RESUMO
Breast cancer is the second leading cause of cancer-related death among women and a major source of brain metastases. Despite the increasing incidence of brain metastasis from breast cancer, the underlying mechanisms remain poorly understood. Altered glycosylation is known to play a role in various diseases including cancer metastasis. However, profiling studies of O-glycans and their isomers in breast cancer brain metastasis (BCBM) are scarce. This study analyzed the expression of O-glycans and their isomers in human breast cancer cell lines (MDA-MB-231, MDA-MB-361, HTB131, and HTB22), a brain cancer cell line (CRL-1620), and a brain metastatic breast cancer cell line (MDA-MB-231BR) using nanoLC-MS/MS, identifying 27 O-glycan compositions. We observed significant upregulation in the expression of HexNAc1Hex1NeuAc2 and HexNAc2Hex3, whereas the expression of HexNAc1Hex1NeuAc1 was downregulated in MDA-MB-231BR compared to other cell lines. In our isomeric analysis, we observed notable alterations in the isomeric forms of the O-glycan structure HexNAc1Hex1NeuAc1 in a comparison of different cell lines. Our analysis of O-glycans and their isomers in cancer cells demonstrated that changes in their distribution can be related to the metastatic process. We believe that our investigation will contribute to an enhanced comprehension of the significance of O-glycans and their isomers in BCBM.
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Neoplasias Encefálicas , Neoplasias da Mama , Feminino , Humanos , Neoplasias da Mama/patologia , Espectrometria de Massas em Tandem , Neoplasias Encefálicas/metabolismo , Células MCF-7 , Linhagem Celular Tumoral , Polissacarídeos/químicaRESUMO
Glycosylation is one of the most significant and abundant posttranslational modifications in mammalian cells. It mediates a wide range of biofunctions, including cell adhesion, cell communication, immune cell trafficking, and protein stability. Also, aberrant glycosylation has been associated with various diseases such as diabetes, Alzheimer's disease, inflammation, immune deficiencies, congenital disorders, and cancers. The alterations in the distributions of glycan and glycopeptide isomers are involved in the development and progression of several human diseases. However, the microheterogeneity of glycosylation brings a great challenge to glycomic and glycoproteomic analysis, including the characterization of isomers. Over several decades, different methods and approaches have been developed to facilitate the characterization of glycan and glycopeptide isomers. Mass spectrometry (MS) has been a powerful tool utilized for glycomic and glycoproteomic isomeric analysis due to its high sensitivity and rich structural information using different fragmentation techniques. However, a comprehensive characterization of glycan and glycopeptide isomers remains a challenge when utilizing MS alone. Therefore, various separation methods, including liquid chromatography, capillary electrophoresis, and ion mobility, were developed to resolve glycan and glycopeptide isomers before MS. These separation techniques were coupled to MS for a better identification and quantitation of glycan and glycopeptide isomers. Additionally, bioinformatic tools are essential for the automated processing of glycan and glycopeptide isomeric data to facilitate isomeric studies in biological cohorts. Here in this review, we discuss commonly employed MS-based techniques, separation hyphenated MS methods, and software, facilitating the separation, identification, and quantitation of glycan and glycopeptide isomers.
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Glicômica , Software , Animais , Humanos , Glicômica/métodos , Espectrometria de Massas , Polissacarídeos/análise , Glicopeptídeos/análise , MamíferosRESUMO
The complexity and heterogeneity of protein glycosylation present an analytical challenge to the studies of characterization and quantitation. Various LC-MS-based quantitation strategies have emerged in recent decades. Metabolic stable isotope labeling has been developed to enhance the accurate LC/MS-based quantitation between different cell lines. Stable isotope labeling by amino acids in a cell culture (SILAC) is the most widely used metabolic labeling method in proteomic analysis. However, it can only label the peptide backbone and is thus limited in glycomic studies. Here, we present a metabolic isotope labeling strategy, named GlyProSILC (Glycan Protein Stable Isotope Labeling in Cell Culture), that can label both the glycan motif and peptide backbone from the same batch of cells. It was performed by feeding cells with a heavy medium containing amide-15N-glutamine, 13C6-arginine (Arg6), and 13C6-15N2-lysine (Lys8). No significant change of cell line metabolism after GlyProSILC labeling was observed based on transcriptomic, glycomic, and proteomic data. The labeling conditions, labeling efficiency, and quantitation accuracy were investigated. After quantitation correction, we simultaneously quantified 62 N-glycans, 574 proteins, and 344 glycopeptides using the same batch of mixed 231BR/231 cell lines. So far, GlyProSILC provides an accurate and effective quantitation approach for glycomics, proteomics, and glycoproteomics in a cell culture system.
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Glicômica , Proteômica , Marcação por Isótopo/métodos , Glicômica/métodos , Proteômica/métodos , Proteínas , Técnicas de Cultura de Células , Glicopeptídeos/metabolismo , Polissacarídeos/químicaRESUMO
Glioblastoma Multiforme (GBM) is the most aggressive form of malignant brain tumor. The median survival rate does not exceed two years, indicating an imminent need to develop novel therapies. The atypical adamantyl retinoid ST1926 induces apoptosis and growth inhibition in different cancer types. We have shown that ST1926 is an inhibitor of the catalytic subunit of DNA polymerase alpha (POLA1), which is involved in initiating DNA synthesis in eukaryotic cells. POLA1 levels are elevated in GBM versus normal brain tissues. Therefore, we studied the antitumor effects of ST1926 in several human GBM cell lines. We further explored the global protein expression profiles in GBM cell lines using liquid chromatography coupled with tandem mass spectrometry to identify new targets of ST1926. Low sub-micromolar concentrations of ST1926 potently decreased cell viability, induced cell damage and apoptosis, and reduced POLA1 protein levels in GBM cells. The proteomics profiles revealed 197 proteins significantly differentially altered upon ST1926 treatment of GBM cells involved in various cellular processes. We explored the differential gene and protein expression of significantly altered proteins in GBM compared to normal brain tissues.
Assuntos
Glioblastoma , Humanos , Glioblastoma/tratamento farmacológico , DNA Polimerase I , Proteômica , Cinamatos , Inibidores da Síntese de Ácido Nucleico , NucleotidiltransferasesRESUMO
Targeted mass spectrometric analysis is widely employed across various omics fields. The approach has been successfully employed for the structural analysis of proteins, glycans, lipids, and small molecules. Selected reaction monitoring and multiple reaction monitoring (MRM) have been a method of choice for targeted structural studies of biomolecules. However, innovations in instrument designs have led to the development of parallel reaction monitoring (PRM). PRM detects all product ions simultaneously rather than optimizing/preselecting the target glycan transitions, simplifying the analytical workflow. By reducing background interference, increasing selectivity/specificity, and improving data quality, PRM allows reliable quantification of target glycans in complex matrices. PRM can also improve sensitivity for detecting low-abundance target glycans and reduce low-level limit of quantification values with an improved S/N ratio. PRM's advantages are attributed to the development of sensitive and highly selective mass analyzers, orbitrap, and time of flight. In this study, we developed a sensitive PRM method for the quantitative analysis of permethylated N-glycans, an important class of disease biomarkers, using a quadrupole-orbitrap hybrid mass spectrometer. Pooled human cerebrospinal fluid was used for the study as a source of permethylated N-glycans. The method illustrates the fragmentation of N-glycans at different collision energies as well as the optimization of collision energy. The method also detects low-abundance N-glycans more efficiently than MRM. This study is the first attempt to develop a sensitive PRM-based method to analyze permethylated N-glycans.
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Proteínas , Proteômica , Humanos , Proteômica/métodos , Espectrometria de Massas/métodos , Íons , PolissacarídeosRESUMO
The wide variety of chemical properties and biological functions found in proteins is attained via post-translational modifications like glycosylation. Covalently bonded to proteins, glycans play a critical role in cell activity. Complex structures with microheterogeneity, the glycan structures that are associated with proteins are difficult to analyze comprehensively. Recent advances in sample preparation methods, separation techniques, and MS have facilitated the quantitation and structural elucidation of glycans. This review focuses on highlighting advances in MS-based techniques for glycomic analysis that occurred over the last 5 years (2017-2021) as an update to the previous review on the subject. The topics of discussion will include progress in glycomic workflow such as glycan release, purification, derivatization, and separation as well as the topics of ionization, tandem MS, and separation techniques that can be coupled with MS. Additionally, bioinformatics tools used for the analysis of glycans will be described.
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Cromatografia , Glicômica , Espectrometria de Massas em Tandem , Glicosilação , PolissacarídeosRESUMO
The emergence of COVID-19 pandemic has engaged the scientific community around the globe in the rapid development of effective therapeutics and vaccines. Owing to its crucial role in the invasion of the host cell, spike (S) glycoprotein is one of the major targets in these studies. The S1 subunit of the S protein (S1 protein) accommodates the receptor-binding domain, which enables the initial binding of the virus to the host cell. Being a heavily glycosylated protein, numerous studies have investigated its glycan composition. However, none of the studies have explored the isomeric glycan distribution of this protein. Furthermore, this isomeric glycan distribution has never been compared to that in S1 proteins of other coronaviruses, severe acute respiratory syndrome coronavirus 1 and Middle East respiratory syndrome coronavirus, which were responsible for past epidemics. This study explores the uncharted territory of the isomeric glycan distribution in the coronaviruses' S1 protein using liquid chromatography coupled to tandem mass spectrometry. We believe that our data would facilitate future investigations to study the role of isomeric glycans in coronavirus viral pathogenesis.
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Polissacarídeos/química , COVID-19 , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio , Pandemias , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
Post-translational modifications are vital aspects of functional proteins. Therefore, it is critical to understand their roles in biological processes. Glycosylation is particularly challenging to study among these modifications due to the heterogeneity displayed by the glycans in terms of their isomers. Thus, researchers continue to strive for the development of efficient liquid chromatography techniques for isomeric separation of glycans. Porous graphitized carbon (PGC) nano column has been one of the most widely used columns for this purpose, but poor stability and lack of reproducibility led to its discontinuation. In our endeavor to find an alternative stationary phase for isomeric glycan separation, we tested the mesoporous graphitized carbon (MGC) material. Unprecedentedly, satisfactory results were obtained with a column only 1 cm long, which was tested on permethylated N-glycans derived from model glycoproteins as well as biological samples. The column was found to be reproducible across months as well as across different column preparations. Additionally, to decrease the dead volume and attain a better resolution, MGC was utilized to pack a 1 cm length of a pulled capillary nanospray emitter and again demonstrated efficient isomeric separation. Thus, MGC proved to be a suitable stationary phase to obtain efficient isomeric separation of permethylated N-glycans with 1 cm-long packing length, in both capillary columns and packed nanospray emitters.
Assuntos
Carbono , Espectrometria de Massas em Tandem , Cromatografia Líquida , Polissacarídeos , Reprodutibilidade dos TestesRESUMO
Glycosylation is a complex and common post-translational modification of proteins. To study glycosylation, liquid chromatography-mass spectrometry (LC-MS) is often used to profile and structurally characterize the glycans in biological systems. While bed packed reverse phase columns are frequently utilized for the separation of permethylated glycans, the use of newly commercialized micro array pillar nanoLC columns (µPAC) have not been demonstrated previously. Owing to its advantages such as low back pressure, reproducibility, and durability, we have investigated the viability of the µPAC for the analysis of permethylated glycans. In this work, we demonstrate the online purification ability of µPAC trapping column compared against PepMap trapping column. We also found that the 50 cm µPAC can be used for the analysis of both permethylated N- and O-glycans. The use of 50 cm µPAC was compared against the previous method. The use of 200 cm µPAC was also investigated for the permethylated glycan analysis. 200 cm µPAC demonstrated efficient separation of oligomannose glycan isomers as well as other complex glycans.
Assuntos
Polissacarídeos , Espectrometria de Massas em Tandem , Cromatografia Líquida , Glicosilação , Reprodutibilidade dos TestesRESUMO
Breast cancer is a leading cancer in women and is considered to be the second-most common metastatic cancer following lung cancer. An estimated 10-16% of breast cancer patients are suffering from brain metastasis, and the diagnostic cases of breast cancer brain metastasis are increasing. Nevertheless, the mechanisms behind this process are still unclear. Aberrant glycosylation has been proved to be related to many diseases and cancer metastasis. However, studies of N-glycan isomer function in breast cancer brain metastasis are limited. In this study, the expressions of N-glycan isomers derived from five breast cancer cell lines and one brain cancer cell line were investigated and compared to a brain-seeking cell line, 231BR, to acquire a better understanding of the role glycan isomers play in breast cancer brain metastasis. The high temperature nanoPGC-LC-MS/MS achieved an efficient isomeric separation and permitted the identification and quantitation of 144 isomers from 50 N-glycan compositions. There were significant expression alterations of these glycan isomers among the different breast cancer cell lines. The increase of total glycan abundance and sialylation level were observed to be associated with breast cancer invasion. With regard to individual isomers, the greatest number of sialylated isomers was observed along with significant expression alterations in 231BR, suggesting a relationship between glycan sialylation and breast cancer brain metastasis. Furthermore, the increase of the α2,6-sialylation level in 231BR likely contributes to the passage of breast cancer cells through the blood-brain barrier, thus facilitating breast cancer brain metastasis. Meanwhile, the upregulation of highly sialylated glycan isomers with α2,6-linked sialic acids were found to be associated with breast cancer metastasis. This investigation of glycan isomer expressions, especially the unique isomeric expression in brain-seeking cell line 231BR, provides new information toward understanding the potential roles glycan isomers play during breast cancer metastasis and more clues for a deeper insight of this bioprocess.
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Neoplasias Encefálicas/secundário , Neoplasias da Mama/patologia , Polissacarídeos/metabolismo , Neoplasias Encefálicas/metabolismo , Carbono , Linhagem Celular Tumoral , Cromatografia Líquida/métodos , Glicosilação , Grafite , Humanos , Isomerismo , Metástase Neoplásica/diagnóstico , Metástase Neoplásica/patologia , Porosidade , Ácidos Siálicos/metabolismo , Espectrometria de Massas em Tandem/métodosRESUMO
Protein glycosylation, an important PTM, plays an essential role in a wide range of biological processes such as immune response, intercellular signaling, inflammation, and host-pathogen interaction. Aberrant glycosylation has been correlated with various diseases. However, studying protein glycosylation remains challenging because of low abundance, microheterogeneities of glycosylation sites, and poor ionization efficiency of glycopeptides. Therefore, the development of sensitive and accurate approaches to characterize protein glycosylation is crucial. The identification and characterization of protein glycosylation by MS is referred to as the field of glycoproteomics. Methods such as enrichment, metabolic labeling, and derivatization of glycopeptides in conjunction with different MS techniques and bioinformatics tools, have been developed to achieve an unequivocal quantitative and qualitative characterization of glycoproteins. This review summarizes the recent developments in the field of glycoproteomics over the past 6 years (2012 to 2018).
Assuntos
Glicopeptídeos/análise , Glicoproteínas/análise , Espectrometria de Massas/métodos , Proteômica/métodos , Animais , Glicosilação , Humanos , CamundongosRESUMO
Glycosylation of proteins is an important post-translational modification that plays a role in a wide range of biological processes, including immune response, intercellular signaling, inflammation, and host-pathogen interaction. Abnormal protein glycosylation has been correlated with various diseases. However, the study of protein glycosylation remains challenging due to its low abundance, microheterogeneity of glycosylation sites, and low ionization efficiency. During the past decade, several methods for enrichment and for isolation of glycopeptides from biological samples have been developed and successfully employed in glycoproteomics research. In this chapter, we discuss the sample preparation protocol and the strategies for effectively isolating and enriching glycopeptides from biological samples, using PolyHYDROXYETHYL A as a hydrophilic interaction liquid chromatography (HILIC) enrichment technique.
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Glicopeptídeos , Processamento de Proteína Pós-Traducional , Glicopeptídeos/análise , Cromatografia Líquida/métodos , Glicosilação , Interações Hidrofóbicas e HidrofílicasRESUMO
The neurological condition known as narcolepsy type 1 (NT1) is an uncommon condition marked by extreme daytime sleepiness, cataplexy, sleep paralysis, hallucinations, disrupted nocturnal sleep, and low or undetectable levels of orexin in the CSF fluid. NT1 has been hypothesized to be an immunological disorder; its treatment is currently only symptomatic, and misdiagnosis is not uncommon. This study compares the N-glycome of NT1 patients with healthy controls in search of potential glycan biomarkers using LC-MS/MS. A total of 121 candidate N-glycans were identified, 55 of which were isomeric N-glycan structures and 65 were not. Seventeen N-glycan biomarker candidates showed significant differences between the NT1 and control cohorts. All of the candidate glycan biomarkers were isomeric except HexNAc6Hex7Fuc0NeuAc1 (6701) and HexNAc6Hex7Fuc1NeuAc2 (6712). Therefore, with isomeric and nonisomeric structures, a total of 20 candidate N-glycan biomarkers are reported in this study, and interestingly, all are either sialylated or sialylated-fucosylated and upregulated in NT1 relative to the control. The distribution levels of all the identified N-glycans show that the sialylated glycan type is the most abundant in NT1 and is majorly disialylated, although the trisialylated subtype is three-fold higher in NT1 compared to the healthy control. The first isomers of HexNAc5Hex6Fuc0NeuAc3 (5603), HexNAc6Hex7Fuc0NeuAc2 (6702), and HexNAc6Hex7Fuc1NeuAc4 (6714) expressed a high level of fold changes (FC) of 1.62, 2.19, and 2.98, respectively. These results suggest a different N-glycome profile of NT1 and a relationship between sialylated glycan isomers in NT1 disease development or progression. The revelation of N-glycan expression alterations in this study may improve NT1 diagnostic methods, understanding of NT1 pathology, and the development of new targeted therapeutics.
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Neuroproteomics, an emerging field at the intersection of neuroscience and proteomics, has garnered significant attention in the context of neurotrauma research. Neuroproteomics involves the quantitative and qualitative analysis of nervous system components, essential for understanding the dynamic events involved in the vast areas of neuroscience, including, but not limited to, neuropsychiatric disorders, neurodegenerative disorders, mental illness, traumatic brain injury, chronic traumatic encephalopathy, and other neurodegenerative diseases. With advancements in mass spectrometry coupled with bioinformatics and systems biology, neuroproteomics has led to the development of innovative techniques such as microproteomics, single-cell proteomics, and imaging mass spectrometry, which have significantly impacted neuronal biomarker research. By analyzing the complex protein interactions and alterations that occur in the injured brain, neuroproteomics provides valuable insights into the pathophysiological mechanisms underlying neurotrauma. This review explores how such insights can be harnessed to advance personalized medicine (PM) approaches, tailoring treatments based on individual patient profiles. Additionally, we highlight the potential future prospects of neuroproteomics, such as identifying novel biomarkers and developing targeted therapies by employing artificial intelligence (AI) and machine learning (ML). By shedding light on neurotrauma's current state and future directions, this review aims to stimulate further research and collaboration in this promising and transformative field.
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Narcolepsy type 1 (NT1) is the most common type of narcolepsy known to be caused by the loss of specific neurons responsible for producing peptide neurotransmitters (orexins/hypocretins), resulting in a sleep-wake cycle disorder. It is characterized by its association with cataplexy and abnormalities in rapid eye movement. To date, no cure has been established for this life-threatening condition. Misdiagnosis of NT1 is also quite common, although it is not exceedingly rare. Therefore, successfully identifying candidate serum biomarkers for NT1 would be a head start for accurate diagnosis and development of therapeutics for this disorder. This study aims to identify such potential serum biomarkers. A depletion protocol was employed for 27 human serum samples (16 NT1 and 11 healthy controls), followed by applying LC-MS/MS bottom-up proteomics analysis, then LC-PRM-MS for validation. The comparison of the proteome profiles of the low-abundant proteins in the samples was then investigated based on age, sex, sample groups, and the presence of the Human Leukocyte Antigen (HLA) DQB1*0602 allele. The results were tracked to gene expression studies as well as system biology to identify key proteins and understand their relationship in the pathogenesis of NT1. Our results revealed 36 proteins significantly and differentially expressed. Among the impaired pathways and bioprocesses, the complement activation pathway is impaired by six of the differentially expressed proteins (DEPs). They are coded by the genes C2, CFB, C5, C1R, C1S, and MASP1, while 11 DEPs are involved in Acute Phase Response Signaling (APRS), which are coded by the genes FN1, AMBP, APOH, CFB, CP, ITIH2, C5, C2, F2, C1, and ITIH4. The combined AUCs of the downregulated and upregulated DEPs are 0.95 and 0.76, respectively. Overall, this study reveals potential serum-protein biomarkers of NT1 and explains the possible correlation between the biomarkers and pathophysiological effects, as well as important biochemical pathways involved in NT1.
Assuntos
Narcolepsia , Proteômica , Humanos , Cromatografia Líquida , Espectrometria de Massas em Tandem , Narcolepsia/etiologia , Narcolepsia/genética , Biomarcadores , OrexinasRESUMO
Column compartments in liquid chromatography (LC) systems house the LC columns. These compartments are responsible for maintaining a suitable column environment for achieving optimal chromatographic performance. However, the advancements in instrument and column designs demand newer technologies. It is a well-established concept that decreasing the dead volume of the column improves the column resolution, thereby providing enhanced chromatographic separation. One of the major contributors in the dead volume is the line connecting the column in the LC compartment to the ion source in the mass spectrometer. Using in-source emitter columns is one strategy to enhance the resolution. However, ion sources without temperature control are not suitable for columns that are used at high temperatures. In this work, we are introducing a nano electrospray ionization source with an integrated Peltier heater designed for pulled capillary nanospray emitter columns. Although the performance of the device is demonstrated by showing the isomeric separation of permethylated glycans using a mesoporous graphitized carbon packed pulled capillary emitter, it can easily be paired with any nanospray emitter column that requires temperature control.
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Polissacarídeos , Espectrometria de Massas por Ionização por Electrospray , Carbono , Cromatografia Líquida/métodos , Isomerismo , Polissacarídeos/química , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
Traumatic brain injury (TBI) is a heterogeneous disease in its origin, neuropathology, and prognosis, with no FDA-approved treatments. The pathology of TBI is complicated and not sufficiently understood, which is the reason why more than 30 clinical trials in the past three decades turned out unsuccessful in phase III. The multifaceted pathophysiology of TBI involves a cascade of metabolic and molecular events including inflammation, oxidative stress, excitotoxicity, and mitochondrial dysfunction. In this study, an open head TBI mouse model, induced by controlled cortical impact (CCI), was used to investigate the chronic protective effects of mitoquinone (MitoQ) administration 30 days post-injury. Neurological functions were assessed with the Garcia neuroscore, pole climbing, grip strength, and adhesive removal tests, whereas cognitive and behavioral functions were assessed using the object recognition, Morris water maze, and forced swim tests. As for molecular effects, immunofluorescence staining was conducted to investigate microgliosis, astrocytosis, neuronal cell count, and axonal integrity. The results show that MitoQ enhanced neurological and cognitive functions 30 days post-injury. MitoQ also decreased the activation of astrocytes and microglia, which was accompanied by improved axonal integrity and neuronal cell count in the cortex. Therefore, we conclude that MitoQ has neuroprotective effects in a moderate open head CCI mouse model by decreasing oxidative stress, neuroinflammation, and axonal injury.
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The proteome represents all the proteins expressed by a genome, a cell, a tissue, or an organism at any given time under defined physiological or pathological circumstances. Proteomic analysis has provided unparalleled opportunities for the discovery of expression patterns of proteins in a biological system, yielding precise and inclusive data about the system. Advances in the proteomics field opened the door to wider knowledge of the mechanisms underlying various post-translational modifications (PTMs) of proteins, including glycosylation. As of yet, the role of most of these PTMs remains unidentified. In this state-of-the-art review, we present a synopsis of glycosylation processes and the pathophysiological conditions that might ensue secondary to glycosylation shortcomings. The dynamics of protein glycosylation, a crucial mechanism that allows gene and pathway regulation, is described. We also explain how-at a biomolecular level-mutations in glycosylation-related genes may lead to neuropsychiatric manifestations and neurodegenerative disorders. We then analyze the shortcomings of glycoproteomic studies, putting into perspective their downfalls and the different advanced enrichment techniques that emanated to overcome some of these challenges. Furthermore, we summarize studies tackling the association between glycosylation and neuropsychiatric disorders and explore glycoproteomic changes in neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntington disease, multiple sclerosis, and amyotrophic lateral sclerosis. We finally conclude with the role of glycomics in the area of traumatic brain injury (TBI) and provide perspectives on the clinical application of glycoproteomics as potential diagnostic tools and their application in personalized medicine.
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Glicômica , Doenças Neurodegenerativas , Biomarcadores/metabolismo , Humanos , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Proteoma , Proteômica/métodosRESUMO
Mild cognitive impairment (MCI) is an early stage of memory loss that affects cognitive abilities, such as language or virtual/spatial comprehension. This cognitive decline is mostly observed with the aging of individuals. Recently, MCI has been considered as a prodromal phase of Alzheimer's disease (AD), with a 10-15% conversion rate. However, the existing diagnostic methods fail to provide precise and well-timed diagnoses, and the pathophysiology of MCI is not fully understood. Alterations of serum N-glycan expression could represent essential contributors to the overall pathophysiology of neurodegenerative diseases and be used as a potential marker to assess MCI diagnosis using non-invasive procedures. Herein, we undertook an LC-MS/MS glycomics approach to determine and characterize potential N-glycan markers in depleted blood serum samples from MCI patients. For the first time, we profiled the isomeric glycome of the low abundant serum glycoproteins extracted from serum samples of control and MCI patients using an LC-MS/MS analytical strategy. Additionally, the MRM validation of the identified data showed five isomeric N-glycans with the ability to discriminate between healthy and MCI patients: the sialylated N-glycans GlcNAc5,Hex6,Neu5Ac3 and GlcNAc6,Hex7,Neu5Ac4 with single AUCs of 0.92 and 0.87, respectively, and a combined AUC of 0.96; and the sialylated-fucosylated N-glycans GlcNAc4,Hex5,Fuc,Neu5Ac, GlcNAc5,Hex6,Fuc,Neu5Ac2, and GlcNAc6,Hex7,Fuc,Neu5Ac3 with single AUCs of 0.94, 0.67, and 0.88, respectively, and a combined AUC of 0.98. According to the ingenuity pathway analysis (IPA) and in line with recent publications, the identified N-glycans may play an important role in neuroinflammation. It is a process that plays a fundamental role in neuroinflammation, an important process in the progression of neurodegenerative diseases.
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Disfunção Cognitiva , Soro , Humanos , Cromatografia Líquida/métodos , Soro/química , Espectrometria de Massas em Tandem/métodos , Polissacarídeos/análise , Glicoproteínas , Biomarcadores , Disfunção Cognitiva/diagnósticoRESUMO
Background: Glycans play essential functional roles in the nervous system and their pathobiological relevance has become increasingly recognized in numerous brain disorders, but not fully explored in traumatic brain injury (TBI). We investigated longitudinal glycome patterns in patients with moderate to severe TBI (Glasgow Coma Scale [GCS] score ≤12) to characterize glyco-biomarker signatures and their relation to clinical features and long-term outcome. Methods: This prospective single-center observational study included 51 adult patients with TBI (GCS ≤12) admitted to the neurosurgical unit of the University Hospital of Pecs, Pecs, Hungary, between June 2018 and April 2019. We used a high-throughput liquid chromatography-tandem mass spectrometry platform to assess serum levels of N-glycans up to 3 days after injury. Outcome was assessed using the Glasgow Outcome Scale-Extended (GOS-E) at 12 months post-injury. Multivariate statistical techniques, including principal component analysis and orthogonal partial least squares discriminant analysis, were used to analyze glycomics data and define highly influential structures driving class distinction. Receiver operating characteristic analyses were used to determine prognostic accuracy. Findings: We identified 94 N-glycans encompassing all typical structural types, including oligomannose, hybrid, and complex-type entities. Levels of high mannose, hybrid and sialylated structures were temporally altered (p<0·05). Four influential glycans were identified. Two brain-specific structures, HexNAc5Hex3DeoxyHex0NeuAc0 and HexNAc5Hex4DeoxyHex0NeuAc1, were substantially increased early after injury in patients with unfavorable outcome (GOS-E≤4) (area under the curve [AUC]=0·75 [95%CI 0·59-0·90] and AUC=0·71 [0·52-0·89], respectively). Serum levels of HexNAc7Hex7DeoxyHex1NeuAc2 and HexNAc8Hex6DeoxyHex0NeuAc0 were persistently increased in patients with favorable outcome, but undetectable in those with unfavorable outcome. Levels of HexNAc5Hex4DeoxyHex0NeuAc1 were acutely elevated in patients with mass lesions and in those requiring decompressive craniectomy. Interpretation: In spite of the exploratory nature of the study and the relatively small number of patients, our results provide to the best of our knowledge initial evidence supporting the utility of glycomics approaches for biomarker discovery and patient phenotyping in TBI. Further larger multicenter studies will be required to validate our findings and to determine their pathobiological value and potential applications in practice. Funding: This work was funded by the Italian Ministry of Health (grant number GR-2013-02354960), and also partially supported by a NIH grant (1R01GM112490-08).