RESUMO
Fragile X syndrome (FXS), the leading cause of inherited intellectual disability and autism, is caused by the transcriptional silencing of the FMR1 gene, which encodes the fragile X messenger ribonucleoprotein (FMRP). FMRP interacts with numerous brain mRNAs that are involved in synaptic plasticity and implicated in autism spectrum disorders. Our published studies indicate that single-source, soy-based diets are associated with increased seizures and autism. Thus, there is an acute need for an unbiased protein marker identification in FXS in response to soy consumption. Herein, we present a spatial proteomics approach integrating mass spectrometry imaging with label-free proteomics in the FXS mouse model to map the spatial distribution and quantify levels of proteins in the hippocampus and hypothalamus brain regions. In total, 1250 unique peptides were spatially resolved, demonstrating the diverse array of peptidomes present in the tissue slices and the broad coverage of the strategy. A group of proteins that are known to be involved in glycolysis, synaptic transmission, and coexpression network analysis suggest a significant association between soy proteins and metabolic and synaptic processes in the Fmr1KO brain. Ultimately, this spatial proteomics work represents a crucial step toward identifying potential candidate protein markers and novel therapeutic targets for FXS.
Assuntos
Síndrome do Cromossomo X Frágil , Proteínas de Soja , Camundongos , Animais , Proteínas de Soja/metabolismo , Proteína do X Frágil da Deficiência Intelectual/genética , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Síndrome do Cromossomo X Frágil/metabolismo , Proteômica , Camundongos Knockout , Modelos Animais de DoençasRESUMO
Protein citrullination is a key post-translational modification (PTM) that leads to the loss of positive charge on arginine and consequent protein structural and functional changes. Though it has been indicated to play critical roles in various physiological and pathological processes, effective analytical tools are largely limited due to a few challenges such as the small mass shift induced by this PTM and its low-abundance nature. Recently, we developed a biotin thiol tag, which enabled large-scale profiling of protein citrullination from complex biological samples via mass spectrometry. However, a high-throughput quantitative approach is still in great need to further improve the understanding of this PTM. In this study, we report an efficient pipeline using our custom-developed N,N-dimethyl leucine isobaric tags to achieve a multiplexed quantitative analysis of citrullination from up to 12 samples for the first time. We then apply this strategy to investigating citrullination alterations in response to DNA damage stress using human cell lines. We unveil important biological functions regulated by protein citrullination and observe hypercitrullination on RNA-binding proteins and DNA repair proteins, respectively. Our results reveal the involvement of citrullination in DNA damage pathways and may provide new insights into DNA-damage-related disease pathogenesis.
Assuntos
Citrulinação , Espectrometria de Massas em Tandem , Dano ao DNA , Humanos , Leucina/análogos & derivados , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Espectrometria de Massas em Tandem/métodosRESUMO
Protein sialylation has been closely linked to many diseases including Alzheimer's disease (AD). It is also broadly implicated in therapeutics operating in a pattern-dependent (e.g., Neu5Ac vs Neu5Gc) manner. However, how the sialylation pattern affects the AD-associated, transferrin-assisted iron/Aß cellular uptake process remains largely ill-defined. Herein, we report the use of native ion mobility-mass spectrometry (IM-MS)-based fast structural probing methodology, enabling well-controlled, synergistic, and in situ manipulation of mature glycoproteins and attached sialic acids. IM-MS-centered experiments enable the combinatorial interrogation of sialylation effects on Aß cytotoxicity and the chemical, conformational, and topological stabilities of transferrin. Cell viability experiments suggest that Neu5Gc replacement enhances the transferrin-assisted, iron loading-associated Aß cytotoxicity. Native gel electrophoresis and IM-MS reveal that sialylation stabilizes transferrin conformation but inhibits its dimerization. Collectively, IM-MS is adapted to capture key sialylation intermediates involved in fine-tuning AD-associated glycoprotein structural microheterogeneity. Our results provide the molecular basis for the importance of sustaining moderate TF sialylation levels, especially Neu5Ac, in promoting iron cellular transportation and rescuing iron-enhanced Aß cytotoxicity.
Assuntos
Doença de Alzheimer , Doença de Alzheimer/metabolismo , Glicoproteínas/metabolismo , Humanos , Espectrometria de Massas/métodos , Ácidos Siálicos , TransferrinaRESUMO
Site-specific O-glycoproteome mapping in complex biological systems provides a molecular basis for understanding the structure-function relationships of glycoproteins and their roles in physiological and pathological processes. Previous O-glycoproteome analysis in cerebrospinal fluid (CSF) focused on sialylated glycoforms, while missing information on other glycosylation types. In order to achieve an unbiased O-glycosylation profile, we developed an integrated strategy combining universal boronic acid enrichment, high-pH fractionation, and electron-transfer and higher-energy collision dissociation (EThcD) for enhanced intact O-glycopeptide analysis. We applied this strategy to analyze the O-glycoproteome in CSF, resulting in the identification of 308 O-glycopeptides from 110 O-glycoproteins, covering both sialylated and nonsialylated glycoforms. To our knowledge, this is the largest data set of O-glycoproteins and O-glycosites reported for CSF to date. We also developed a peptidomics workflow that utilized the EThcD and a three-step database searching strategy for comprehensive PTM analysis of endogenous peptides, including N-glycosylation, O-glycosylation, and other common peptide PTMs. Interestingly, among the 1411 endogenous peptides identified, 89 were O-glycosylated, and only one N-glycosylated peptide was found, indicating that CSF endogenous peptides were predominantly O-glycosylated. Analyses of the O-glycoproteome and endogenous peptidome PTMs were also conducted in the CSF of MCI and AD patients to provide a landscape of glycosylation patterns in different disease states. Our results showed a decreasing trend in fucosylation and an increasing trend of endogenous peptide O-glycosylation, which may play an important role in AD progression.
Assuntos
Doença de Alzheimer , Disfunção Cognitiva , Humanos , Glicosilação , Glicoproteínas/química , Glicopeptídeos/química , Proteoma/metabolismo , PeptídeosRESUMO
As the body fluid that directly interchanges with the extracellular fluid of the central nervous system (CNS), cerebrospinal fluid (CSF) serves as a rich source for CNS-related disease biomarker discovery. Extensive proteome profiling has been conducted for CSF, but studies aimed at unraveling site-specific CSF N-glycoproteome are lacking. Initial efforts into site-specific N-glycoproteomics study in CSF yield limited coverage, hindering further experimental design of glycosylation-based disease biomarker discovery in CSF. In the present study, we have developed an N-glycoproteomic approach that combines enhanced N-glycopeptide sequential enrichment by hydrophilic interaction chromatography (HILIC) and boronic acid enrichment with electron transfer and higher-energy collision dissociation (EThcD) for large-scale intact N-glycopeptide analysis. The application of the developed approach to the analyses of human CSF samples enabled identifications of a total of 2893 intact N-glycopeptides from 511 N-glycosites and 285 N-glycoproteins. To our knowledge, this is the largest site-specific N-glycoproteome dataset reported for CSF to date. Such dataset provides molecular basis for a better understanding of the structure-function relationships of glycoproteins and their roles in CNS-related physiological and pathological processes. As accumulating evidence suggests that defects in glycosylation are involved in Alzheimer's disease (AD) pathogenesis, in the present study, a comparative in-depth N-glycoproteomic analysis was conducted for CSF samples from healthy control and AD patients, which yielded a comparable N-glycoproteome coverage but a distinct expression pattern for different categories of glycoforms, such as decreased fucosylation in AD CSF samples. Altered glycosylation patterns were detected for a number of N-glycoproteins including alpha-1-antichymotrypsin, ephrin-A3 and carnosinase CN1 etc., which serve as potentially interesting targets for further glycosylation-based AD study and may eventually lead to molecular elucidation of the role of glycosylation in AD progression.
Assuntos
Doença de Alzheimer/líquido cefalorraquidiano , Glicopeptídeos/líquido cefalorraquidiano , Glicoproteínas/líquido cefalorraquidiano , Proteoma/análise , Linhagem Celular , Glicosilação , HumanosRESUMO
The extracellular matrix (ECM) is unique to each tissue and capable of guiding cell differentiation, migration, morphology, and function. The ECM proteome of different developmental stages has not been systematically studied in the human pancreas. In this study, we apply mass spectrometry-based quantitative proteomics strategies using N,N-dimethyl leucine isobaric tags to delineate proteome-wide and ECM-specific alterations in four age groups: fetal (18-20 weeks gestation), juvenile (5-16 years old), young adults (21-29 years old) and older adults (50-61 years old). We identify 3,523 proteins including 185 ECM proteins and quantify 117 of them. We detect previously unknown proteome and matrisome features during pancreas development and maturation. We also visualize specific ECM proteins of interest using immunofluorescent staining and investigate changes in ECM localization within islet or acinar compartments. This comprehensive proteomics analysis contributes to an improved understanding of the critical roles that ECM plays throughout human pancreas development and maturation.
Assuntos
Proteínas da Matriz Extracelular/genética , Regulação da Expressão Gênica no Desenvolvimento , Pâncreas/metabolismo , Proteoma/genética , Adolescente , Adulto , Criança , Pré-Escolar , Cromatografia Líquida , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/classificação , Proteínas da Matriz Extracelular/metabolismo , Feminino , Feto , Imunofluorescência , Ontologia Genética , Humanos , Masculino , Pessoa de Meia-Idade , Anotação de Sequência Molecular , Organogênese/genética , Pâncreas/crescimento & desenvolvimento , Proteoma/classificação , Proteoma/metabolismo , Proteômica/métodos , Espectrometria de Massas em TandemRESUMO
The unbiased selection of peptide precursors makes data-independent acquisition (DIA) an advantageous alternative to data-dependent acquisition (DDA) for discovery proteomics, but traditional multiplexed quantification approaches employing mass difference labeling or isobaric tagging are incompatible with DIA. Here, we describe a strategy that permits multiplexed quantification by DIA using mass defect-based N,N-dimethyl leucine (mdDiLeu) tags and high-resolution tandem mass spectrometry (MS2) analysis. Millidalton mass differences between mdDiLeu isotopologues produce fragment ion multiplet peaks separated in mass by as little as 5.8 mDa, enabling up to 4-plex quantification in DIA MS2 spectra. Quantitative analysis of yeast samples displayed comparable accuracy and precision for MS2-based DIA and MS1-based DDA methods. Multiplexed DIA analysis of cerebrospinal fluid revealed the dynamic proteome changes in Alzheimer's disease, demonstrating its utility for discovery of potential clinical biomarkers. We show that the mdDiLeu tagging approach for multiplexed DIA is a viable methodology for investigating proteome changes, particularly for low-abundance proteins, in different biological matrices.
Assuntos
Leucina/análogos & derivados , Proteoma/análise , Proteômica/métodos , Proteínas de Saccharomyces cerevisiae/análise , Doença de Alzheimer/líquido cefalorraquidiano , Sequência de Aminoácidos , Biomarcadores/líquido cefalorraquidiano , Biomarcadores/química , Proteínas do Líquido Cefalorraquidiano/líquido cefalorraquidiano , Proteínas do Líquido Cefalorraquidiano/química , Humanos , Pessoa de Meia-Idade , Estudo de Prova de Conceito , Proteoma/química , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química , Espectrometria de Massas em TandemRESUMO
The use of mass spectrometry for protein identification and quantification in cerebrospinal fluid (CSF) is at the forefront of research efforts to identify and explore biomarkers for the early diagnosis and prognosis of neurologic disorders. Here we implemented a 4-plex N,N-dimethyl leucine (DiLeu) isobaric labeling strategy in a longitudinal study aiming to investigate protein dynamics in children with B-cell acute lymphoblastic leukemia (B-cell ALL) undergoing chemotherapy. The temporal profile of CSF proteome during chemotherapy treatment at weeks 5, 10-14, and 24-28 highlighted many differentially expressed proteins, such as neural cell adhesion molecule, neuronal growth regulator 1, and secretogranin-3, all of which play important roles in neurodegenerative diseases. A total of 63 proteins were significantly altered across all of the time points investigated. The most over-represented biological processes from gene ontology analysis included platelet degranulation, complement activation, cell adhesion, fibrinolysis, neuron projection, regeneration, and regulation of neuron death. We expect that results from this and future studies will provide a means to monitor neurotoxicity and develop strategies to prevent central nervous system injury in response to chemotherapy in children.
Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras , Proteômica , Linfócitos B , Criança , Humanos , Leucina , Estudos Longitudinais , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Espectrometria de Massas em TandemRESUMO
N-linked glycosylation, featuring various glycoforms, is one of the most common and complex protein post-translational modifications (PTMs) controlling protein structures and biological functions. It has been revealed that abnormal changes of protein N-glycosylation patterns are associated with many diseases. Hence, unraveling the disease-related alteration of glycosylation, especially the glycoforms, is crucial and beneficial to improving our understanding about the pathogenic mechanisms of various diseases. In past decades, given the capability of in situ mapping of biomolecules and their region-specific localizations, matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has been widely applied to the discovery of potential biomarkers for many diseases. In this study, we coupled a novel subatmospheric pressure (SubAP)/MALDI source with a Q Exactive HF hybrid quadrupole-orbitrap mass spectrometer for in situ imaging of N-linked glycans from formalin-fixed paraffin-embedded (FFPE) tissue sections. The utility of this new platform for N-glycan imaging analysis was demonstrated with a variety of FFPE tissue sections. A total of 55 N-glycans were successfully characterized and visualized from a FFPE mouse brain section. Furthermore, 29 N-glycans with different spatial distribution patterns could be identified from a FFPE mouse ovarian cancer tissue section. High-mannose N-glycans exhibited elevated expression levels in the tumor region, indicating the potential association of this type of N-glycans with tumor progression.
Assuntos
Encéfalo/metabolismo , Formaldeído/química , Neoplasias Ovarianas/metabolismo , Polissacarídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Feminino , Glicosilação , Humanos , Camundongos , Neoplasias Ovarianas/patologia , Fixação de TecidosRESUMO
Absolute quantification in targeted proteomics is challenging due to a variety of factors, including low specificity in complex backgrounds, limited analytical throughput, and wide dynamic range. To address these problems, we developed a hybrid offset-triggered multiplex absolute quantification (HOTMAQ) strategy that combines cost-effective mass difference and isobaric tags to enable simultaneous construction of an internal standard curve in the MS1 precursor scan, real-time identification of peptides at the MS2 level, and mass offset-triggered accurate quantification of target proteins in synchronous precursor selection (SPS)-MS3 spectra. This approach increases the analytical throughput of targeted quantitative proteomics by up to 12-fold. The HOTMAQ strategy was employed to verify candidate protein biomarkers in preclinical Alzheimer's disease with high accuracy. The greatly enhanced throughput and quantitative performance, paired with sample flexibility, makes HOTMAQ broadly applicable to targeted peptidomics, proteomics, and phosphoproteomics.
Assuntos
Doença de Alzheimer/diagnóstico , Proteínas/análise , Proteômica , Doença de Alzheimer/líquido cefalorraquidiano , Biomarcadores/análise , Biomarcadores/líquido cefalorraquidiano , Cromatografia Líquida , Humanos , Espectrometria de MassasRESUMO
Lipids are highly diverse biomolecules associated with several biological functions including structural constituent, energy storage, and signal transduction. It is essential to characterize lipid structural isomers and further understand their biological roles. Unsaturated lipids contain one or multiple carbon-carbon double bonds. Identifying double bond position presents a major challenge in unsaturated lipid characterization. Recently, several advancements have been made for double bond localization by mass spectrometry (MS) analysis. However, many of these studies require complex chemical reactions or advanced mass spectrometers with special fragmentation techniques, which limits the application in lipidomics study. Here, an innovative meta-chloroperoxybenzoic acid ( m-CPBA) epoxidation reaction coupling with collision-induced dissociation (CID)-MS/MS strategy provides a new tool for unsaturated lipidomics analysis. The rapid epoxidation reaction was carried out by m-CPBA with high specificity. Complete derivatization was achieved in minutes without overoxidized byproduct. Moreover, diagnostic ion pair with 16 Da mass difference indicated localization of carbon-carbon double bond in MS/MS spectra. Multiple lipid classes were evaluated with this strategy and generated abundant fragments for structural analysis. Unsaturated lipid analysis of yeast extract using this strategy took less than 30 min, demonstrating the potential for high-throughput lipidomics analysis by this approach. This study opens a door for high throughput unsaturated lipid analysis with minimal requirement for instrumentation, which could be widely applied in lipidomics analysis.
Assuntos
Fosfatidilcolinas/análise , Fosfatidilcolinas/química , Clorobenzoatos/química , Ácidos Graxos Insaturados/análise , Ácidos Graxos Insaturados/química , Isomerismo , Lipidômica/métodos , Oxirredução , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Leveduras/químicaRESUMO
Glycans are highly complex entities with multiple building units and different degrees of branched polymerization. Intensive research efforts have been directed to mass spectrometry (MS)-based qualitative and quantitative glycomic analysis due to the important functions of glycans. Among various strategies, isobaric labeling has become popular because of its higher multiplexing capacity. Over the past few years, several isobaric chemical tags have been developed for quantitative glycomics. However, caveats also exist for these tags, such as relatively low reporter ion yield for aminoxyTMT-labeled complex glycans. To overcome the limitations of existing isobaric chemical tags, we designed a class of novel isobaric multiplex reagents for carbonyl-containing compound (SUGAR) tags that can be used to label glycans for quantitative glycomic analysis. The quantitative performance including labeling efficiency, quantification accuracy, and dynamic range of these SUGAR tags has been evaluated, showing promising results. Finally, the 4-plex SUGAR tags have been utilized to investigate N-glycan changes of B-cell acute lymphoblastic leukemia (ALL) pediatric patients before and after chemotherapy.