Focal clusters of peri-synaptic matrix contribute to activity-dependent plasticity and memory in mice.

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.

RAMZIS: a bioinformatic toolkit for rigorous assessment of the alterations to glycoprotein composition that occur during biological processes.

Motivation: Glycosylation elaborates the structures and functions of glycoproteins; glycoproteins are common post-translationally modified proteins and are heterogeneous and non-deterministically synthesized as an evolutionarily driven mechanism that elaborates the functions of glycosylated gene products. Glycoproteins, accounting for approximately half of all proteins, require specialized proteomics data analysis methods due to micro- and macro-heterogeneities as a given glycosite can be divided into several glycosylated forms, each of which must be quantified. Sampling of heterogeneous glycopeptides is limited by mass spectrometer speed and sensitivity, resulting in missing values. In conjunction with the low sample size inherent to glycoproteomics, a specialized toolset is needed to determine if observed changes in glycopeptide abundances are biologically significant or due to data quality limitations. Results: We developed an R package, Relative Assessment of m/z Identifications by Similarity (RAMZIS), that uses similarity metrics to guide researchers to a more rigorous interpretation of glycoproteomics data. RAMZIS uses a permutation test to generate contextual similarity, which assesses the quality of mass spectral data and outputs a graphical demonstration of the likelihood of finding biologically significant differences in glycosylation abundance datasets. Investigators can assess dataset quality, holistically differentiate glycosites, and identify which glycopeptides are responsible for glycosylation pattern change. RAMZIS is validated by theoretical cases and a proof-of-concept application. RAMZIS enables comparison between datasets too stochastic, small, or sparse for interpolation while acknowledging these issues in its assessment. Using this tool, researchers will be able to rigorously define the role of glycosylation and the changes that occur during biological processes. Availability and implementation: https://github.com/WillHackett22/RAMZIS.

A critical evaluation of ultrasensitive single-cell proteomics strategies.

Success of mass spectrometry characterization of the proteome of single cells allows us to gain a greater understanding than afforded by transcriptomics alone but requires clear understanding of the tradeoffs between analytical throughput and precision. Recent advances in mass spectrometry acquisition techniques, including updated instrumentation and sample preparation, have improved the quality of peptide signals obtained from single cell data. However, much of the proteome remains uncharacterized, and higher throughput techniques often come at the expense of reduced sensitivity and coverage, which diminish the ability to measure proteoform heterogeneity, including splice variants and post-translational modifications, in single cell data analysis. Here, we assess the growing body of ultrasensitive single-cell approaches and their tradeoffs as researchers try to balance throughput and precision in their experiments.

Direct and Detailed Site-Specific Glycopeptide Characterization by Higher-Energy Electron-Activated Dissociation Tandem Mass Spectrometry.

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.

Akt may associate with insulin-responsive vesicles via interaction with sortilin.

Insulin-responsive vesicles (IRVs) deliver the glucose transporter Glut4 to the plasma membrane in response to activation of the insulin signaling cascade: insulin receptor-IRS-PI3 kinase-Akt-TBC1D4-Rab10. Previous studies have shown that Akt, TBC1D4, and Rab10 are compartmentalized on the IRVs. Although functionally significant, the mechanism of Akt association with the IRVs remains unknown. Using pull-down assays, immunofluorescence microscopy, and cross-linking, we have found that Akt may be recruited to the IRVs via the interaction with the juxtamembrane domain of the cytoplasmic C terminus of sortilin, a major IRV protein. Overexpression of full-length sortilin increases insulin-stimulated phosphorylation of TBC1D4 and glucose uptake in adipocytes, while overexpression of the cytoplasmic tail of sortilin has the opposite effect. Our findings demonstrate that the IRVs represent both a scaffold and a target of insulin signaling.

Proteomics-based mass spectrometry profiling of SARS-CoV-2 infection from human nasopharyngeal samples.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the on-going global pandemic of coronavirus disease 2019 (COVID-19) that continues to pose a significant threat to public health worldwide. SARS-CoV-2 encodes four structural proteins namely membrane, nucleocapsid, spike, and envelope proteins that play essential roles in viral entry, fusion, and attachment to the host cell. Extensively glycosylated spike protein efficiently binds to the host angiotensin-converting enzyme 2 initiating viral entry and pathogenesis. Reverse transcriptase polymerase chain reaction on nasopharyngeal swab is the preferred method of sample collection and viral detection because it is a rapid, specific, and high-throughput technique. Alternate strategies such as proteomics and glycoproteomics-based mass spectrometry enable a more detailed and holistic view of the viral proteins and host-pathogen interactions and help in detection of potential disease markers. In this review, we highlight the use of mass spectrometry methods to profile the SARS-CoV-2 proteome from clinical nasopharyngeal swab samples. We also highlight the necessity for a comprehensive glycoproteomics mapping of SARS-CoV-2 from biological complex matrices to identify potential COVID-19 markers.

Progressive mechanical and structural changes in anterior cerebral arteries with Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disease and the main cause for dementia. The irreversible neurodegeneration leads to a gradual loss of brain function characterized predominantly by memory loss. Cerebrovascular changes are common neuropathologic findings in aged subjects with dementia. Cerebrovascular integrity is critical for proper metabolism and perfusion of the brain, as cerebrovascular remodeling may render the brain more susceptible to pulse pressure and may be associated with poorer cognitive performance and greater risk of cerebrovascular events. The objective of this study is to provide understanding of cerebrovascular remodeling with AD progression. Anterior cerebral arteries (ACAs) from a total of 19 brain donor participants from controls and pathologically diagnosed AD groups (early-Braak stages I-II; intermediate-Braak stages III-IV; and advanced-Braak stages V-VI) were included in this study. Mechanical testing, histology, advanced optical imaging, and mass spectrometry were performed to study the progressive structural and functional changes of ACAs with AD progression. Biaxial extension-inflation tests showed that ACAs became progressively less compliant, and the longitudinal stress in the intermediate and advanced AD groups was significantly higher than that from the control group. With pathological AD development, the inner and outer diameters of the ACAs remained almost unchanged; however, histology study revealed progressive smooth muscle cell atrophy and loss of elastic fibers which led to compromised structural integrity of the arterial wall. Multiphoton imaging demonstrated elastin degradation at the media-adventitia interface, which led to the formation of an empty band of 21.0 ± 15.4 µm and 32.8 ± 9.24 µm in width for the intermediate and advanced AD groups, respectively. Furthermore, quantitative birefringence microscopy showed disorganized adventitial collagen with AD development. Mass spectrometry analysis provided further evidence of altered collagen content and other extracellular matrix (ECM) molecule and smooth muscle cell changes that were consistent with the mechanical and structural alterations. Collectively, our study provides understanding of the mechanical and structural cerebrovascular deterioration in cerebral arteries with AD, which may be related to neurodegenration and pathology in the brain.

Progressive Mechanical and Structural Changes in Anterior Cerebral Arteries with Alzheimer's Disease.

Alzheimer disease (AD) is a neurodegenerative disease and the main cause for dementia. The irreversible neurodegeneration leads to a gradual loss of brain function characterized predominantly by memory loss. Cerebrovascular changes are common neuropathologic findings in aged subjects with dementia. Cerebrovascular integrity is critical for proper metabolism and perfusion of the brain, as cerebrovascular remodeling may render the brain more susceptible to pulse pressure and may be associated with poorer cognitive performance and greater risk of cerebrovascular events. The objective of this study is to provide understanding of cerebrovascular remodeling with AD progression. A total of 28 brain donor participants with human anterior cerebral artery (ACA) from controls and pathologically diagnosed AD groups (early - Braak stages I-II; intermediate - Braak stages III-IV; and advanced - Braak stages V-VI) were included in this study. Mechanical testing, histology, advanced optical imaging, and mass spectrometry were performed to study the progressive structural and functional changes of ACAs with AD progression. Biaxial extension-inflation tests showed that ACAs became progressively less compliant, and the longitudinal stress in the intermediate& advanced AD groups was significantly higher than that from the control group. With pathological AD development, the inner and outer diameter of ACA remained almost unchanged; however, histology study revealed progressive smooth muscle cell atrophy and loss of elastic fibers which led to compromised structural integrity of the arterial wall. Multiphoton imaging demonstrated elastin degradation at the media-adventitia interface, which led to the formation of an empty band of 21.0 ± 15.4 µm and 32.8 ± 9.24 µm in width for the intermediate& advanced AD groups, respectively. Furthermore, quantitative birefringence microscopy showed disorganized adventitial collagen with AD development. Mass spectrometry analysis provided further evidence of altered collagen content and other extracellular matrix (ECM) molecule and smooth muscle cell changes that were consistent with the mechanical and structural alterations. Collectively, our study provides understanding of the mechanical and structural cerebrovascular deterioration in cerebral arteries with AD, which may be related to neurodegenration and pathology in the brain.

Analysis of complex proteoglycans using serial proteolysis and EThcD provides deep N- and O-glycoproteomic coverage.

Proteoglycans are a small but diverse family of proteins that play a wide variety of roles at the cell surface and in the extracellular matrix. In addition to their glycosaminoglycan (GAG) chains, they are N- and O-glycosylated. All of these types of glycosylation are crucial to their function but present a considerable analytical challenge. We describe the combination of serial proteolysis followed by the application of higher-energy collisional dissociation (HCD) and electron transfer/higher-energy collisional dissociation (EThcD) to optimize protein sequence coverage and glycopeptide identification from proteoglycans. In many cases, the use of HCD alone allows the identification of more glycopeptides. However, the localization of glycoforms on multiply glycosylated peptides has remained elusive. We demonstrate the use of EThcD for the confident assignment of glycan compositions on multiply glycosylated peptides. Dense glycosylation on proteoglycans is key to their biological function; thus, developing tools to identify and quantify doubly glycosylated peptides is of interest. Additionally, glycoproteomics searches identify glycopeptides in otherwise poorly covered regions of proteoglycans. The development of these and other analytical tools may permit glycoproteomic similarity comparisons in biological samples.

RAMZIS: a bioinformatic toolkit for rigorous assessment of the alterations to glycoprotein structure that occur during biological processes.

Motivation: Glycosylation elaborates the structures and functions of glycoproteins; glycoproteins are common post-translationally modified proteins and are heterogeneous and non-deterministically syn-thesized as an evolutionarily driven mechanism that elaborates the functions of glycosylated gene products. While glycoproteins account for approximately half of all proteins, their macro- and micro-heterogeneity requires specialized proteomics data analysis methods as a given glycosite can be divided into several glycosylated forms, each of which must be quantified. Sampling of heterogeneous glycopeptides is limited by mass spectrometer speed and sensitivity, resulting in missing values. In conjunction with the low sample size inherent to glycoproteomics, this necessitated specialized statistical metrics to identify if observed changes in glycopeptide abundances are biologically significant or due to data quality limitations. Results: We developed an R package, Relative Assessment of m/z Identifications by Similarity (RAMZIS), that uses similarity metrics to guide biomedical researchers to a more rigorous interpretation of glycoproteomics data. RAMZIS uses contextual similarity to assess the quality of mass spectral data and generates graphical output that demonstrates the likelihood of finding biologically significant differences in glycosylation abundance dataset. Investigators can assess dataset quality, holistically differentiate glycosites, and identify which glycopeptides are responsible for glycosylation pattern expression change. Herein RAMZIS approach is validated by theoretical cases and by a proof-of-concept application. RAMZIS enables comparison between datasets too stochastic, small, or sparse for interpolation while acknowledging these issues in its assessment. Using our tool, researchers will be able to rigor-ously define the role of glycosylation and the changes that occur during biological processes.

De novo glycan sequencing by electronic excitation dissociation MS2-guided MS3 analysis on an Omnitrap-Orbitrap hybrid instrument.

Comprehensive de novo glycan sequencing remains an elusive goal due to the structural diversity and complexity of glycans. Present strategies employing collision-induced dissociation (CID) and higher energy collisional dissociation (HCD)-based multi-stage tandem mass spectrometry (MSn) or MS/MS combined with sequential exoglycosidase digestions are inherently low-throughput and difficult to automate. Compared to CID and HCD, electron transfer dissociation (ETD) and electron capture dissociation (ECD) each generate more cross-ring cleavages informative about linkage positions, but electronic excitation dissociation (EED) exceeds the information content of all other methods and is also applicable to analysis of singly charged precursors. Although EED can provide extensive glycan structural information in a single stage of MS/MS, its performance has largely been limited to FTICR MS, and thus it has not been widely adopted by the glycoscience research community. Here, the effective performance of EED MS/MS was demonstrated on a hybrid Orbitrap-Omnitrap QE-HF instrument, with high sensitivity, fragmentation efficiency, and analysis speed. In addition, a novel EED MS2-guided MS3 approach was developed for detailed glycan structural analysis. Automated topology reconstruction from MS2 and MS3 spectra could be achieved with a modified GlycoDeNovo software. We showed that the topology and linkage configurations of the Man9GlcNAc2 glycan can be accurately determined from first principles based on one EED MS2 and two CID-EED MS3 analyses, without reliance on biological knowledge, a structure database or a spectral library. The presented approach holds great promise for autonomous, comprehensive and de novo glycan sequencing.

The 2022 Nobel Prize in Chemistry for the development of click chemistry and bioorthogonal chemistry.

The 2022 Nobel Prize in Chemistry recognized the development of biorthogonal chemical ligation reactions known as click chemistry in biomedicine. This concept has catalyzed significant progress in sensing and diagnosis, chemical biology, materials chemistry, and drug discovery and delivery. In proteomics, the ability to incorporate a click tag into proteins has propelled development of powerful new methods for selective enrichment of protein complexes that inform understanding of protein networks. It also has had a strong influence on the ability to enrich for protein post-translational modifications. This feature article summarizes the impacts of biorthogonal click chemistry on proteomics.

Glycoproteomic Analyses of Influenza A Viruses Using timsTOF Pro MS.

N-Linked glycosylation in hemagglutinin and neuraminidase glycoproteins of influenza viruses affects antigenic and receptor binding properties, and precise analyses of site-specific glycoforms in these proteins are critical in understanding the antigenic and immunogenic properties of influenza viruses. In this study, we developed a glycoproteomic approach by using a timsTOF Pro mass spectrometer (MS) to determine the abundance and heterogeneity of site-specific glycosylation for influenza glycoproteins. Compared with a Q Exactive HF MS, the timsTOF Pro MS method without the hydrophilic interaction liquid chromatography column enrichment achieved similar glycopeptide coverage and quantities but was more effective in identifying low-abundance glycopeptides. We quantified the distributions of intact site-specific glycopeptides in hemagglutinin of A/chicken/Wuxi/0405005/2013 (H7N9) and A/mute swan/Rhode Island/A00325125/2008 (H7N3). Results showed that hemagglutinin for both viruses had complex N-glycans at N22, N38, N240, and N483 but only high-mannose glycans at N411 and, however, that the type and quantities of glycans were distinct between these viruses. Collisional cross section (CCS) provided by the ion mobility spectrometry from the timsTOF Pro MS data differentiated sialylation linkages of the glycopeptides. In summary, timsTOF Pro MS method can quantify intact site-specific glycans for influenza glycoproteins without enrichment and thus facilitate influenza vaccine development and production.

Mass spectrometry methods for analysis of extracellular matrix components in neurological diseases.

The brain extracellular matrix (ECM) is a highly glycosylated environment and plays important roles in many processes including cell communication, growth factor binding, and scaffolding. The formation of structures such as perineuronal nets (PNNs) is critical in neuroprotection and neural plasticity, and the formation of molecular networks is dependent in part on glycans. The ECM is also implicated in the neuropathophysiology of disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), and Schizophrenia (SZ). As such, it is of interest to understand both the proteomic and glycomic makeup of healthy and diseased brain ECM. Further, there is a growing need for site-specific glycoproteomic information. Over the past decade, sample preparation, mass spectrometry, and bioinformatic methods have been developed and refined to provide comprehensive information about the glycoproteome. Core ECM molecules including versican, hyaluronan and proteoglycan link proteins, and tenascin are dysregulated in AD, PD, and SZ. Glycomic changes such as differential sialylation, sulfation, and branching are also associated with neurodegeneration. A more thorough understanding of the ECM and its proteomic, glycomic, and glycoproteomic changes in brain diseases may provide pathways to new therapeutic options.

Improving Statistical Certainty of Glycosylation Similarity between Influenza A Virus Variants Using Data-Independent Acquisition Mass Spectrometry.

Amino acid sequences of immunodominant domains of hemagglutinin (HA) on the surface of influenza A virus (IAV) evolve rapidly, producing viral variants. HA mediates receptor recognition, binding and cell entry, and serves as the target for IAV vaccines. Glycosylation, a post-translational modification that places large branched polysaccharide molecules on proteins, can modulate the function of HA and shield antigenic regions allowing for viral evasion from immune responses. Our previous work showed that subtle changes in the HA protein sequence can have a measurable change in glycosylation. Thus, being able to quantitatively measure glycosylation changes in variants is critical for understanding how HA function may change throughout viral evolution. Moreover, understanding quantitatively how the choice of viral expression systems affects glycosylation can help in the process of vaccine design and manufacture. Although IAV vaccines are most commonly expressed in chicken eggs, cell-based vaccines have many advantages, and the adoption of more cell-based vaccines would be an important step in mitigating seasonal influenza and protecting against future pandemics. Here, we have investigated the use of data-independent acquisition (DIA) mass spectrometry for quantitative glycoproteomics. We found that DIA improved the sensitivity of glycopeptide detection for four variants of A/Switzerland/9715293/2013 (H3N2): WT and mutant, each expressed in embryonated chicken eggs and Madin-Darby canine kidney cells. We used the Tanimoto similarity metric to quantify changes in glycosylation between WT and mutant and between egg-expressed and cell-expressed virus. Our DIA site-specific glycosylation similarity comparison of WT and mutant expressed in eggs confirmed our previous analysis while achieving greater depth of coverage. We found that sequence variations and changing viral expression systems affected distinct glycosylation sites of HA. Our methods can be applied to track glycosylation changes in circulating IAV variants to bolster genomic surveillance already being done, for a more complete understanding of IAV evolution.

O-Glycoproteomic analysis of engineered heavily glycosylated fusion proteins using nanoHILIC-MS.

Recombinant protein engineering design affects therapeutic properties including protein efficacy, safety, and immunogenicity. Importantly, glycosylation modulates glycoprotein therapeutic pharmacokinetics, pharmacodynamics, and effector functions. Furthermore, the development of fusion proteins requires in-depth characterization of the protein integrity and its glycosylation to evaluate their critical quality attributes. Fc-fusion proteins can be modified by complex glycosylation on the active peptide, the fragment crystallizable (Fc) domain, and the linker peptides. Moreover, the type of glycosylation and the glycan distribution at a given glycosite depend on the host cell line and the expression system conditions that significantly impact safety and efficacy. Because of the inherent heterogeneity of glycosylation, it is necessary to assign glycan structural detail for glycoprotein quality control. Using conventional reversed-phase LC-MS methods, the different glycoforms at a given glycosite elute over a narrow retention time window, and glycopeptide ionization is suppressed by co-eluting non-modified peptides. To overcome this drawback, we used nanoHILIC-MS to characterize the complex glycosylation of UTI-Fc, a fusion protein that greatly increases the half-life of ulinastatin. By this methodology, we identified and characterized ulinastatin glycopeptides at the Fc domain and linker peptide. The results described herein demonstrate the advantages of nanoHILIC-MS to elucidate glycan features on glycotherapeutics that fail to be detected using traditional reversed-phase glycoproteomics.

Measuring change in glycoprotein structure.

Biosynthetic enzymes in the secretory pathway create distributions of glycans at each glycosite that elaborate the biophysical properties and biological functions of glycoproteins. Because the biosynthetic glycosylation reactions do not go to completion, each protein glycosite is heterogeneous with respect to glycosylation. This heterogeneity means that it is not sufficient to measure protein abundance in omics experiments. Rather, it is necessary to sample the distribution of glycosylation at each glycosite to quantify the changes that occur during biological processes. On the one hand, the use of data-dependent acquisition methods to sample glycopeptides is limited by the instrument duty cycle and the missing value problem. On the other, stepped window data-independent acquisition samples all precursors, but ion abundances are limited by duty cycle. Therefore, the ability to quantify accurately the flux in glycoprotein glycosylation that occurs during biological processes requires the exploitation of emerging mass spectrometry technologies capable of deep, comprehensive sampling and selective high confidence assignment of the complex glycopeptide mixtures. This review summarizes recent technical advances and mass spectral glycoproteomics analysis strategies and how these developments impact our ability to quantify the changes in glycosylation that occur during biological processes. We highlight specific improvements to glycopeptide characterization through activated electron dissociation, ion mobility trends and instrumentation, and efficient algorithmic approaches for glycopeptide assignment. We also discuss the emerging need for unified standards to enable interlaboratory collaborations and effective monitoring of structural changes in glycoproteins.