Antiviral innate immune networks in human cells comprise core components that serve as central signaling hubs and several context-dependent modulators whose role may be virus- or tissue-specific. One such modulator is LSM14A, which potentiates innate immune response but is not essential. We recently showed that enteroviruses deploy their protease activity to cleave LSM14A, thereby disabling its antiviral function. In this study, we probe the molecular mechanism by which LSM14A contributes to innate immunity. We show that although LSM14A predominantly localizes to processing bodies (P-bodies; PBs), this localization is not essential for its innate immune function. Likewise, association with peroxisomes does not contribute to its immune activity. Instead, an unbiased systems-level interactomic analysis reveals a distinct cohort of LSM14A-associated proteins that assemble outside canonical PBs and peroxisomes following infection with Sendai virus, a robust inducer of innate immunity. Functional interrogation of these interactors demonstrate that several are essential for LSM14A-dependent amplification of antiviral signaling. Together, these findings uncover a functional axis of LSM14A that operates independently of its canonical subcellular localizations and is mediated through a specialized interaction network, improving our understanding of how this protein reinforces the antiviral innate immune system.

Glycoproteomics presents a challenge for analysis by conventional proteomics methods due to the size, complexity, and heterogeneity of glycans. Many published methods use higher-energy collisional dissociation (HCD) that preferentially fragments the glycan, preventing its confident localization on the peptide due to low abundances of peptide backbone fragmentation. However, the glycan fragment ions resulting from collisional dissociation have limited value for determining glycan topologies in part due to rearrangements that occur to protonated glycan groups during vibrational excitation. To overcome this problem, we investigated the use of electron-activated dissociation (ExD) methods that preferentially dissociate the peptide with low levels of vibrational excitation. Hot electron capture dissociation (hECD) has previously been demonstrated for glycosylated peptides using a Fourier-transform ion cyclotron resonance mass spectrometer, but its slower scan speed and lower sensitivity make it ineffective for larger-scale glycoproteomics studies. In this work, we employed an Omnitrap platform integrated with a Q Exactive-class mass spectrometer to perform hECD with enhanced speed and sensitivity. We evaluated a set of higher energy (he) ExD conditions and observed useful glycan fragmentation, including the cross-ring cleavages that define glycan topologies. Using this approach, we detected isomeric glycopeptides differing in glycan topology or glycosylation sites, highlighting the power of on-line LC-MS heExD analysis.

Glycosaminoglycans (GAGs) are linear, negatively charged polysaccharides composed of repeating disaccharide units. Heparan sulfate (HS) and chondroitin sulfate (CS) are highly sulfated GAG classes, ubiquitously expressed in mammalian tissues, that play critical roles in cellular signaling, tissue homeostasis, and disease progression. Aside from their biological importance, the structural analysis of HS and CS remains limited to bulk tissue analysis due to their extensive heterogeneity, structural complexity, and the presence of isomers and epimers. In this work, we developed an integrated workflow combining laser microdissection (LMD), hydrophilic interaction liquid chromatography (HILIC), and cyclic ion mobility mass spectrometry (cIM-MS) for the identification and quantification of HS and CS disaccharides from small-scale and spatially resolved mouse brain tissue sections. Through sequential enzymatic digestion of HS and CS chains from the same sample, we profiled not only the common disaccharides that serve as structural signatures for HS and CS, but also rarely detected HS disaccharides containing saturated uronic acid residues, as well as lyase-resistant 3-O-sulfated HS tetrasaccharides. HILIC enabled the separation of HS and CS disaccharides based on their composition and hydrophilicity, while cIM-MS further enhanced the resolution of positional isomers. Quantitative analysis using linear calibration curves revealed disaccharide abundances in small-scale tissue sections collected by LMD. Overall, our finding highlighted the merit of the LMD-HILIC-cIM-MS workflow for HS and CS analysis in spatial GAGomics and its potential for biomarker discovery and therapeutic application studies.

Neurodegenerative disorders such as Alzheimer’s disease (AD) and Lewy body disease (LBD) are typically diagnosed after irreversible pathology has developed. Aging, the strongest risk factor, drives molecular changes that predispose the brain to synaptic dysfunction and proteinopathy. Glycosylation and extracellular matrix (ECM) remodeling represent underexplored mechanisms linking aging to neurodegeneration, opening avenues for biomarker discovery, yet mass spectrometry-based glycoproteomics and glycomics studies remain limited. Here, we optimized an on-slide tissue digestion workflow for integrated glycome and proteome profiling from 5-mm brain regions from mice and humans using liquid chromatography data-independent acquisition–tandem mass spectrometry (LC-DIA-MS/MS). This workflow was applied to whole brains from age- and sex-stratified wild-type mice (n = 12) and to human postmortem prefrontal cortex tissue from individuals (n = 14) with brainstem- (n = 8) or limbic-predominant (n = 6) LBD, with or without AD co-pathology. DIA substantially increased protein, glycosylated protein, and ECM coverage by two- to threefold relative to traditional data-dependent acquisition (DDA), while library-free DIA searches further improved detection of low-abundance or region-specific proteins. In aged mouse brains, we observed increased levels of synapse-related proteins—including SYNPR, ZNT3, and HPCA—and enrichment of glutamatergic and postsynaptic pathways, reflecting age-associated synaptic remodeling. Glycomics revealed subtle shifts in the sulfation of chondroitin sulfate (CS) disaccharides with age. In human samples, AD-LBD brainstem tissue exhibited significantly reduced unsulfated, 4-O-sulfated, and total CS levels, along with differential expression of ECM, glycosylated, and mitochondrial proteins, and enrichment of mitochondrial pathways compared with LBD brainstem and AD-LBD limbic tissues. These findings indicate that AD co-pathology exerts a region-specific influence on the proteomic and glycomic landscape of LBD. Collectively, this study establishes a robust DIA-based on-slide digestion platform for high-resolution spatial glycomics and proteomics from minimal tissue, revealing aging- and pathology-specific molecular alterations relevant to neurodegeneration and providing a framework for biomarker discovery.

Hyperglycosylated proteins with a high sialic acid content show great promise in the development of long-acting biotherapeutics. However, their structural complexity and heterogeneity pose significant challenges to traditional analytical methods, which often fail to provide comprehensive glycan information across all glycosylation sites, leading to ambiguities in characterization. Despite the fact that long-acting hyperglycosylated erythropoietin (hyperEPO) has been available on the market for over two decades, its site-specific glycosylation profile remains ambiguous due to closely spaced glycosylation sites and large glycans that contain labile sialic acids substituents. Here, using hyperEPO as an example, we developed an integrated workflow that incorporates an experimentally cross-validated glycan database, optimized nonspecific digestion, and a streamlined glycopeptide derivatization method to enhance sialylated peptide detection, enabling comprehensive site-specific characterization of both N- and O-glycosylation. We applied this strategy to compare the glycosylation profiles of the commercial hyperEPO drug darbepoetin alfa and a novel high-potency analogue, EPO-XL. We found that both proteins exhibited high site occupancy, large tetra-sialylated glycans, and similar types of sialic acid linkages but differed markedly in site-specific glycoform distributions. Notably, EPO-XL contained extensive LacNAc structures at all five N-glycosylation sites, and a previously unreported O-glycosylation site at S120 was identified alongside the canonical S126 site. Collectively, this study presents the first site-specific N- and O- glycosylation profiles of hyperEPO proteins, offering valuable guidance for the quality control and rational design of these therapeutics. The state-of-the-art analytical strategy introduced here holds great potential to advance the site-specific glycosylation characterization of proteins with complex glycosylation.

Flamenco (Flam) is a prominent Piwi-interacting RNA (piRNA) locus expressed in Drosophila ovarian follicle cells that silences gypsy/mdg4 transposons to ensure female fertility. Promoter-bashing reporter assays in ovarian somatic sheet (OSS) cells uncover compact enhancer sequences within Flam. We confirm the enhancer sequence relevance in vivo with Drosophila Flam deletion mutants that compromise Flam piRNA levels and female fertility. Proteomic analysis of proteins associated with Flam enhancer sequences discover the transcription factor Traffic Jam (TJ). Tj knockdown in OSS cells causes a decrease in Flam transcripts, Flam piRNAs, and multiple Piwi pathway genes. TJ chromatin immunoprecipitation sequencing (ChIP-seq) analysis confirms TJ binding at enhancer sequences deleted in our distinct Flam mutants. TJ also binds multiple Piwi pathway gene enhancers and long terminal repeats of transposons that decrease in expression after Tj knockdown. TJ plays an integral role in the ongoing arms race between selfish transposons and their suppression by the host Piwi pathway and Flam piRNA locus.

Mass spectrometry-based investigation of the heterogeneous glycoproteome from complex biological specimens is a robust approach to mapping the structure, function, and dynamics of the glycome and proteome. Sampling whole wet tissues often provides a large amount of starting material; however, there is a reasonable variability in tissue handling prior to downstream processing steps, and it is difficult to capture all the different biomolecules from a specific region. The on-slide tissue digestion approach, outlined in this protocol chapter, is a simple and cost-effective method that allows comprehensive mapping of the glycoproteome from a single spot of tissue of 1 mm or greater diameter. It provides a selection of target areas on tissue slides appropriate for tissue volumes of 10 nL or greater, corresponding to a 1 μL droplet of enzyme solution applied to a 1-mm diameter target on a 10-μm-thick tissue slice. Sequential enzymatic digestions and desalting of the biomolecules without any prior derivatization from the surface of fresh frozen or formalin-fixed paraffin-embedded tissue slides enable the simultaneous identification of glycosaminoglycan disaccharides such as hyaluronan, chondroitin sulfate and heparan sulfate, asparagine or N-linked glycans, and intact (glyco)peptides using liquid chromatography-tandem mass spectrometry. The in-depth information obtained from this method including the disaccharide compositions, glycan structures, peptide abundances, and site-specific glycan occupancies provides a detailed profiling of a single spot of tissue which has the potential to be disseminated to biomedical laboratories.

In this review, we describe the methods used for the extraction and mass spectrometry proteomics analysis of amyloid plaques and neurofibrillary tangles (NFTs), the two primary pathological hallmarks of Alzheimer’s disease (AD). We also provide a comprehensive overview of the mass spectrometry-based studies conducted to analyze these pathological features. AD is the most prevalent form of dementia and the sixth leading cause of death in the United States. While the current treatments can alleviate early-stage memory and cognitive symptoms, they do not offer a cure. Thus, there is a pressing need to deepen our understanding of the neuropathological mechanisms underlying AD and to develop more effective therapeutics. In-depth mass spectrometry-based proteomics analyses of AD pathology—specifically, extracellular the Aβ plaques found in extracellular spaces and blood vessel walls and intraneuronal NFTs composed of the microtubule-associated protein tau—may offer molecular-level observations that contribute to the understanding of the biological context of plaque and NFT formation and support the discovery of potential biomarkers and therapeutic targets for AD.

The cerebrovasculature is responsible for supplying oxygenated blood and nutrients to the brain and removing neurotoxic buildup. With age, trauma, and disease, the structural constituents of cerebral arteries including the extracellular matrix and smooth muscle cells are subject to remodeling and degradation. Cerebrovascular dysfunction can have detrimental impacts on the brain and is closely associated with cognitive impairment. Clinical studies have found that cerebrovascular dysfunction is correlated with cognitive decline in neurodegenerative diseases including Alzheimer’s disease (AD) and chronic traumatic encephalopathy (CTE). However, cerebrovascular changes during the progression of neurological disorders remain to be understood. Using matched and parallel studies of cerebrovasculature and brain tissue, this study set out to determine the temporal development of cerebrovascular remodeling and neurodegenerative disease progression. We examined changes to human anterior cerebral arteries (ACAs) from subjects with various degrees of AD and CTE neuropathology. Using biaxial inflation-extension testing, histological staining, and multiphoton imaging, we examined changes to the mechanical response and to the ACA wall structure. We found circumferential stiffening of the ACA with age. Furthermore, a minor relationship was reported between ACA stiffening and elevated levels of tau-based neuropathologies including neurofibrillary tangles, characteristic of both AD and CTE. Histological and multiphoton structural studies of the ACAs revealed smooth muscle cell atrophy at the media-adventitia interface and disorganization and straightening of adventitial collagen with age and disease. Our study reveals changes to the extracellular and cellular components of cerebral arteries that help describe the functional alterations of cerebrovasculature. Results from this study shed light on the complex relationship between cerebrovascular remodeling and neurodegenerative disease progression.

The MIRAGE (Minimum Information Required for A Glycomics Experiment) guidelines for mass spectrometry (MS) data were initially developed to standardize the reporting of instrumentation, data acquisition and analytical details of the MS-based identification of released glycans. However, the growing interest in the study of intact glycoproteins and recent advances in MS-based glycoproteomics now necessitate a revision and expansion of these guidelines. This update includes an enhanced section focused on glycan structure analysis (glycomics) and introduces a new component tailored to the specific requirements of glycoproteomics. It addresses both shared and unique aspects of each approach and highlights glycoinformatics resources designed to facilitate data submission in compliance with the updated standards.

Glycosylation is an abundant post-translational modification that impacts a wide variety of functions, including protein regulation, cell adhesion, and structural integrity. The application of proteomics methods to glycopeptide assignment faces unique challenges due to high heterogeneity, which results in complex populations with low overall abundance per glycopeptide. In addition, glycans dissociate at a lower collision energy compared to their attached peptide component. The resulting mass spectral data require specialized assignment software, which has caused glycoproteomics to lag traditional proteomics. Existing software primarily focuses on data-dependent acquisition (DDA), but manual validation is frequently required, and experiments are necessarily limited by the stochastic nature of DDA ion-selection. Data-independent acquisition (DIA) allows for a more complete and robust analysis of glycopeptide samples, but analysis software is still sparse. In this review, we discuss the current state of DDA analysis software, the limitations, and how it can inform our forays into DIA glycoproteomics.

Heparan sulfate (HS) is a linear, highly sulfated, and heterogeneous polysaccharide that covalently attaches to core proteins to form heparan sulfate proteoglycans (HSPGs). HSPGs are widely expressed in mammalian cells and are found on the cell surface and within the extracellular matrix (ECM). Structurally, HS consists of repeating disaccharide units composed of hexuronic acid (HexA) (either glucuronic acid (GlcA) or iduronic acid (IdoA)) linked to glucosamine (GlcN) units. The HS chain undergoes extensive post-polymerization modifications, including N-deacetylation of GlcN, C5-epimerization of HexA, and sulfation at various positions like 2-O-sulfation of HexA, as well as 3-O-, 6-O-, and N-sulfation of GlcN. Among these modifications, 3-O-sulfation of HS, produced by HS 3-O-sulfotransferase (HS3OST), is the rarest and most functionally significant. While 3-O-sulfated HS is well known for its anticoagulant properties through the activation of antithrombin, it also plays a critical role in various physiological and pathological processes, including cell differentiation, cancer progression, herpes simplex virus entry, and neuronal development. However, the precise mechanisms underlying these functions and their pathological implications remain inadequately characterized. This knowledge gap is primarily due to the low abundance of 3-O-sulfated HS and the lack of standardized analytical methods for its detection in biological samples. In this review, we summarize recent advancements in analytical techniques for the analysis of 3-O-sulfated HS and highlight potential future directions to improve its characterization and advance our understanding of its biological roles.

Heparan sulfate (HS) is a linear polysaccharide that modifies proteoglycans. HS biosynthesis is regulated in a spatiotemporal manner, leading to structural diversity, including variable de-N-acetylation, N-sulfation, hexuronic acid C5 epimerization, and 2-O-, 6-O-, and 3-O-sulfation. Specific structural motifs within HS chains offer multiple specific binding sites for protein partners. The occurrence of HS 3-O-sulfation is relatively rare; however, there is accumulating evidence identifying the importance of this low-abundance modification in many different biological scenarios. Initially described as a key determinant for binding and activation of antithrombin, and more recently, as a coreceptor for viral infection, 3-O-sulfation has been associated with the progression of several neurological disorders. The analytical ability to study the biological roles of HS 3-O-sulfation is hindered by its low abundance within HS chains and the complex isomeric nature of highly sulfated HS, which places a burden on the tandem mass spectrometry step for assigning saccharide structures. In this context, we developed a specific cationic peptide-affinity method for 3-O-sulfation enrichment, followed by hydrophilic interaction liquid chromatography-cyclic ion mobility mass spectrometry analysis (HILIC-cIM-MS). We first demonstrated the high specificity of this approach to capture 3-O-sulfated HS oligosaccharides within complex mixtures. We next showed the influence of specific sulfate and epimerization patterns on HS binding selectivity. Finally, we used the enrichment strategy to analyze 3-O-sulfated HS oligosaccharides from heparin lyase III-digested HS from porcine intestinal mucosa (HSPIM). We concluded that this enrichment method was useful to guide new studies to reveal the biological roles of 3-O-sulfation and to elucidate new HS structural motifs.

Glycosylation is widely recognized as the most complex post-translational modification due to the widespread presence of macro- and microheterogeneities, wherein its biological consequence is closely related to both the glycosylation sites and the glycan fine structures. Yet, efficient site-specific detailed glycan characterization remains a significant analytical challenge. Here, utilizing an Orbitrap-Omnitrap platform, higher-energy electron-activated dissociation (heExD) tandem mass spectrometry (MS/MS) revealed extraordinary efficacy for the structural characterization of intact glycopeptides. HeExD produced extensive fragmentation within both the glycan and the peptide, including A-/B-/C-/Y-/Z-/X-ions from the glycan motif and a-/b-/c-/x-/y-/z-type peptide fragments (with or without the glycan). The intensity of cross-ring cleavage and backbone fragments retaining the intact glycan was highly dependent on the electron energy. Among the four electron energy levels investigated, electronic excitation dissociation (EED) provided the most comprehensive structural information, yielding a complete series of glycosidic fragments for accurate glycan topology determination, a wealth of cross-ring fragments for linkage definition, and the most extensive peptide backbone fragments for accurate peptide sequencing and glycosylation site localization. The glycan fragments observed in the EED spectrum correlated well with the fragmentation patterns observed in EED MS/MS of the released glycans. The advantages of EED over higher-energy collisional dissociation (HCD), stepped collision energy HCD (sceHCD), and electron-transfer/higher-energy collisional dissociation (EThcD) were demonstrated for the characterization of a glycopeptide bearing a biantennary disialylated glycan. EED can produce a complete peptide backbone and glycan sequence coverage even for doubly protonated precursors. The exceptional performance of heExD MS/MS, particularly EED MS/MS, in site-specific detailed glycan characterization on an Orbitrap-Omnitrap hybrid instrument presents a novel option for in-depth glycosylation analysis.

Recent findings show that effective integration of novel information in the brain requires coordinated processes of homo- and heterosynaptic plasticity. In this work, we hypothesize that activity-dependent remodeling of the peri-synaptic extracellular matrix (ECM) contributes to these processes. We show that clusters of the peri-synaptic ECM, recognized by CS56 antibody, emerge in response to sensory stimuli, showing temporal and spatial coincidence with dendritic spine plasticity. Using CS56 co-immunoprecipitation of synaptosomal proteins, we identify several molecules involved in Ca2+ signaling, vesicle cycling, and AMPA-receptor exocytosis, thus suggesting a role in long-term potentiation (LTP). Finally, we show that, in the CA1 hippocampal region, the attenuation of CS56 glycoepitopes, through the depletion of versican as one of its main carriers, impairs LTP and object location memory in mice. These findings show that activity-dependent remodeling of the peri-synaptic ECM regulates the induction and consolidation of LTP, contributing to hippocampal-dependent memory.

Substance use disorder is a major concern, with few therapeutic options. Heparan sulfate (HS) and chondroitin sulfate (CS) interact with a plethora of growth factors and their receptors and have profound effects on cellular signaling. Thus, targeting these dynamic interactions might represent a potential novel therapeutic modality. In the present study, we performed mass spectrometry- based glycomic and proteomic analysis to understand the effects of cocaine and methamphetamine (METH) on HS, CS, and the proteome of two brain regions critically involved in drug addiction: the lateral hypothalamus and the striatum. We observed that cocaine and METH significantly alter HS and CS abundances as well as sulfate contents and composition. In particular, repeated METH or cocaine treatments reduced CS 4-O-sulfation and increased CS 6-O-sulfation. Since C4S and C6S exercise differential effects on axon growth, regeneration, and plasticity, these changes likely contribute to drug- induced neural plasticity in these brain regions. Notably, we observed that restoring these alterations by increasing CS 4-0 levels in the lateral hypothalamus by adenoassociated virus delivery of an shRNA to arylsulfatase B (N-acetylgalactosamine-4-sulfatase) ameliorated anxiety and prevented the expression of preference for cocaine in a novelty induced conditioned place preference test during cocaine withdrawal. Finally, proteomics analyses revealed a number of aberrant proteins in METH- and cocaine-treated versus saline-treated mice, including myelin proteolipid protein, calcium/calmodulin-dependent protein kinase type II subunit alpha, synapsin-2, tenascinR, calnexin, annexin A7, hepatoma-derived growth factor, neurocan, and CSPG5, and oxidative phosphorylation among the top perturbed pathway. Taken together, these data support the role of HS, CS, and associated proteins in stimulants abuse and suggest that manipulation of HSPGs can represent a novel therapeutic strategy.