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Solid organ transplant recipients with immunosuppressant regimens to prevent rejection are less able to mount effective immune responses to pathogenic infection. Here, we apply a recently reported mass spectrometry-based serological approach known as Ig-MS to characterize immune responses against infection with SARS-CoV-2 in cohorts of transplant recipients and immunocompetent controls, both at a single early time point following COVID-19 diagnosis as well as over the course of one-month postdiagnosis. We found that the antibody repertoires generated by transplant recipients against SARS-CoV-2 do not differ significantly compared to immunocompetent individuals with regard to repertoire titer, clonality, or glycan composition. Importantly, our study is the first to characterize the evolution of antibody glycan profiles in transplant recipients with COVID-19 disease, presenting evidence that the evolution of glycan composition in these immunocompromised individuals is similar to that in immunocompetent people.
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Proteoform-resolved information, obtained by top-down (TD) "intact protein" proteomics, is expected to contribute substantially to the understanding of molecular pathogenic mechanisms and, in turn, identify novel therapeutic and diagnostic targets. However, the robustness of mass spectrometry (MS) analysis of intact proteins in complex biological samples is hindered by the high dynamic range in protein concentration and mass, protein instability, and buffer complexity. Here, we describe an evolutionary step for intact protein investigations through the online implementation of tandem microflow size-exclusion chromatography with nanoflow reversed-phase liquid chromatography and MS (µSEC2-nRPLC-MS). Online serial high-/low-pass SEC filtration overcomes the aforementioned hurdles to intact proteomic analysis through automated sample desalting/cleanup and enrichment of target mass ranges (5-155 kDa) prior to nRPLC-MS. The coupling of µSEC to nRPLC is achieved through a novel injection volume control (IVC) strategy of inserting protein trap columns, pre- and post-µSEC columns, to enable injection of dilute samples in high volumes without loss of sensitivity or resolution. Critical characteristics of the approach are tested via rigorous investigations on samples of varied complexity and chemical background. Application of the platform to cerebrospinal fluid (CSF) prefractionated by OFFGEL isoelectric focusing drastically increases the number of intact mass tags (IMTs) detected within the target mass range (5-30 kDa) in comparison to one-dimensional nRPLC-MS with approximately 100× less CSF than previous OFFGEL studies. Furthermore, the modular design of the µSEC2-nRPLC-MS platform is robust and promises significant flexibility for large-scale TDMS analysis of diverse samples either directly or in concert with other multidimensional fractionation steps.
Assuntos
Proteínas do Líquido Cefalorraquidiano , Proteômica , Animais , Focalização Isoelétrica , Espectrometria de Massas , PrimatasRESUMO
Glycomics represents one of the last frontiers and most challenging in omic analysis. Glycosylation occurs in the endoplasmic reticulum and the Golgi organelle and its control is neither well-understood nor predictable based on proteomic or genomic analysis. One of the most structurally complex classes of glycoconjugates is the proteoglycans (PGs) and their glycosaminoglycan (GAG) side chains. Previously, our laboratory solved the structure of the chondroitin sulfate chain of the bikunin PG. The current study examines the much more complex structure of the dermatan sulfate GAG chain of decorin PG. By utilizing sophisticated separation methods followed by compositional analysis, domain mapping, and tandem mass spectrometry coupled with analysis by a modified genetic algorithm approach, the structural motif for the decorin dermatan sulfate chain was determined. This represents the second example of a GAG with a prominent structural motif, suggesting that the structural variability of this class of glycoconjugates is somewhat simpler than had been expected.
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Decorina/química , Dermatan Sulfato/química , Algoritmos , Animais , Decorina/isolamento & purificação , Dermatan Sulfato/isolamento & purificação , SuínosRESUMO
Interactions between chemokines such as CCL5 and glycosaminoglycans (GAGs) are essential for creating haptotactic gradients to guide the migration of leukocytes into inflammatory sites, and the GAGs that interact with CCL5 with the highest affinity are heparan sulfates/heparin. The interaction between CCL5 and its receptor on monocytes, CCR1, is mediated through residues Arg-17 and -47 in CCL5, which overlap with the GAG-binding (44)RKNR(47) "BBXB" motifs. Here we report that heparin and tetrasaccharide fragments of heparin are able to inhibit CCL5-CCR1 binding, with IC50 values showing strong dependence on the pattern and extent of sulfation. Modeling of the CCL5-tetrasaccharide complexes suggested that interactions between specific sulfate and carboxylate groups of heparin and residues Arg-17 and -47 of the protein are essential for strong inhibition; tetrasaccharides lacking the specific sulfation pattern were found to preferentially bind CCL5 in positions less favorable for inhibition of the interaction with CCR1. Simulations of a 12-mer heparin fragment bound to CCL5 indicated that the oligosaccharide preferred to interact simultaneously with both (44)RKNR(47) motifs in the CCL5 homodimer and engaged residues Arg-47 and -17 from both chains. Direct engagement of these residues by the longer heparin oligosaccharide provides a rationalization for its effectiveness as an inhibitor of CCL5-CCR1 interaction. In this mode, histidine (His-23) may contribute to CCL5-GAG interactions when the pH drops just below neutral, as occurs during inflammation. Additionally, an examination of the contribution of pH to modulating CCL5-heparin interactions suggested a need for careful interpretation of experimental results when experiments are performed under non-physiological conditions.
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Quimiocina CCL5/química , Heparina/química , Oligossacarídeos/química , Motivos de Aminoácidos , Quimiocina CCL5/genética , Quimiocina CCL5/metabolismo , Heparina/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Oligossacarídeos/metabolismo , Ligação Proteica , Receptores CCR1/química , Receptores CCR1/genética , Receptores CCR1/metabolismoRESUMO
Proton-transfer reaction mass spectrometry (PTR-MS) allows for real-time, on-line determination of absolute concentrations of volatile organic compounds (VOCs) with a high sensitivity and low detection limits (in the pptv range). The technique utilizes H3O⺠ions for proton-transfer reactions with many common VOCs while having little to no reaction with any constituents commonly present in air. Over the past decades, research has greatly improved the applications and instrumental design of PTR-MS. In this article, we give an overview of the development of PTR-MS in recent years and its application in medical research. The theory of PTR-MS and various methods for discriminating isobaric VOCs are also described. We also show several specialized designs of sample inlet system, some of those may make PTR-MS suitable for the detection of aqueous solution and/or non-volatile samples.
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Compostos Orgânicos Voláteis/análise , Animais , Pesquisa Biomédica/métodos , Testes Respiratórios , Humanos , Espectrometria de Massas/instrumentação , Oniocompostos/química , Compostos Orgânicos Voláteis/química , Compostos Orgânicos Voláteis/metabolismoRESUMO
A recent study of substituted face-to-face benzene dimers by Lewis and co-workers [J. Am. Chem. Soc. 2011, 133, 3854-3862] indicated a surprising enhancement of electrostatic interactions for both electron-withdrawing and electron-donating substituents. Here we demonstrate that charge penetration (an attractive electrostatic interaction arising from the overlap of the electron densities on the two monomers) is the cause of this counterintuitive effect. These charge penetration effects are significant at typical π-π interaction distances, and they are not easily described by multipole models. A simple measure of a substituent's electron-donating or electron-withdrawing character, such as the Hammett parameter σ(m), is unlikely to capture subtle charge penetration effects. Indeed, correlation of the relative total energies or relative electrostatic energies with ∑σ(m) breaks down for multiply substituted face-to-face benzene dimers.
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Benzeno/química , Elétrons , Dimerização , Modelos Moleculares , Eletricidade EstáticaRESUMO
The role of glycosaminoglycans (GAGs) in major biological functions is numerous and diverse, yet structural characterization of them by mass spectrometric techniques proves to be challenging. Characterization of GAG structure from tandem mass spectrometry is a tedious and time-consuming process but one that can be automated in a database-independent, high-throughput fashion through the assistance of software implementing a genetic algorithm (J. Am. Soc. Mass Spectrom. 29, 1802-1911, 2018). This work presents the manner in which this data is interpreted by the software, specifically addressing the development of a scoring algorithm. The significance of glycosidic and cross-ring fragment ions and the implications that specific fragments provide for assigning the positions of modifications are discussed. The scoring algorithm is tested for statistical merit using the widely accepted expectation value as the criterion for quality. Using MS/MS data for well-characterized standards, this scoring approach is shown to assign the correct structure, with a low likelihood (1 in 1012 chances) that the assigned structure matches the data due to random chance. The integrated software that automates the structure assignment is called Glycosaminoglycan-Unambiguous Identification Technology (G-UNIT).
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Algoritmos , Glicosaminoglicanos/química , Espectrometria de Massas em Tandem/métodos , Análise de Fourier , SoftwareRESUMO
The biological interactions between glycosaminoglycans (GAGs) and other biomolecules are heavily influenced by structural features of the glycan. The structure of GAGs can be assigned using tandem mass spectrometry (MS2), but analysis of these data, to date, requires manually interpretation, a slow process that presents a bottleneck to the broader deployment of this approach to solving biologically relevant problems. Automated interpretation remains a challenge, as GAG biosynthesis is not template-driven, and therefore, one cannot predict structures from genomic data, as is done with proteins. The lack of a structure database, a consequence of the non-template biosynthesis, requires a de novo approach to interpretation of the mass spectral data. We propose a model for rapid, high-throughput GAG analysis by using an approach in which candidate structures are scored for the likelihood that they would produce the features observed in the mass spectrum. To make this approach tractable, a genetic algorithm is used to greatly reduce the search-space of isomeric structures that are considered. The time required for analysis is significantly reduced compared to an approach in which every possible isomer is considered and scored. The model is coded in a software package using the MATLAB environment. This approach was tested on tandem mass spectrometry data for long-chain, moderately sulfated chondroitin sulfate oligomers that were derived from the proteoglycan bikunin. The bikunin data was previously interpreted manually. Our approach examines glycosidic fragments to localize SO3 modifications to specific residues and yields the same structures reported in literature, only much more quickly. Graphical Abstract á .