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1.
Mass Spectrom Rev ; 2021 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-34608657

RESUMO

Glycosaminoglycans (GAGs) are heterogeneous acidic polysaccharides involved in a range of biological functions. They have a significant influence on the regulation of cellular processes and the development of various diseases and infections. To fully understand the functional roles that GAGs play in mammalian systems, including disease processes, it is essential to understand their structural features. Despite having a linear structure and a repetitive disaccharide backbone, their structural analysis is challenging and requires elaborate preparative and analytical techniques. In particular, the extent to which GAGs are sulfated, as well as variation in sulfate position across the entire oligosaccharide or on individual monosaccharides, represents a major obstacle. Here, we summarize the current state-of-the-art methodologies used for GAG sample preparation and analysis, discussing in detail liquid chromatograpy and mass spectrometry-based approaches, including advanced ion activation methods, ion mobility separations and infrared action spectroscopy of mass-selected species.

2.
Anal Bioanal Chem ; 2021 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-34342671

RESUMO

Structural determination of N-glycans by mass spectrometry is ideally performed by negative ion collision-induced dissociation because the spectra are dominated by cross-ring fragments leading to ions that reveal structural details not available by many other methods. Most glycans form [M - H]- or [M + adduct]- ions but larger ones (above approx. m/z 2000) typically form doubly charged ions. Differences have been reported between the fragmentation of singly and doubly charged ions but a detailed comparison does not appear to have been reported. In addition to [M + adduct]- ions (this paper uses phosphate as the adduct) other doubly, triply, and quadruply charged ions of composition [Mn + (H2PO4)n]n- have been observed in mixtures of N-glycans released from viral and other glycoproteins. This paper explores the formation and fragmentation of these different types of multiply charged ions with particular reference to the presence of diagnostic fragments in the CID spectra and comments on how these ions can be used to characterize these glycans.

3.
Phys Chem Chem Phys ; 23(31): 16488-16500, 2021 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-34342317

RESUMO

Protein-protein interactions are involved in the regulation and function of the majority of cellular processes. As a result, much effort has been aimed at the development of methodologies capable of quantifying protein-protein interactions, with label-free methods being of particular interest due to the associated simplified workflows and minimisation of label-induced perturbations. Here, we review recent advances in optical technologies providing label-free in vitro measurements of affinities and kinetics. We provide an overview and comparison of existing techniques and their principles, discussing advantages, limitations, and recent applications.


Assuntos
Proteínas/química , Cinética , Fenômenos Ópticos , Ligação Proteica , Proteínas/metabolismo
4.
Anal Chem ; 93(30): 10435-10443, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34279906

RESUMO

Glycoproteins are inherently heterogeneous and therefore resolving structures in their entirety remains a major challenge in structural biology. Native mass spectrometry has transformed our ability to study glycoproteins, and despite advances in high-resolution instrumentation, there are comparatively a few studies demonstrating its potential with data largely limited to an overall measure of monosaccharide composition for all glycans across glycosylation sites for a given protein. Clearly, these readouts lack glycan topology information, namely, monosaccharide linkage and glycan branching. To address this deficiency, we developed a new approach that joins native mass spectrometry with glycan exoglycosidase sequencing, the combination of which provides remarkable glycoprotein structural details. We show how N-glycan branching, terminal fucosylation, LacNAc extensions, and N- and O-glycan occupancy (i.e., total number of glycans) can be directly characterized on intact glycoproteins with minimal sample preparation. Taken together, native exoglycosidase sequencing mass spectrometry (NES-MS) notably improves our ability to characterize protein glycosylation, addressing a significant need in structural biology that will enable new routes to understand glycoprotein function.


Assuntos
Glicômica , Glicoproteínas , Glicoproteínas/metabolismo , Glicosilação , Espectrometria de Massas , Polissacarídeos
5.
Anal Bioanal Chem ; 2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34327564

RESUMO

Negative ion collision-induced dissociation (CID) of underivatized N-glycans has proved to be a simple, yet powerful method for their structural determination. Recently, we have identified a series of such structures with GalNAc rather than the more common galactose capping the antennae of hybrid and complex glycans. As part of a series of publications describing the negative ion fragmentation of different types of N-glycan, this paper describes their CID spectra and estimated nitrogen cross sections recorded by travelling wave ion mobility mass spectrometry (TWIMS). Most of the glycans were derived from the recombinant glycoproteins gp120 and gp41 from the human immunodeficiency virus (HIV), recombinantly derived from human embryonic kidney (HEK 293T) cells. Twenty-six GalNAc-capped hybrid and complex N-glycans were identified by a combination of TWIMS, negative ion CID, and exoglycosidase digestions. They were present as the neutral glycans and their sulfated and α2→3-linked sialylated analogues. Overall, negative ion fragmentation of glycans generates fingerprints that reveal their structural identity.

6.
ACS Cent Sci ; 7(4): 586-593, 2021 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-34056088

RESUMO

Severe acute respiratory syndrome coronavirus 2 is the causative pathogen of the COVID-19 pandemic which as of March 29, 2021, has claimed 2 776 175 lives worldwide. Vaccine development efforts focus on the viral trimeric spike glycoprotein as the main target of the humoral immune response. Viral spikes carry glycans that facilitate immune evasion by shielding specific protein epitopes from antibody neutralization, and antigen efficacy is influenced by spike glycoprotein production in vivo. Therefore, immunogen integrity is important for glycoprotein-based vaccine candidates. Here, we show how site-specific glycosylation differs between virus-derived spikes, wild-type, non-stabilized spikes expressed from a plasmid with a CMV promoter and tPA signal sequence, and commonly used recombinant, engineered spike glycoproteins. Furthermore, we show that their distinctive cellular secretion pathways result in different protein glycosylation and secretion patterns, including shedding of spike monomeric subunits for the non-stabilized wild-type spike tested, which may have implications for the resulting immune response and vaccine design.

7.
Chem ; 7(1): 224-236, 2021 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-33511302

RESUMO

Integral membrane proteins (IMPs) are biologically highly significant but challenging to study because they require maintaining a cellular lipid-like environment. Here, we explore the application of mass photometry (MP) to IMPs and membrane-mimetic systems at the single-particle level. We apply MP to amphipathic vehicles, such as detergents and amphipols, as well as to lipid and native nanodiscs, characterizing the particle size, sample purity, and heterogeneity. Using methods established for cryogenic electron microscopy, we eliminate detergent background, enabling high-resolution studies of membrane-protein structure and interactions. We find evidence that, when extracted from native membranes using native styrene-maleic acid nanodiscs, the potassium channel KcsA is present as a dimer of tetramers-in contrast to results obtained using detergent purification. Finally, using lipid nanodiscs, we show that MP can help distinguish between functional and non-functional nanodisc assemblies, as well as determine the critical factors for lipid nanodisc formation.

8.
J Biol Chem ; 2020 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-33273015

RESUMO

Myeloperoxidase (MPO) plays essential roles in neutrophil-mediated immunity via the generation of reactive oxidation products. Complex carbohydrates decorate MPO at discrete sites, but their functional relevance remain elusive. To this end, we have characterised the structure-biosynthesis-activity relationship of neutrophil MPO (nMPO). Mass spectrometry demonstrated that nMPO carries both characteristic under-processed and hyper-truncated glycans. Occlusion of the Asn355/Asn391-glycosylation sites and the Asn323-/Asn483-glycans, located in the MPO dimerisation zone, was found to affect the local glycan processing, thereby providing a molecular basis of the site-specific nMPO glycosylation. Native mass spectrometry, mass photometry, and glycopeptide profiling revealed significant molecular complexity of diprotomeric nMPO arising from heterogeneous glycosylation, oxidation, chlorination and polypeptide truncation variants, and a previously unreported low-abundance monoprotomer. Longitudinal profiling of maturing, mature, granule-separated, and pathogen-stimulated neutrophils demonstrated that nMPO is dynamically expressed during granulopoiesis, unevenly distributed across granules and degranulated upon activation. We also show that proMPO-to-MPO maturation occurs during early/mid-stage granulopoiesis. While similar global MPO glycosylation was observed across conditions, the conserved Asn355-/Asn391-sites displayed elevated glycan hyper-truncation, which correlated with higher enzyme activities of MPO in distinct granule populations. Enzymatic trimming of the Asn355-/Asn391-glycans recapitulated the activity gain and showed that nMPO carrying hyper-truncated glycans at these positions exhibits increased thermal stability, polypeptide accessibility, and ceruloplasmin-mediated inhibition potential relative to native nMPO. Finally, molecular modelling revealed that hyper-truncated Asn355-glycans positioned in the MPO-ceruloplasmin interface are critical for uninterrupted inhibition. Here, through an innovative and comprehensive approach, we report novel functional roles of MPO glycans, providing new insight into neutrophil-mediated immunity.

9.
Nucleic Acids Res ; 48(17): e97, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32756898

RESUMO

Mass photometry is a recently developed methodology capable of measuring the mass of individual proteins under solution conditions. Here, we show that this approach is equally applicable to nucleic acids, enabling their facile, rapid and accurate detection and quantification using sub-picomoles of sample. The ability to count individual molecules directly measures relative concentrations in complex mixtures without need for separation. Using a dsDNA ladder, we find a linear relationship between the number of bases per molecule and the associated imaging contrast for up to 1200 bp, enabling us to quantify dsDNA length with up to 2 bp accuracy. These results introduce mass photometry as an accurate, rapid and label-free single molecule method complementary to existing DNA characterization techniques.


Assuntos
DNA/química , Espectrometria de Massas/métodos , Fotometria/métodos , Imagem Individual de Molécula/métodos , DNA/análise
10.
Nat Commun ; 11(1): 1772, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32286308

RESUMO

Sample purity is central to in vitro studies of protein function and regulation, and to the efficiency and success of structural studies using techniques such as x-ray crystallography and cryo-electron microscopy (cryo-EM). Here, we show that mass photometry (MP) can accurately characterize the heterogeneity of a sample using minimal material with high resolution within a matter of minutes. To benchmark our approach, we use negative stain electron microscopy (nsEM), a popular method for EM sample screening. We include typical workflows developed for structure determination that involve multi-step purification of a multi-subunit ubiquitin ligase and chemical cross-linking steps. When assessing the integrity and stability of large molecular complexes such as the proteasome, we detect and quantify assemblies invisible to nsEM. Our results illustrate the unique advantages of MP over current methods for rapid sample characterization, prioritization and workflow optimization.


Assuntos
Microscopia Crioeletrônica/métodos , Espectrometria de Massas/métodos , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Animais , Bovinos , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/ultraestrutura , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica
11.
Nat Commun ; 11(1): 1481, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-32198425

RESUMO

Despite evident regulatory roles of heparan sulfate (HS) saccharides in numerous biological processes, definitive information on the bioactive sequences of these polymers is lacking, with only a handful of natural structures sequenced to date. Here, we develop a "Shotgun" Ion Mobility Mass Spectrometry Sequencing (SIMMS2) method in which intact HS saccharides are dissociated in an ion mobility mass spectrometer and collision cross section values of fragments measured. Matching of data for intact and fragment ions against known values for 36 fully defined HS saccharide structures (from di- to decasaccharides) permits unambiguous sequence determination of validated standards and unknown natural saccharides, notably including variants with 3O-sulfate groups. SIMMS2 analysis of two fibroblast growth factor-inhibiting hexasaccharides identified from a HS oligosaccharide library screen demonstrates that the approach allows elucidation of structure-activity relationships. SIMMS2 thus overcomes the bottleneck for decoding the informational content of functional HS motifs which is crucial for their future biomedical exploitation.


Assuntos
Heparitina Sulfato/química , Íons , Espectrometria de Massas/métodos , Oligossacarídeos/química , Epitopos , Fatores de Crescimento de Fibroblastos/metabolismo , Ácido Glucurônico/química , Heparina , Heparitina Sulfato/metabolismo , Análise de Sequência/métodos , Relação Estrutura-Atividade , Sulfotransferases/metabolismo
12.
Angew Chem Int Ed Engl ; 59(27): 10774-10779, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32167227

RESUMO

Interactions between biomolecules control the processes of life in health and their malfunction in disease, making their characterization and quantification essential. Immobilization- and label-free analytical techniques are desirable because of their simplicity and minimal invasiveness, but they struggle with quantifying tight interactions. Here, we show that mass photometry can accurately count, distinguish by molecular mass, and thereby reveal the relative abundances of different unlabelled biomolecules and their complexes in mixtures at the single-molecule level. These measurements determine binding affinities over four orders of magnitude at equilibrium for both simple and complex stoichiometries within minutes, as well as the associated kinetics. These results introduce mass photometry as a rapid, simple and label-free method for studying sub-micromolar binding affinities, with potential for extension towards a universal approach for characterizing complex biomolecular interactions.


Assuntos
Proteínas/química , Espectrofotometria Ultravioleta/métodos , Cinética
13.
Nat Struct Mol Biol ; 27(1): 78-83, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31907454

RESUMO

The human integral membrane protein SERINC5 potently restricts HIV-1 infectivity and sensitizes the virus to antibody-mediated neutralization. Here, using cryo-EM, we determine the structures of human SERINC5 and its orthologue from Drosophila melanogaster at subnanometer and near-atomic resolution, respectively. The structures reveal a novel fold comprised of ten transmembrane helices organized into two subdomains and bisected by a long diagonal helix. A lipid binding groove and clusters of conserved residues highlight potential functional sites. A structure-based mutagenesis scan identified surface-exposed regions and the interface between the subdomains of SERINC5 as critical for HIV-1-restriction activity. The same regions are also important for viral sensitization to neutralizing antibodies, directly linking the antiviral activity of SERINC5 with remodeling of the HIV-1 envelope glycoprotein.


Assuntos
Infecções por HIV/imunologia , HIV-1/imunologia , Proteínas de Membrana/química , Proteínas de Membrana/imunologia , Animais , Proteínas de Drosophila/química , Proteínas de Drosophila/ultraestrutura , Drosophila melanogaster/química , Humanos , Proteínas de Membrana/ultraestrutura , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Multimerização Proteica
14.
Methods Mol Biol ; 2084: 203-219, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31729663

RESUMO

Glycoconjugates are diverse biomolecules that are dynamically assembled to regulate and fine-tune numerous cellular processes. Their biosynthesis is nontemplate-driven, achieved stepwise in discrete locations within the cell, giving rise to a range of complex branched structures that pose a significant challenge in structural biology. Mass spectrometry is the leading method for analysis of glycoconjugates, and the addition of ion mobility has proven valuable for improving structural assignments of individual glycans in complex biological mixtures. In this chapter, we briefly discuss recent applications of IM for glycomics and describe how to acquire, interpret, and analyze IM-MS data for the analysis of glycans.


Assuntos
Glicoconjugados/química , Glicômica , Espectrometria de Mobilidade Iônica , Espectrometria de Massas , Glicômica/métodos , Polissacarídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
15.
Angew Chem Int Ed Engl ; 59(36): 15560-15564, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-33462887

RESUMO

The immune scavenger protein DC-SIGN interacts with glycosylated proteins and has a putative role in facilitating viral infection. How these recognition events take place with different viruses is not clear and the effects of glycosylation on the folding and stability of DC-SIGN have not been reported. Herein, we report the development and application of a mass-spectrometry-based approach to both uncover and characterise the effects of O-glycans on the stability of DC-SIGN. We first quantify the Core 1 and 2 O-glycan structures on the carbohydrate recognition and extracellular domains of the protein using sequential exoglycosidase sequencing. Using ion mobility mass spectrometry, we show how specific O-glycans, and/or single monosaccharide substitutions, alter both the overall collision cross section and the gas-phase stability of the DC-SIGN isoforms. We find that rather than the mass or length of glycoprotein modifications, the stability of DC-SIGN is better correlated with the number of glycosylation sites.


Assuntos
Moléculas de Adesão Celular/química , Lectinas Tipo C/química , Polissacarídeos/química , Receptores de Superfície Celular/química , Moléculas de Adesão Celular/análise , Glicosilação , Células HEK293 , Humanos , Espectrometria de Mobilidade Iônica/métodos , Lectinas Tipo C/análise , Espectrometria de Massas/métodos , Polissacarídeos/análise , Isoformas de Proteínas/análise , Isoformas de Proteínas/química , Estabilidade Proteica , Receptores de Superfície Celular/análise
16.
Analyst ; 144(17): 5292-5298, 2019 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-31380551

RESUMO

The analysis of complex oligosaccharides is traditionally based on multidimensional workflows where liquid chromatography is coupled to tandem mass spectrometry (LC-MS/MS). Due to the presence of multiple isomers, which cannot be distinguished easily using tandem MS, a detailed structural elucidation is still challenging in many cases. Recently, ion mobility spectrometry (IMS) showed great potential as an additional structural parameter in glycan analysis. While the time-scale of the IMS separation is fully compatible to that of LC-MS-based workflows, there are very few reports in which both techniques have been directly coupled for glycan analysis. As a result, there is little knowledge on how the derivatization with fluorescent labels as common in glycan LC-MS affects the mobility and, as a result, the selectivity of IMS separations. Here, we address this problem by systematically analyzing six isomeric glycans derivatized with the most common fluorescent tags using ion mobility spectrometry. We report >150 collision cross-sections (CCS) acquired in positive and negative ion mode and compare the quality of the separation for each derivatization strategy. Our results show that isomer separation strongly depends on the chosen label, as well as on the type of adduct ion. In some cases, fluorescent labels significantly enhance peak-to-peak resolution which can help to distinguish isomeric species.

17.
Anal Chem ; 91(16): 10604-10613, 2019 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-31298840

RESUMO

Glycosylation is one of the most important post-translational modifications essential for modulating biological functions on cellular surfaces and within cells. Glycan structures are not predictable from the genome since their biosynthesis is nontemplate driven and subject to multiple sequential and competitive glycosyltransferases/glycosidases. From a structural viewpoint, their analysis presents a particular challenge in terms of sensitivity and structural characterization. Porous graphitized carbon liquid chromatography coupled mass spectrometry (PGCLC-MS) is arguably the gold-standard for the structural characterization of glycoconjugates, especially complex mixtures typical in biological samples. This high performance is due in large part to chromatographic separation of isomers and the information delivered by collision induced fragmentation of each glycan in the mass spectrometer. More recently, ion mobility mass spectrometry (IM-MS) has emerged as an effective tool for gas-phase separation of isomeric oligosaccharides that has been demonstrated with small oligosaccharides and N-glycans. Here, we present a direct comparison of the IM- and LC-separation of O-glycans from porcine gastric and human salivary mucins. Our results identify structures, which are resolved by PGCLC and/or IM, validating the combination of the two methods. Taken together, the incorporation of both techniques into a single platform would be powerful and undoubtedly valuable for determining the full glycome of unknown samples.


Assuntos
Mucinas Gástricas/química , Mucosa Bucal/química , Polissacarídeos/isolamento & purificação , Animais , Cromatografia Líquida , Humanos , Espectrometria de Mobilidade Iônica , Espectrometria de Massas , Tamanho da Partícula , Polissacarídeos/química , Porosidade , Propriedades de Superfície , Suínos
18.
Curr Opin Struct Biol ; 58: 241-248, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31326232

RESUMO

Glycosylation is the most complex and prevalent protein modification that influences attributes ranging from cellular localization and signaling to half-life and proteolysis. Glycoconjugates are fundamental for cellular function and alterations in their structure are often observed in pathological states. Most biotherapeutic proteins are glycosylated, which influences drug safety and efficacy. Therefore, the ability to characterize glycoproteins is important in all areas of biomolecular and medicinal research. Here we discuss recent advances in native mass spectrometry that have significantly improved our ability to characterize heterogeneous glycoproteins and to relate glycan structure to protein function.


Assuntos
Glicoproteínas/química , Glicoproteínas/metabolismo , Espectrometria de Massas/métodos , Polissacarídeos/metabolismo
19.
Nat Commun ; 10(1): 3275, 2019 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-31332201

RESUMO

The mass spectrometry (MS)-based analysis of free polysaccharides and glycans released from proteins, lipids and proteoglycans increasingly relies on databases and software. Here, we review progress in the bioinformatics analysis of protein-released N- and O-linked glycans (N- and O-glycomics) and propose an e-infrastructure to overcome current deficits in data and experimental transparency. This workflow enables the standardized submission of MS-based glycomics information into the public repository UniCarb-DR. It implements the MIRAGE (Minimum Requirement for A Glycomics Experiment) reporting guidelines, storage of unprocessed MS data in the GlycoPOST repository and glycan structure registration using the GlyTouCan registry, thereby supporting the development and extension of a glycan structure knowledgebase.


Assuntos
Biologia Computacional/métodos , Glicômica/métodos , Glicoproteínas/metabolismo , Polissacarídeos/metabolismo , Animais , Biologia Computacional/normas , Bases de Dados Factuais/normas , Bases de Dados Factuais/estatística & dados numéricos , Humanos , Espectrometria de Massas/métodos , Padrões de Referência
20.
Chem Sci ; 10(19): 5146-5155, 2019 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-31183067

RESUMO

Lectins are carbohydrate binding proteins that recognize specific epitopes present on target glycoproteins. Changes in lectin-reactive carbohydrate repertoires are related to many biological signaling pathways and recognized as hallmarks of several pathological processes. Consequently, lectins are valuable probes, commonly used for examining glycoprotein structural and functional microheterogeneity. However, the molecular interactions between a given lectin and its preferred glycoproteoforms are largely unknown due to the inherent complexity and limitations of methods used to investigate intact glycoproteins. Here, we apply a lectin-affinity purification procedure coupled with native mass spectrometry to characterize lectin-reactive glycoproteoforms at the intact protein level. We investigate the interactions between the highly fucosylated and highly branched glycoproteoforms of haptoglobin and α1-acid glycoprotein using two different lectins Aleuria aurantia lectin (AAL) and Phaseolus vulgaris leucoagglutinin (PHA-L), respectively. Firstly we show a co-occurrence of fucosylation and N-glycan branching on haptoglobin, particularly among highly fucosylated glycoproteoforms. Secondly, we analyze the global heterogeneity of highly branched glycoproteoforms of haptoglobin and α1-acid glycoprotein and reveal that while multi-fucosylation attenuates the lectin PHA-L binding to haptoglobin, it has no impact on AGP. Taken together, our lectin affinity purification native MS approach elucidates lectin specificities between intact glycoproteins, not achievable by other methods. Moreover, since aberrant glycosylation of Hp and AGP are potential markers for many diseases, including pancreatic, hepatic and ovarian cancers, understanding their interactions with lectins will help the development of carbohydrate-centric monitoring methods to understand their pathophysiological implications.

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