Joseph Zaia

• Boston University

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 un...

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 pri...

Accurate glycopeptide identification in mass spectrometry-based glycoproteomics is a challenging problem at scale. Recent innovation has been made in increasing the scope and accuracy of glycopeptide identifications, with more precise uncertainty estimates for each part of the structure. We present a dynamically adapting relative retention time mod...

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 s...

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 a...

Ion mobility-mass spectrometry (IM-MS) is a powerful analytical tool for structural characterization. IM measurement provides collision cross section (CCS) values that facilitate analyte identification. While CCS values can be directly calculated from mobility measurements obtained using drift tube ion mobility spectrometry (DT-IMS), this method ha...

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...

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...

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 des...

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 m...

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...

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. While glycoproteins account for approximatel...

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 digestion are...

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 a...

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 digestion are...

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...

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 mole...

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 inte...

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 depe...

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...

The Minimum Information Required for a Glycomics Experiment (MIRAGE) is an initiative to standardize the reporting of glycoanalytical methods and to assess their reproducibility. To date, the MIRAGE Commission has published several reporting guidelines that describe what information should be provided for sample preparation methods, mass spectromet...

Glioblastoma (GBM) is the most common and malignant primary brain tumor. The extracellular matrix (ECM), also known as the matrisome, helps determine glioma invasion, adhesion, and growth. Little attention, however, has been paid to glycosylation of the ECM components that constitute the majority of glycosylated protein mass and presumed biological...

Extracellular matrix (ECM) proteins, collectively known as the matrisome, include collagens, glycoproteins, and proteoglycans. Alterations in the matrisome have been implicated in the neurodegenerative pathologies including Parkinson's disease (PD). In this work, we utilized our previously published PD and control proteomics data from human prefron...

Glycoproteomics is a powerful yet analytically challenging research tool. Software packages aiding the interpretation of complex glycopeptide tandem mass spectra have appeared, but their relative performance remains untested. Conducted through the HUPO Human Glycoproteomics Initiative, this community study, comprising both developers and users of g...

The spike protein of SARS-CoV-2, the virus responsible for the global pandemic of COVID-19, is an abundant, heavily glycosylated surface protein that plays a key role in receptor binding and host cell fusion, and is the focus of all current vaccine development efforts. Variants of concern are now circulating worldwide that exhibit mutations in the...

Protein glycosylation that mediates interactions among viral proteins, host receptors, and immune molecules is an important consideration for predicting viral antigenicity. Viral spike proteins, the proteins responsible for host cell invasion, are especially important to be examined. However, there is a lack of consensus within the field of glycopr...

The glycosaminoglycan, heparan sulphate (HS), orchestrates many developmental processes. Yet its biological role has not yet fully been elucidated. Small molecule chemical inhibitors can be used to perturb HS function and these compounds provide cheap alternatives to genetic manipulation methods. However, existing chemical inhibition methods for HS...

The glycosaminoglycan, heparan sulphate (HS), orchestrates many developmental processes. Yet its biological role has not yet fully been elucidated. Small molecule chemical inhibitors can be used to perturb HS function and these compounds pro-vide cheap alternatives to genetic manipulation methods. However, existing chemical inhibition methods for H...

The sulfated glycosaminoglycans (GAGs) are long, linear polysaccharide chains that are typically found as the glycan portion of proteoglycans. These GAGs are characterized by repeating disaccharide units with variable sulfation and acetylation patterns along the chain. GAG length and modification patterns have profound impacts on growth factor sign...

A library of 23 synthetic heparan sulfate (HS) oligosaccharides, varying in chain length, types and positions of modifications, was used to analyze the substrate specificities of heparin lyase III enzymes from both Flavobacterium heparinum and Bacteroides eggerthii. The influence of specific modifications, including N-substitution, 2-O sulfation, 6...

Advances in mass spectrometry instrumentation, methods development, and bioinformatics have greatly improved the ease and accuracy of site‐specific, quantitative glycoproteomics analysis. Data‐dependent acquisition is the most popular method for identification and quantification of glycopeptides; however, complete coverage of glycosylation site gly...

Glycoproteome profiling (glycoproteomics) remains a considerable analytical challenge that hinders rapid progress in glycobiology. The complex tandem mass spectra generated from glycopeptide mixtures require sophisticated analysis pipelines for structural determination. Diverse informatics solutions aiding the process have appeared, but their relat...

Identification of N ‐ and O ‐glycosylation on specific sites of proteins, along with glycan structural information, is necessary to determine the roles glycoproteins play in normal and pathologic cellular functions. Because such glycosylation is macro‐ and micro‐heterogeneous and alters the dissociation behavior of glycopeptides, specific sample pr...

Significance Although heparan sulfate (HS) modified by a 3- O -sulfate (3-OS) has been implicated in many biological processes, there is only a rudimentary understanding of ligand requirements of HS-binding proteins requiring this type of modification. We developed a modular synthetic approach that provided a structurally diverse collection of HS o...

Sulfation of heparan sulfate proteoglycans (HSPG) regulates signaling of growth factor receptors via specific interactions with the sulfate groups. 6-O-Sulfation of HSPG is an impactful modification regulated by the activities of dedicated extracellular endosulfatases. Specifically, extracellular sulfatase Sulf-2 (SULF2) removes 6-O-sulfate from HS...

The ECM is a complex molecular network that surrounds all cells and consists of proteins, glycoproteins, hyaluronan, glycosaminoglycans (GAGs), and proteoglycans (PGs). GAGs and PGs play vital roles in ECM-related processes such as cell migration, proliferation, adhesion, and differentiation. Among the omics technologies, including genomics, transc...

Wild-type transthyretin-associated (ATTRwt) amyloidosis is an age-related disease that causes heart failure in older adults. This disease frequently features cardiac amyloid fibril deposits that originate from dissociation of the tetrameric protein, transthyretin (TTR). Unlike hereditary TTR (ATTRm) amyloidosis, where amino acid replacements destab...

Complex protein glycosylation occurs through biosynthetic steps in the secretory pathway that create macro- and microheterogeneity of structure and function. Required for all life forms, glycosylation diversifies and adapts protein interactions with binding partners that underpin interactions at cell surfaces and pericellular and extracellular envi...

Previous studies on Parkinson’s disease mechanisms have shown dysregulated extracellular transport of α-synuclein and growth factors in the extracellular space. In the human brain these consist of perineuronal nets, interstitial matrices, and basement membranes, each composed of a set of collagens, non-collagenous glycoproteins, proteoglycans, and...

Influenza A virus (IAV) mutates rapidly, resulting in antigenic drift and poor year-to-year vaccine effectiveness. One challenge in designing effective vaccines is that genetic mutations frequently cause amino acid variations in IAV envelope protein hemagglutinin (HA) that create new N-glycosylation sequons; resulting N-glycans cause antigenic shie...

Widely-available LC-MS instruments and methods allow users to acquire glycoproteomics data. Complex glycans, however, add a dimension of complexity to the data analysis workflow. In a sense, complex glycans are post-translationally modified post-translational modifications, reflecting a series of biosynthetic reactions in the secretory pathway that...

The GlycReSoft software tool allows users to process glycoproteomics LC-MS data sets. The tool accepts proteomics database search results or a user-defined list of proteins in the sample. GlycReSoft processes LC-MS data to yield deconvoluted exact mass values. The user has the option to import a list of theoretical glycans from an external database...

Enteroviruses support cell-to-cell viral transmission prior to their canonical lytic spread of virus. Poliovirus (PV), a prototype for human pathogenic positive-sense RNA enteroviruses, and picornaviruses in general, transport multiple virions en bloc via infectious extracellular vesicles, 100~1000 nm in diameter, secreted from host cells. Using bi...

Background: In addition to causing the pandemic influenza outbreaks of 1918 and 2009, subtype H1N1 influenza A viruses (IAVs) have caused seasonal epidemics since 1977. Antigenic property of influenza viruses are determined by both protein sequence and N-linked glycosylation of influenza glycoproteins, especially hemagglutinin (HA). The currently...

Introduction: Extracellular vesicles (EVs) from human Alzheimer's disease (AD) biospecimens contain amyloid beta (Aβ) peptide and tau. While AD EVs are known to affect brain disease pathobiology, their biochemical and molecular characterizations remain ill defined. Methods: EVs were isolated from the cortical gray matter of 20 AD and 18 control...

Advancement in mass spectrometry has revolutionized the field of proteomics. However, there remains a gap in the analysis of protein post-translational modifications (PTMs), particularly for glycosylation. Glycosylation, the most common form of PTM, is involved in most biological processes; thus, analysis of glycans along with proteins is crucial t...

Introduction Glycans are the most abundant biopolymers found in nature; they play crucial roles in a wide range of biological activities. Glycan biosynthesis does not follow genetic blueprints, and the resultant glycome is often a mixture of glycoforms with varying compositions, branching patterns, linkages, and stereochemical configurations. Subtl...

Glycopeptides identified by tandem mass spectrometry rely on identification of the peptide backbone sequence and attached glycan(s) by incomplete fragmentation of both moieties. This may lead to ambiguous identifications where multiple structures could explain the same spectrum equally well, due to missing information in the mass spectrum or incorr...

Influenza A virus (IAV) mutates rapidly, resulting in antigenic drift and poor year-to-year vaccine effectiveness. One challenge in designing effective vaccines is that genetic mutations frequently cause amino acid variations in IAV envelope protein hemagglutinin (HA) that create new N -glycosylation sequons; resulting N -glycans cause antigenic sh...

Protein glycosylation, resulting from glycosyl transferase reactions under complex control in the secretory pathway, consists of a distribution of related glycoforms at each glycosylation site. Because the biosynthetic substrate concentration and transport rates depend on architecture and other aspects of cellular phenotypes, site specific glycosyl...

Despite the recent advances in mass spectrometry (MS)-based methods for glycan structural analysis, characterization of glycomes remains a significant analytical challenge, in part due to the widespread presence of isomeric structures and the need to define the many structural variables for each glycan. Interpretation of the complex tandem mass spe...

Low vaccine efficacy against seasonal influenza A virus (IAV) stems from the ability of the virus to evade existing immunity while maintaining fitness. While most potent neutralizing antibodies bind antigenic sites on the globular head domain of the IAV envelope glycoprotein hemagglutinin (HA), the error-prone IAV polymerase enables rapid evolution...

Glycosylation is a topic of intense current interest in the development of biopharmaceuticals since it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy‑six laboratories from industry, universit...

In the Atlantic salmon (Salmo salar) aquaculture industry, gaping (the separation of muscle bundles from the connective tissue) is a major quality problem. This study characterized chondroitin sulfate (CS) and heparan sulfate (HS) in the connective tissue of intact and gaping salmon fillets from 30 salmon by mass spectrometry. Statistical differenc...

We describe a protocol for glycomic and proteomic profiling that uses serial enzyme digestions from the surface of fresh frozen or fixed tissue slides. The abundances of the extracted glycans and peptides are determined using liquid chromatography-mass spectrometry. In a typical experiment, our method quantifies 14 heparan sulfate disaccharides, 11...

The most straightforward applications of proteomics database searching involve intracellular proteins. While intracellular gene products number in the thousands, their relatively well-defined post-translational modifications (PTMs) makes database searching practical. By contrast, cell surface and extracellular matrisome proteins pass through the se...

Heparan sulfate (HS) mediates a wide range of protein binding interactions key to normal and pathological physiology. Though liquid chromatography coupled with mass spectrometry (LC-MS) based disaccharide composition analysis is able to profile changes in HS composition, the heterogeneity of modifications and the labile sulfate group present major...

Glycoinformatics is a critical resource for the study of glycobiology, and glycobiology is a necessary component for understanding the complex interface between intra- and extra-cellular spaces. Despite this, there is limited software available to scientists studying these topics, requiring each to create fundamental data structures and representat...

Glycosaminoglycans (GAGs) play vital roles in many biological processes, and are naturally present as complex mixtures of poly-saccharides with tremendous structural heterogeneity, including many structural isomers. Mass spectrometric analysis of GAG isomers, in particular highly sulfated heparin (Hep) and heparan sulfate (HS), is challenging becau...

mzML and mzIdentML are commonly used, powerful tools for representing mass spectrometry data and derived identification information. These formats are complex, requiring non-trivial logic to translate data into the appropriate representation. Most published implementations are tightly coupled to data structures. The most complete implementations ar...

Parkinson's disease (PD) is a neurological disorder characterized by the progressive loss of functional dopaminergic neurons in the nigrostriatal pathway in the brain. While current treatments provide only symptomatic relief, gene therapy has the potential to slow or halt the degeneration of nigrostriatal dopamine neurons in PD patients. Adeno-asso...

Motivation: Glycosylation is one of the most heterogeneous and complex protein post-translational modifications. Liquid chromatography coupled mass spectrometry (LC-MS) is a common high throughput method for analyzing complex biological samples. Accurate study of glycans require high resolution mass spectrometry. Mass spectrometry data contains in...

Proteoglycans are distributed in all animal tissues and play critical, multifaceted, physiological roles. Expressed in a spatially and temporally regulated manner, these molecules regulate interactions among growth factors and cell surface receptors and play key roles in basement membranes and other extracellular matrices. Because of the high degre...

Innate immunity is critical in the early containment of influenza A virus (IAV) infection, and surfactant protein D (SP-D) plays a crucial role in the pulmonary defense against IAV. In pigs, which are important intermediate hosts during the generation of pandemic IAVs, SP-D uses its unique carbohydrate recognition domain (CRD) to interact with IAV....

Analysis of singly glycosylated peptides has evolved to a point where large-scale LC-MS analyses can be performed at almost the same scale as proteomics experiments. While collisionally activated dissociation (CAD) remains the mainstay of bottom-up analyses, it performs poorly for the middle-down analysis of multiply glycosylated peptides. With imp...

Glycosaminoglycans (GAGs) covalently linked to proteoglycans (PGs) are characterized by repeating disaccharide units and variable sulfation patterns along the chain. GAG length and sulfation patterns impact disease etiology, cellular signaling, and structural support for cells. We and others have demonstrated the usefulness of tandem mass spectrome...

Among dissociation methods, negative electron transfer dissociation (NETD) has been proven the most useful for glycosaminoglycan (GAG) sequencing because it produces informative fragmentation, a low degree of sulfate losses, high sensitivity, and translatability to multiple instrument types. The challenge, however, is to distinguish positional sulf...

A simple, isocratic HPLC method based on HILIC-WAX separation, has been developed for analyzing sulfated disaccharides of glycosaminoglycans (GAGs). To our best knowledge, this is the first successful attempt using this special phase in nano-HPLC-MS analysis. Mass spectrometry was based on negative ionization, improving both sensitivity and specifi...

Heparan sulfate (HS) is a polysaccharide fundamentally important for biologically activities. T/Tn antigens are universal carbohydrate cancer markers. Here, we report the specific imaging of these carbohydrates using a mesenchymal stem cell line and human umbilical vein endothelial cells (HUVEC). The staining specificities were demonstrated by comp...

Glycomics and glycoproteomics analyses by mass spectrometry require efficient front-end separation methods to enable deep characterization of heterogeneous glycoform populations. Chromatography methods are generally limited in their ability to resolve glycoforms using mobile phases that are compatible with online liquid chromatography-mass spectrom...

An ion mobility quadrupole time-of-flight mass spectrometer was used to examine the gas-phase structures of a set of glycopeptides resulting from proteolytic digestion of the well-characterized glycoproteins bovine ribonuclease B, human transferrin, bovine fetuin and human α1-acid glycoprotein, the corresponding deglycosylated peptides and the glyc...

Heparan sulfate (HS) is a linear polysaccharide found in the extracellular matrix (ECM) and on the cell membrane. It plays numerous roles in cellular events, including cell growth, migration and differentiation through binding to various growth factors, cytokines and other ECM proteins. Heparanase (HPSE) is an endoglycosidase that cleaves HS in the...

MIRAGE (Minimum Information Required for A Glycomics Experiment) is an initiative that was created by experts in the fields of glycobiology, glycoanalytics and glycoinformatics to produce guidelines for reporting results from the diverse types of experiments and analyses used in structural and functional studies of glycans in the scientific literat...

Cell surface heparan sulfates modulate many signaling pathways by binding growth factors and growth factor receptors. Expressed in a spatially and temporally regulated manner, these highly sulfated polysaccharides play important roles in all aspects of animal physiology. To understand heparan sulfate-protein binding, it is necessary to develop inst...

Low molecular weight heparins (LMWH) prepared by partial depolymerization of unfractionated heparin are used globally to treat coagulation disorders on an outpatient basis. Patent protection for several LMWH has expired and abbreviated new drug applications have been approved by the Food and Drug Administration. As a result, reverse engineering of...

Proteoglycans are proteins that carry sulfated glycosaminoglycans (GAGs). They help form and maintain morphogen gradients, guiding cell migration and differentiation during animal development. While no sulfated GAGs have been found in marine sponges, chondroitin sulfate (CS) and heparan sulfate (HS) have been identified in Cnidarians, Lophotrocozoa...

In order to interpret glycopeptide tandem mass spectra, it is necessary to estimate the theoretical glycan compositions and peptide sequences, known as the search space. The simplest way to do this is to build a naïve search space from sets of glycan compositions from public databases and to assume that the target glycoprotein is pure. Often, howev...

The minimum information required for a glycomics experiment (MIRAGE) project was established in 2011 to provide guidelines to aid in data reporting from all types of experiments in glycomics research including mass spectrometry (MS), liquid chromatography, glycan arrays, data handling and sample preparation. MIRAGE is a concerted effort of the wide...

Despite the publication of several software tools for analysis of glycopeptide tandem mass spectra, there remains a lack of consensus regarding the most effective and appropriate methods. In part, this reflects problems with applying standard methods for proteomics database searching and false discovery rate calculation. While the analysis of small...

Despite sustained biomedical research effort, influenza A virus remains an imminent threat to the world population and a major healthcare burden. The challenge in developing vaccines against influenza is the ability of the virus to mutate rapidly in response to selective immune pressure. Hemagglutinin is the predominant surface glycoprotein and the...

Glycoproteomics involves the study of glycosylation events on protein sequences ranging from purified proteins to whole proteome scales. Understanding these complex post-translational modification (PTM) events requires elucidation of the glycan moieties (monosaccharide sequences and glycosidic linkages between residues), protein sequences, as well...

Distinct from template-directed biosynthesis of nucleic acids and proteins, the enzymatic synthesis of heterogeneous polysaccharides is a complex process that is difficult to study using common analytical tools. Therefore, the mode of action and processivity of those enzymes are largely unknown. Dermatan sulfate epimerase 1 (DS-epi1) is the predomi...

Glycans are known as the third major class of biopolymers, next to DNA and proteins. They cover the surfaces of many cells, serving as the ‘face’ of cells, whereby other biomolecules and viruses interact. The structure of glycans, however, differs greatly from DNA and proteins in that they are branched, as opposed to linear sequences of amino acids...

An library implementing data structures describing sugar biomolecules. Provides several tools for manipulating structures and compositions, including in-silico derivatization, theoretical fragment generation, mass calculations, motif finding, topological analysis, and more. It can understand popular text formats such as GlycoCT, IUPAC 3-Letter Code...