Simultaneous Protein Quantitation and Glycosylation Profiling of Antigen-Specific Immunoglobulin G1 in Large Clinical Studies.

Antibodies have a key role in the immune system, making their characterization essential to biomedical, biopharmaceutical, and clinical research questions. Antibody effector functions are mainly controlled by quantity, subclass, and Fc glycosylation. We describe an integrated method to measure these three critical dimensions simultaneously.

Hematopoietic stem cell metabolism within the bone marrow niche - insights and opportunities.

Hematopoiesis unfolds within the bone marrow niche where hematopoietic stem cells (HSCs) play a central role in continually replenishing blood cells. The hypoxic bone marrow environment imparts peculiar metabolic characteristics to hematopoietic processes. Here, we discuss the internal metabolism of HSCs and describe external influences exerted on HSC metabolism by the bone marrow niche environment.

Single high-fat challenge and trained innate immunity: A randomized controlled cross-over trial.

Brief exposure of monocytes to atherogenic molecules, such as oxidized lipoproteins, triggers a persistent pro-inflammatory phenotype, named trained immunity. In mice, transient high-fat diet leads to trained immunity, which aggravates atherogenesis. We hypothesized that a single high-fat challenge in humans induces trained immunity.

Apolipoprotein E aggregation in microglia initiates Alzheimer's disease pathology by seeding β-amyloidosis.

The seeded growth of pathogenic protein aggregates underlies the pathogenesis of Alzheimer's disease (AD), but how this pathological cascade is initiated is not fully understood. Sporadic AD is linked genetically to apolipoprotein E (APOE) and other genes expressed in microglia related to immune, lipid, and endocytic functions. We generated a transgenic knockin mouse expressing HaloTag-tagged APOE and optimized experimental protocols for the biochemical purification of APOE, which enabled us to identify fibrillary aggregates of APOE in mice with amyloid-β (Aβ) amyloidosis and in human AD brain autopsies.

Transgenic sensors reveal compartment-specific effects of aggregation-prone proteins on subcellular proteostasis during aging.

Loss of proteostasis is a hallmark of aging that underlies many age-related diseases. Different cell compartments experience distinctive challenges in maintaining protein quality control, but how aging regulates subcellular proteostasis remains underexplored. Here, by targeting the misfolding-prone Fluc luciferase to the cytoplasm, mitochondria, and nucleus, we established transgenic sensors to examine subcellular proteostasis in Drosophila.

Characterization of the Clostridioides difficile 630Δerm putative Pro-Pro endopeptidase CD1597.

is the leading cause of antibiotic-associated infections worldwide. Within the host, can transition from a sessile to a motile state by secreting PPEP-1, which releases the cells from the intestinal epithelium by cleaving adhesion proteins. PPEP-1 belongs to the group of Pro-Pro endopeptidases (PPEPs), which are characterized by their unique ability to cleave proline-proline bonds.

Targeted Bmal1 restoration in muscle prolongs lifespan with systemic health effects in aging model.

Disruption of the circadian clock in skeletal muscle worsens local and systemic health, leading to decreased muscle strength, metabolic dysfunction, and aging-like phenotypes. Whole-body knockout mice that lack Bmal1, a key component of the molecular clock, display premature aging. Here, by using adeno-associated viruses, we rescued Bmal1 expression specifically in the skeletal muscle fibers of Bmal1-KO mice and found that this engaged the circadian clock and clock output gene expression, contributing to extended lifespan.