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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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Numerous Aß proteoforms, identified in the human brain, possess differential neurotoxic and aggregation propensities. These proteoforms contribute in unknown ways to the conformations and resultant pathogenicity of oligomers, protofibrils, and fibrils in Alzheimer's disease (AD) manifestation owing to the lack of molecular-level specificity to the exact chemical composition of underlying protein products with widespread interrogating techniques, like immunoassays. We evaluated Aß proteoform flux using quantitative top-down mass spectrometry (TDMS) in a well-studied 5xFAD mouse model of age-dependent Aß-amyloidosis. Though the brain-derived Aß proteoform landscape is largely occupied by Aß1-42, 25 different forms of Aß with differential solubility were identified. These proteoforms fall into three natural groups defined by hierarchical clustering of expression levels in the context of mouse age and proteoform solubility, with each group sharing physiochemical properties associated with either N/C-terminal truncations or both. Overall, the TDMS workflow outlined may hold tremendous potential for investigating proteoform-level relationships between insoluble fibrils and soluble Aß, including low-molecular-weight oligomers hypothesized to serve as the key drivers of neurotoxicity. Similarly, the workflow may also help to validate the utility of AD-relevant animal models to recapitulate amyloidosis mechanisms or possibly explain disconnects observed in therapeutic efficacy in animal models vs humans.
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Doença de Alzheimer , Amiloidose , Camundongos , Humanos , Animais , Peptídeos beta-Amiloides/metabolismo , Doença de Alzheimer/metabolismo , Camundongos Transgênicos , Modelos Animais de Doenças , Espectrometria de MassasRESUMO
The effectiveness of any proteomics database search depends on the theoretical candidate information contained in the protein database. Unfortunately, candidate entries from protein databases such as UniProt rarely contain all the post-translational modifications (PTMs), disulfide bonds, or endogenous cleavages of interest to researchers. These omissions can limit discovery of novel and biologically important proteoforms. Conversely, searching for a specific proteoform becomes a computationally difficult task for heavily modified proteins. Both situations require updates to the database through user-annotated entries. Unfortunately, manually creating properly formatted UniProt Extensible Markup Language (XML) files is tedious and prone to errors. ProSight Annotator solves these issues by providing a graphical interface for adding user-defined features to UniProt-formatted XML files for better informed proteoform searches. It can be downloaded from http://prosightannotator.northwestern.edu.
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Idioma , Proteínas , Bases de Dados de Proteínas , Processamento de Proteína Pós-Traducional , Proteínas/química , Proteômica , SoftwareRESUMO
Human amyloid beta peptide (Aß) is a brain catabolite that at nanomolar concentrations can form neurotoxic oligomers (AßOs), which are known to accumulate in Alzheimer's disease. Because a predisposition to form neurotoxins seems surprising, we have investigated whether circumstances might exist where AßO accumulation may in fact be beneficial. Our investigation focused on the embryonic chick retina, which expresses the same Aß as humans. Using conformation-selective antibodies, immunoblots, mass spectrometry, and fluorescence microscopy, we discovered that AßOs are indeed present in the developing retina, where multiple proteoforms are expressed in a highly regulated cell-specific manner. The expression of the AßO proteoforms was selectively associated with transiently expressed phosphorylated Tau (pTau) proteoforms that, like AßOs, are linked to Alzheimer's disease (AD). To test whether the AßOs were functional in development, embryos were cultured ex ovo and then injected intravitreally with either a beta-site APP-cleaving enzyme 1 (BACE-1) inhibitor or an AßO-selective antibody to prematurely lower the levels of AßOs. The consequence was disrupted histogenesis resulting in dysplasia resembling that seen in various retina pathologies. We suggest the hypothesis that embryonic AßOs are a new type of short-lived peptidergic hormone with a role in neural development. Such a role could help explain why a peptide that manifests deleterious gain-of-function activity when it oligomerizes in the aging brain has been evolutionarily conserved.
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Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Retina/metabolismo , Animais , Encéfalo/metabolismo , Galinhas/metabolismo , Espaço Extracelular/metabolismo , Sinapses/metabolismoRESUMO
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.
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Proteínas do Líquido Cefalorraquidiano , Proteômica , Animais , Focalização Isoelétrica , Espectrometria de Massas , PrimatasRESUMO
The combined use of electrospray ionization run in so-called "native mode" with top-down mass spectrometry (nTDMS) is enhancing both structural biology and discovery proteomics by providing three levels of information in a single experiment: the intact mass of a protein or complex, the masses of its subunits and non-covalent cofactors, and fragment ion masses from direct dissociation of subunits that capture the primary sequence and combinations of diverse post-translational modifications (PTMs). While intact mass data are readily deconvoluted using well-known software options, the analysis of fragmentation data that result from a tandem MS experiment - essential for proteoform characterization - is not yet standardized. In this tutorial, we offer a decision-tree for the analysis of nTDMS experiments on protein complexes and diverse bioassemblies. We include an overview of strategies to navigate this type of analysis, provide example data sets, and highlight software for the hypothesis-driven interrogation of fragment ions for localization of PTMs, metals, and cofactors on native proteoforms. Throughout we have emphasized the key features (deconvolution, search mode, validation, other) that the reader can consider when deciding upon their specific experimental and data processing design using both open-access and commercial software.
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Despite decades of accumulated knowledge about proteins and their post-translational modifications (PTMs), numerous questions remain regarding their molecular composition and biological function. One of the most fundamental queries is the extent to which the combinations of DNA-, RNA- and PTM-level variations explode the complexity of the human proteome. Here, we outline what we know from current databases and measurement strategies including mass spectrometry-based proteomics. In doing so, we examine prevailing notions about the number of modifications displayed on human proteins and how they combine to generate the protein diversity underlying health and disease. We frame central issues regarding determination of protein-level variation and PTMs, including some paradoxes present in the field today. We use this framework to assess existing data and to ask the question, "How many distinct primary structures of proteins (proteoforms) are created from the 20,300 human genes?" We also explore prospects for improving measurements to better regularize protein-level biology and efficiently associate PTMs to function and phenotype.
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Genoma Humano , Processamento de Proteína Pós-Traducional , Proteínas/química , Proteoma/química , Proteômica/métodos , Bases de Dados de Proteínas , Humanos , Espectrometria de Massas , Fenótipo , Biossíntese de Proteínas , Isoformas de Proteínas/química , Ubiquitina/químicaRESUMO
Cocaine addiction afflicts nearly 1 million adults in the United States, and to date, there are no known treatments approved for this psychiatric condition. Women are particularly vulnerable to developing a cocaine use disorder and suffer from more serious cardiac consequences than men when using cocaine. Estrogen is one biological factor contributing to the increased risk for females to develop problematic cocaine use. Animal studies have demonstrated that estrogen (17ß-estradiol or E2) enhances the rewarding properties of cocaine. Although E2 affects the dopamine system, the molecular and cellular mechanisms of E2-enhanced cocaine reward have not been characterized. In this study, quantitative top-down proteomics was used to measure intact proteins in specific regions of the female mouse brain after mice were trained for cocaine-conditioned place preference, a behavioral test of cocaine reward. Several proteoform changes occurred in the ventral tegmental area after combined cocaine and E2 treatments, with the most numerous proteoform alterations on myelin basic protein, indicating possible changes in white matter structure. There were also changes in histone H4, protein phosphatase inhibitors, cholecystokinin, and calmodulin proteoforms. These observations provide insight into estrogen signaling in the brain and may guide new approaches to treating women with cocaine use disorder.
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Encéfalo/efeitos dos fármacos , Cocaína/farmacologia , Estradiol/farmacologia , Proteoma/metabolismo , Proteômica/métodos , Animais , Encéfalo/metabolismo , Condicionamento Clássico/efeitos dos fármacos , Dopamina/metabolismo , Inibidores da Captação de Dopamina/farmacologia , Estrogênios/farmacologia , Feminino , Camundongos Endogâmicos C57BL , Ovariectomia , Recompensa , Área Tegmentar Ventral/efeitos dos fármacos , Área Tegmentar Ventral/metabolismoRESUMO
Protein N-myristoylation is a 14-carbon fatty-acid modification that is conserved across eukaryotic species and occurs on nearly 1% of the cellular proteome. The ability of the myristoyl group to facilitate dynamic protein-protein and protein-membrane interactions (known as the myristoyl switch) makes it an essential feature of many signal transduction systems. Thus pathogenic strategies that facilitate protein demyristoylation would markedly alter the signalling landscape of infected host cells. Here we describe an irreversible mechanism of protein demyristoylation catalysed by invasion plasmid antigen J (IpaJ), a previously uncharacterized Shigella flexneri type III effector protein with cysteine protease activity. A yeast genetic screen for IpaJ substrates identified ADP-ribosylation factor (ARF)1p and ARF2p, small molecular mass GTPases that regulate cargo transport through the Golgi apparatus. Mass spectrometry showed that IpaJ cleaved the peptide bond between N-myristoylated glycine-2 and asparagine-3 of human ARF1, thereby providing a new mechanism for host secretory inhibition by a bacterial pathogen. We further demonstrate that IpaJ cleaves an array of N-myristoylated proteins involved in cellular growth, signal transduction, autophagasome maturation and organelle function. Taken together, these findings show a previously unrecognized pathogenic mechanism for the site-specific elimination of N-myristoyl protein modification.
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Antígenos de Bactérias/metabolismo , Ácido Mirístico/metabolismo , Processamento de Proteína Pós-Traducional , Proteólise , Shigella flexneri/metabolismo , Fatores de Virulência/metabolismo , Fator 1 de Ribosilação do ADP/química , Fator 1 de Ribosilação do ADP/metabolismo , Fatores de Ribosilação do ADP/metabolismo , Sequência de Aminoácidos , Animais , Asparagina/metabolismo , Autofagia , Biocatálise , Cisteína Proteases/metabolismo , Disenteria Bacilar , Feminino , Glicina/metabolismo , Complexo de Golgi/metabolismo , Complexo de Golgi/patologia , Células HEK293 , Células HeLa , Humanos , Listeria monocytogenes/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Fagossomos/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Shigella flexneri/enzimologia , Transdução de Sinais , Especificidade por Substrato , VirulênciaRESUMO
Over the past decade, advances in mass spectrometry-based proteomics have accelerated brain proteome research aimed at studying the expression, dynamic modification, interaction and function of proteins in the nervous system that are associated with physiological and behavioral processes. With the latest hardware and software improvements in top-down mass spectrometry, the technology has expanded from mere protein profiling to high-throughput identification and quantification of intact proteoforms. Murine systems are broadly used as models to study human diseases. Neuroscientists specifically study the mouse brain from inbred strains to help understand how strain-specific genotype and phenotype affect development, functioning, and disease progression. This work describes the first application of label-free quantitative top-down proteomics to the analysis of the mouse brain proteome. Operating in discovery mode, we determined physiochemical differences in brain tissue from four healthy inbred strains, C57BL/6J, DBA/2J, FVB/NJ, and BALB/cByJ, after probing their intact proteome in the 3.5-30 kDa mass range. We also disseminate these findings using a new tool for top-down proteomics, TDViewer and cataloged them in a newly established Mouse Brain Proteoform Atlas. The analysis of brain tissues from the four strains identified 131 gene products leading to the full characterization of 343 of the 593 proteoforms identified. Within the results, singly and doubly phosphorylated ARPP-21 proteoforms, known to inhibit calmodulin, were differentially expressed across the four strains. Gene ontology (GO) analysis for detected differentially expressed proteoforms also helps to illuminate the similarities and dissimilarities in phenotypes among these inbred strains.
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Química Encefálica , Espectrometria de Massas/métodos , Camundongos Endogâmicos , Proteoma/análise , Proteômica/métodos , Animais , Encéfalo/metabolismo , Cromatografia Líquida/métodos , Feminino , Camundongos Endogâmicos BALB C/metabolismo , Camundongos Endogâmicos C57BL/metabolismo , Camundongos Endogâmicos DBA/metabolismo , Camundongos Endogâmicos/metabolismo , Proteoma/metabolismo , SoftwareRESUMO
Myelin basic protein (MBP) plays an important structural and functional role in the neuronal myelin sheath. Translated MBP exhibits extreme microheterogeneity with numerous alternative splice variants (ASVs) and post-translational modifications (PTMs) reportedly tied to central nervous system maturation, myelin stability, and the pathobiology of various de- and dys-myelinating disorders. Conventional bioanalytical tools cannot efficiently examine ASV and PTM events simultaneously, which limits understanding of the role of MBP microheterogeneity in human physiology and disease. To address this need, we report on a top-down proteomics pipeline that combines superficially porous reversed-phase liquid chromatography (SPLC), Fourier transform mass spectrometry (FTMS), data-independent acquisition (DIA) with nozzle-skimmer dissociation (NSD), and aligned data processing resources to rapidly characterize abundant MBP proteoforms within murine tissue. The three-tier proteoform identification and characterization workflow resolved four known MBP ASVs and hundreds of differentially modified states from a single 90 min SPLC-FTMS run on â¼0.5 µg of material. This included 323 proteoforms for the 14.1 kDa ASV alone. We also identified two novel ASVs from an alternative transcriptional start site (ATSS) of the MBP gene as well as a never before characterized S-acylation event linking palmitic acid, oleic acid, and stearic acid at C78 of the 17.125 kDa ASV.
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Cromatografia de Fase Reversa/métodos , Proteína Básica da Mielina/análise , Animais , Análise de Fourier , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Porosidade , Propriedades de SuperfícieRESUMO
This chapter highlights many of the fundamental concepts and technologies in the field of top-down mass spectrometry (TDMS), and provides numerous examples of contributions that TD is making in biology, biophysics, and clinical investigations. TD workflows include variegated steps that may include non-specific or targeted preparative strategies, orthogonal liquid chromatography techniques, analyte ionization, mass analysis, tandem mass spectrometry (MS/MS) and informatics procedures. This diversity of experimental designs has evolved to manage the large dynamic range of protein expression and diverse physiochemical properties of proteins in proteome investigations, tackle proteoform microheterogeneity, as well as determine structure and composition of gas-phase proteins and protein assemblies.
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Cromatografia/métodos , Espectrometria de Massas/métodos , Proteínas/análise , Proteoma , Proteômica/métodos , Animais , Biologia Computacional , Bases de Dados de Proteínas , Ensaios de Triagem em Larga Escala , Humanos , Fluxo de TrabalhoRESUMO
(13)C Methyl TROSY NMR spectroscopy has emerged as a powerful method for studying the dynamics of large systems such as macromolecular assemblies and membrane proteins. Specific (13)C labeling of aliphatic methyl groups and perdeuteration has been limited primarily to proteins expressed in E. coli, preventing studies of many eukaryotic proteins of physiological and biomedical significance. We demonstrate the feasibility of efficient (13)C isoleucine δ1-methyl labeling in a deuterated background in an established eukaryotic expression host, Pichia pastoris, and show that this method can be used to label the eukaryotic protein actin, which cannot be expressed in bacteria. This approach will enable NMR studies of previously intractable targets.
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Isótopos de Carbono/química , Ressonância Magnética Nuclear Biomolecular/métodos , Pichia/metabolismo , Proteínas Recombinantes/química , Actinas/química , Actinas/metabolismo , Isótopos de Carbono/metabolismo , Isoleucina/química , Isoleucina/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
Lipocalin-type prostaglandin D-synthase (L-PGDS) in cerebrospinal fluid contributes to the maturation and maintenance of the CNS. L-PGDS PTMs may contribute to pathobiology of different CNS diseases, but methods to monitor its proteoforms are limited. Herein, we combined off-gel IEF and superficially porous LC (SPLC) with Fourier transform MS to characterize common cerebrospinal fluid L-PGDS proteoforms. Across 3D physiochemical space (pI, hydrophobicity, and mass), 217 putative proteoforms were observed from 21 to 24 kDa and pI 5-10. Glycoprotein accurate mass information, combined with MS/MS analysis of peptides generated from 2D-fractionated proteoforms, enabled the putative assignment of 208 proteoforms with varied PTM positional occupants. Fifteen structurally related N-glycans at N29 and N56 were observed, with different N-glycan compositional variants being preferred on each amino acid. We also observed that sialic acid content was a major factor for pI shifts between L-PGDS proteoforms. Other putative PTMs characterized include a core-1 HexHexNAc-O-glycan at S7, acetylation at K16 and K138, sulfonation at S41 and T142, and dioxidation at C43 and C145. The IEF-SPLC-MS platform presented provides 30-40× improved peak capacity versus conventional 2DE and shows potential for repeatable proteoform analysis of surrogate PTM-based biomarkers.
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Cromatografia Líquida/métodos , Oxirredutases Intramoleculares/líquido cefalorraquidiano , Oxirredutases Intramoleculares/química , Focalização Isoelétrica/métodos , Lipocalinas/líquido cefalorraquidiano , Lipocalinas/química , Espectrometria de Massas/métodos , Humanos , Isoformas de Proteínas/líquido cefalorraquidiano , Isoformas de Proteínas/química , Processamento de Proteína Pós-TraducionalRESUMO
We report novel ligand binding assay (LBA) surface modalities that permit plasma protease catalytic efficiency (kcat/km) determination by MALDI-TOF MS without the use of liquid chromatography or internal standards such as chemical or metalized labels. Two model LBAs were constructed on planar self-assembled monolayers (SAMs) and used to evaluate the clinically relevant metalloprotease ADAMTS-13 kinetics in plasma. The SAM chemistries were designed to improve biosampling efficiency by minimization of nonspecific adsorption of abundant proteins present at ~100,000× the concentration of the endogenous enzyme. In the first protocol, in-solution digestion of the ADAMTS-13 substrate (vWFh) was performed with immunoaffinity enrichment of the reaction substrate and product to SAM arrays. The second configuration examined protease kcat/km via a surface digestion modality where different substrates were covalently immobilized to the SAM at controlled surface density for optimized protease screens. The results show the MALDI-TOF MS LBA platforms provide limits of quantitation to ~1% protease activity (~60 pM enzyme concentration) in <1 h analysis time, a ~16× improvement over other MS-based LBA formats. Implementation of a vacuum-sublimed MALDI matrix provided good MALDI-TOF MS intra- and interday repeatability, ~1.2 and ~6.6% RSD, respectively. Platform reliability permitted kcat/km determination without internal standards with observed values ~10× improved versus conventional fluorophoric assays. Application of the assays to 12 clinical plasma samples demonstrated proof-of-concept for clinical applications. Overall, this work demonstrates that rationally designed surface chemistries for MALDI-TOF MS may serve as an alternative, label-free methodology with potential for a wide range of biotechnology applications related to targeted enzyme molecular diagnostics.
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Peptídeo Hidrolases/sangue , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sequência de Aminoácidos , Cinética , Ligantes , Dados de Sequência Molecular , Peptídeo Hidrolases/química , Reprodutibilidade dos TestesRESUMO
Top-down mass spectrometry (MS) has emerged as a powerful complement to peptide-based proteomics. Despite advancements, the field has had limited application to clinical proteomics investigations due to the complexity and poor dynamic range of chromatography used to separate intact proteins from tissue and biofluids. To address these limitations, we developed a two-dimensional (2D) chromatography platform that includes isoelectric focusing (IEF) through immobilized pH gradient and superficially porous liquid chromatography (SPLC). Analysis of standard proteins demonstrates compatibility of IEF-SPLC processing and high resolving-power MS analysis with results showing ~7.0 femtomole detection limits and linear spectral response for proteins fractionated over ~4 log sample loads. For proteins from heart myofibrils and cerebrospinal fluid (CSF), compared to one-dimensional SPLC-MS, the 2D IEF-SPLC-MS platform resulted in a 5-6× increase in the number of unique monoisotopic masses observed <30 kDa and an ~4× improved mass range enabling the observation of proteins >200 kDa. In the heart myofibrils, common protein proteoforms observed were associated with phosphorylation of contractile proteins with results showing that quantitative evaluation of their PTM stoichiometry was possible despite differentially modified forms being fractionated into separate pI compartments. In CSF, diverse protein mutations and PTM classes were also observed, including differentially glycosylated protein forms separated to different pI. Results also demonstrate that by the generation of IEF-SPLC protein libraries by fraction collection, the platform enables prospective protein identification and proteoform analysis investigations by complementary top-down and bottom-up strategies. Overall, the 2D platform presented may provide the speed, dynamic range, and detection limits necessary for routine characterization of proteoform-based biomarkers from biofluids and tissues.
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Líquidos Corporais/química , Cromatografia Líquida/métodos , Focalização Isoelétrica/métodos , Espectrometria de Massas/métodos , Dióxido de Silício , Animais , CamundongosRESUMO
Immunoassays are widely used in biochemical/clinical laboratories owing to their simplicity, speed, and sensitivity. We combined self-assembled monolayer-based immunoassays with MALDI-TOF MS to show that high-fidelity surface preparations with a novel matrix deposition/crystallization technique permits quantitative analysis of monolayer-bound antigens at picomolar detection limits. Calibration curves for intact proteins are possible over a broad concentration range and improved specificity of MS-immunoassays is highlighted by simultaneous label-free quantitation of ligand-bound protein complexes.
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Imunoensaio/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Anticorpos Imobilizados/metabolismo , Antígenos/análise , Antígenos/química , Antígenos/metabolismo , Imunoensaio/instrumentação , Limite de Detecção , Ligação Proteica , Proteínas/análise , Proteínas/química , Proteínas/metabolismoRESUMO
The compatibility of superficially porous (SP) resin for label-free intact protein analysis with online capillary LC/MS is demonstrated to give improved chromatographic resolution, sensitivity, and reproducibility. The robustness of the platform was measured against several samples of varying complexity and sample loading amount. The results indicate that capillary SP columns provide high loading capacities and that â¼6 s chromatographic peak widths are typical for standard proteins in simple mixtures and proteins isolated from cell and tissue lysates. Subfemtomole detection limits for standard proteins were consistently observed, with the lowest levels at 12 amol for ubiquitin. The analysis of total heart homogenates shows that capillary SP columns provide theoretical peak capacity of 106 protein forms with 30 min total analysis time and enabled detection of proteins from complex mixtures with a single high-resolution scan. The SPLC/MS platform also detected 343 protein forms from two HeLa acid extract replicate analyses that consumed 5 × 10(4) cells and 30 min analysis time, each. Comparison of all the species observed in each HeLa replicate showed 90% overlap (309 forms) with a Pearson correlation coefficient of 89.9% for the common forms observed in the replicates. Efficient acid extract of 1 × 10(4) HeLa cells allowed reproducible detection of common modification states and members from all five of the histone families and demonstrated that capillary SPLC/MS supports reproducible label-free profiling of histones in <15 min total analysis time. The data presented demonstrate that a capillary LC/MS platform utilizing superficially porous stationary phase and a LTQ-Orbitrap FT-MS is fast, sensitive, and reproducible for intact protein profiling from small tissue and cell amounts.
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Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Espectrometria de Massas , Proteínas/análise , Células HeLa , Histonas/análise , Humanos , Peso Molecular , Ubiquitina/análiseRESUMO
Terminal sialic acid residues often mediate the interactions of cell surface glycoconjugates. Sialic acid-dependent interactions typically exhibit rapid dissociation rates, precluding the use of traditional biological techniques for complex isolation. To stabilize these transient interactions, we employ a targeted photo-cross-linking approach in which a diazirine photo-cross-linker is incorporated into cell surface sialylated glycoconjugates through the use of metabolic oligosaccharide engineering. We describe three diazirine-modified N-acetylmannosamine (ManNAc) analogues in which the length of the linker between the pyranose ring and the diazirine was varied. These analogues were each metabolized to their respective sialic acid counterparts, which were added to both glycoproteins and glycolipids. Diazirine-modified sialic acid analogues could be incorporated into both α2-3 and α2-6 linkages. Upon exposure to UV irradiation, diazirine-modified glycoconjugates were covalently cross-linked to their interaction partners. We demonstrate that all three diazirine-modified analogues were capable of competing with endogeneous sialic acid, albeit to varying degrees. We found that larger analogues were less efficiently metabolized, yet could still function as effective cross-linkers. Notably, the addition of the diazirine substituent interferes with metabolism of ManNAc analogues to glycans other than sialosides, providing fidelity to selectively incorporate the cross-linker into sialylated molecules. These compounds are nontoxic and display only minimal growth inhibition at the concentrations required for cross-linking studies. This report provides essential information for the deployment of photo-cross-linking analogues to capture and study ephemeral, yet essential, sialic acid-mediated interactions.