RESUMO
The embryonic cerebrospinal fluid (eCSF) influences neuroepithelial cell behavior, affecting proliferation, differentiation, and survival. One major question to resolve in the field is to precisely describe the eCSF molecules responsible and to understand how these molecules interact in order to exert their functions. Here we describe an in vitro protocol to analyze the influence of eCSF components on neuroepithelium development.
Assuntos
Técnicas de Cultura de Células/métodos , Proteínas do Líquido Cefalorraquidiano/metabolismo , Células Neuroepiteliais/citologia , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Proteínas do Líquido Cefalorraquidiano/isolamento & purificação , Proteínas do Líquido Cefalorraquidiano/fisiologia , Embrião de Galinha , Imuno-Histoquímica/métodos , Neurogênese , Técnicas de Cultura de Órgãos/métodos , Tegmento Mesencefálico/citologia , Tegmento Mesencefálico/embriologiaRESUMO
Data-independent acquisition (DIA) is becoming more prominent as a method for comprehensive proteomic analysis of clinical samples due to its ability to acquire essentially all fragment ion spectra in a single LC-ESI-MS/MS experiment. Since the direct correlation between a precursor and its fragment ions is lost when acquiring all ions in a defined m/z range, one data analysis strategy is using so-called peptide spectral libraries. These are usually generated by measuring similar biological samples in data-dependent (DDA) mode. The peptide spectral library content is a major limitation for the successful identification from DIA data. This is because a fragment ion spectrum from the sample can only be matched, and thus identified, when it is present in the peptide spectral library. In order to enhance peptide spectral library size, the sample for generating the peptide spectral library can be subjected to extended separation strategies prior to DDA. These strategies are of special relevance for biological samples containing a few very high-abundant proteins, such as CSF, as they enlarge the identification of low-abundant proteins. In instances of CSF separation, suitable methods include the 1D SDS-PAGE of proteins and high-pH reversed-phase peptide fractionation. Both methods are based on different protein/peptide characteristics, are complementary with one another, and are inexpensive and easy to establish. Ideally, DDA spectra from samples generated with both methods combine to achieve a comprehensive spectral library.
Assuntos
Proteínas do Líquido Cefalorraquidiano/isolamento & purificação , Fracionamento Químico/métodos , Peptídeos/líquido cefalorraquidiano , Peptídeos/isolamento & purificação , Proteínas do Líquido Cefalorraquidiano/análise , Proteínas do Líquido Cefalorraquidiano/química , Fracionamento Químico/instrumentação , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , Íons/líquido cefalorraquidiano , Íons/química , Biblioteca de Peptídeos , Peptídeos/química , Proteólise , Proteômica , Software , Espectrometria de Massas em TandemRESUMO
Proteomics is an indispensable tool for disease biomarker discovery. It is widely used for the analysis of biological fluids such as cerebrospinal fluid (CSF), blood, and saliva, which further aids in our understanding of disease incidence and progression. CSF is often the biospecimen of choice in case of intracranial tumors, as rapid changes in the tumor microenvironment can be easily assessed due to its close proximity to the brain. On the contrary studies comprising of serum or plasma samples do not truly reflect the underlying molecular alterations due to the presence of protective blood-brain barrier. We have described in here the detailed workflows for two advanced proteomics techniques, namely, 2D-DIGE (two-dimensional difference in-gel electrophoresis) and iTRAQ (isobaric tag for relative and absolute quantitation), for CSF analysis. Both of these techniques are very sensitive and widely used for quantitative proteomics analysis.
Assuntos
Neoplasias Encefálicas/líquido cefalorraquidiano , Proteínas do Líquido Cefalorraquidiano/análise , Proteínas do Líquido Cefalorraquidiano/isolamento & purificação , Fracionamento Químico/métodos , Glioma/líquido cefalorraquidiano , Proteômica/métodos , Neoplasias Encefálicas/química , Proteínas do Líquido Cefalorraquidiano/química , Fracionamento Químico/instrumentação , Glioma/química , Humanos , Espectrometria de Massas , Proteoma/química , Proteoma/metabolismo , Proteoma/normas , Proteômica/normas , Software , Coloração e Rotulagem/métodos , Microambiente Tumoral/genética , Eletroforese em Gel Diferencial Bidimensional/métodos , Fluxo de TrabalhoRESUMO
Proteo-peptidomic profiling of biofluids is used to identify disease biomarkers and to study molecular mechanisms of pathology development. Previously, we studied changes in cerebrospinal fluid (CSF) and blood plasma associated with Guillain-Barre syndrome (GBS)-a rare and severe disorder of the peripheral nervous system with an unknown etiology. Here, we describe the workflow for the analysis of endogenous peptides from CSF. The procedure covers sample preparation, liquid chromatography-mass spectrometry (LC-MS) analysis, and bioinformatics analysis and allows identification of more than 1100 peptides from 181 protein groups in ~3 h from a single CSF sample derived from non-neurological, non-oncological patients.
Assuntos
Proteínas do Líquido Cefalorraquidiano/isolamento & purificação , Peptídeos/líquido cefalorraquidiano , Peptídeos/isolamento & purificação , Proteômica/métodos , Biomarcadores/líquido cefalorraquidiano , Proteínas do Líquido Cefalorraquidiano/análise , Proteínas do Líquido Cefalorraquidiano/química , Proteínas do Líquido Cefalorraquidiano/metabolismo , Cromatografia Líquida/métodos , Biologia Computacional , Humanos , Espectrometria de Massas em Tandem , Fluxo de TrabalhoRESUMO
Molecular analysis of cerebrospinal fluid (CSF) provides comprehensive information on physiological and pathological processes related to the brain. In particular, proteomic studies give insights into the pathogenesis of many brain diseases which still pose diagnostic and therapeutic challenges. The identification of reliable biomarkers is an important step to meet these challenges. Mass spectrometry is an essential proteomic tool, not only for highly sensitive identification of proteins and posttranslational modifications, but also for their reliable quantification. Here, 18O labeling of tryptic peptides was employed to qualitative and quantitative analyses of protein fractions obtained by depletion of highly abundant proteins from cerebrospinal fluid. It was found that the execution of the investigated depletion protocols may cause the loss of potential protein biomarkers of neurological diseases.
Assuntos
Proteínas do Líquido Cefalorraquidiano/análise , Radioisótopos de Oxigênio , Biomarcadores/líquido cefalorraquidiano , Proteínas do Líquido Cefalorraquidiano/química , Proteínas do Líquido Cefalorraquidiano/isolamento & purificação , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Humanos , Marcação por Isótopo/métodos , Peptídeos/líquido cefalorraquidiano , Peptídeos/química , Peptídeos/isolamento & purificação , Proteólise , Proteômica/métodos , Software , Espectrometria de Massas em TandemRESUMO
Cerebrospinal fluid (CSF) is in direct contact with the brain and represents a valuable source of mediators that reflect metabolic processes occurring in the central nervous system (CNS). In this sense, mass spectrometry (MS) methods have proven to be sensitive in quantifying the proteomic profiles of CSF, therefore being able to detect biomarker candidates for neurological disorders. In particular, a key development has been the use of multiplexing technologies to easily identify and quantify complex protein mixtures. This chapter describes a workflow suitable for the analysis of CSF proteome using isobaric labeling coupled to strong cation-exchange chromatography fractionation for its potential use as a biomarker discovery platform. In this case, the isobaric tags for relative and absolute quantitation (iTRAQ) label all proteins in a sample via free amines at the N-terminus and on the side chain of lysine residues. Then, the labeled samples are pooled and chromatographically fractionated. These fractions with the pooled samples are afterward analyzed by tandem mass spectrometry (MS/MS), and proteins are quantified by the relative intensities of the reporter ions in the MS/MS spectra, simultaneously obtaining the amino acid sequence. This method complements the neuroproteomic toolbox to identify new protein biomarkers not only for the early clinical diagnosis and disease staging of CNS-related disorders but also to elucidate the molecular mechanisms related to the pathophysiology of these symptoms.
Assuntos
Proteínas do Líquido Cefalorraquidiano/análise , Proteínas do Líquido Cefalorraquidiano/isolamento & purificação , Proteoma/metabolismo , Proteômica/métodos , Cátions/química , Proteínas do Líquido Cefalorraquidiano/sangue , Fracionamento Químico/instrumentação , Fracionamento Químico/métodos , Cromatografia por Troca Iônica/métodos , Cromatografia Líquida , Humanos , Proteólise , Coloração e Rotulagem/métodos , Espectrometria de Massas em Tandem , Fluxo de TrabalhoRESUMO
Mass spectrometry (MS) has become the gold standard method for proteomics by allowing the simultaneous identification and/or quantification of thousands of proteins of a given sample. Over time, mass spectrometry has evolved into newer quantitative approaches with increased sensitivity and accuracy, such as the sequential windows acquisition of all theoretical fragment-ion spectra (SWATH)-MS approach. Moreover, in the past few years, some improvements were made in the SWATH-acquisition algorithm, allowing the design of sample-customized acquisition methods by adjusting the Q1 windows' width in order to reduce it in the most populated m/z regions. This customization results in an increase in the specificity and a reduction in the interferences, ultimately leading to an improvement in the amount of quantitative data extracted to eventually increase the proteome coverage. These improvements are especially relevant for clinical neuroproteomics, which is mainly based on the analysis of circulatory biofluids, in particular the cerebrospinal fluid (CSF) due to its close connection with the brain.In the present chapter, a detailed description of the methodologies necessary to perform a whole-proteome relative quantification of CSF samples by SWATH-MS is presented, starting with the isolation of the protein fraction, its preparation for MS analysis, with all the necessary information for the design of a SWATH-MS method specific for each sample batch, and finally providing different methodologies for the analysis of the quantitative data obtained.
Assuntos
Proteínas do Líquido Cefalorraquidiano/análise , Cromatografia Líquida/métodos , Proteoma/metabolismo , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Animais , Proteínas do Líquido Cefalorraquidiano/química , Proteínas do Líquido Cefalorraquidiano/isolamento & purificação , Humanos , Íons/química , Peptídeos/líquido cefalorraquidiano , Peptídeos/química , Peptídeos/isolamento & purificação , Proteólise , Proteoma/química , Ratos , SoftwareRESUMO
Cerebrospinal fluid (CSF) is the fluid of choice to study pathologies and disorders of the central nervous system (CNS). Its composition, especially its proteins and peptides, holds the promise that it may reflect the pathological state of an individual. Traditionally, proteins and peptides in CSF have been analyzed using bottom-up proteomics technologies in the search of high proteome coverage. However, the limited protein sequence coverage of this technology means that information regarding post-translational modifications (PTMs) and alternative splice variants is lost. As an alternative technology, top-down proteomics offers low to medium proteome coverage, but high protein coverage enabling almost a full characterization of the proteins' primary structure. This allows us to precisely identify distinct molecular forms of proteins (proteoforms) as well as naturally occurring bioactive peptide fragments, which could be of critical biological relevance and would otherwise remain undetected with a classical proteomics approach.Here, we describe various strategies including sample preparation protocols, off-line intact protein prefractionation, and LC-MS/MS methods together with data analysis pipelines to analyze cerebrospinal fluid (CSF) by top-down proteomics. However, there is not a unique or standardized method and the selection of the top-down strategy will depend on the exact goal of the study. Here, we describe various top-down proteomics methods that enable rapid protein characterization and may be an excellent companion analytical workflow in the search for new protein biomarkers in neurodegenerative diseases.
Assuntos
Proteínas do Líquido Cefalorraquidiano/análise , Proteoma/metabolismo , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Biomarcadores/metabolismo , Proteínas do Líquido Cefalorraquidiano/química , Proteínas do Líquido Cefalorraquidiano/isolamento & purificação , Fracionamento Químico/métodos , Cromatografia Líquida/métodos , Humanos , Fragmentos de Peptídeos/química , Peptídeos/líquido cefalorraquidiano , Peptídeos/química , Processamento de Proteína Pós-Traducional , Proteoma/química , Software , Fluxo de TrabalhoRESUMO
Cerebrospinal fluid (CSF) has been considered the key source for the search of biomarkers, in particular for neurological diseases, such as Alzheimer's and Parkinson's disease, since it reflects the state of the central nervous system (CNS). Finding biomarkers in the earliest stages of neurodegenerative diseases has become imperative, since, at the moment, there are no drugs that can reverse these pathological processes. Untargeted metabolomics analysis by liquid chromatography combined with SWATH-MS relative quantification is an emerging approach to search for potential biomarkers. In this chapter, we describe a method for untargeted metabolomics analysis of CSF samples that can also be used in parallel to a proteomics approach. The analysis is focused on the SWATH acquisition mode, where beyond precursor's relative quantification, the information of the MS/MS relative quantification is also used to help in the search of new potential biomarkers.
Assuntos
Biomarcadores/líquido cefalorraquidiano , Metabolômica/métodos , Biomarcadores/química , Proteínas do Líquido Cefalorraquidiano/química , Proteínas do Líquido Cefalorraquidiano/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Análise por Conglomerados , Humanos , Software , Extração em Fase Sólida , Espectrometria de Massas em Tandem , Fluxo de TrabalhoRESUMO
In the field of neurodegeneration, it is important to identify biomarkers that enable early disease prediction, since these disorders start decades before clinical symptoms manifest. Cerebrospinal fluid (CSF) is considered an excellent source for biomarker discovery since it is in direct contact with the extracellular space of the brain and directly reflects disease-specific changes.While the liquor drainage is no major risk factor for patients, it is still not as easy and popular as simple blood sampling and less liquid can be collected. Especially when a variety of experiments for one cohort is planned, the volume of CSF can be a limiting factor. Therefore, it is essential that extraction and analytical methods are adapted to low amounts of liquor. If in follow-up studies, additional replicates to increase statistical significance or different extraction approaches are planned, the required amounts have to be minimized.With this extraction method, a combined proteomics and metabolomics approach is possible. This opportunity implies a variety of advantages. First, a classification matrix based on the comprehensive data set has a potentially higher accuracy even without a deeper understanding of the biological meaning of the different omics changes. If the proteome and metabolome differences can be linked to each other, this approach can conceivably open so far unknown doors regarding the cause or progression of different diseases like Alzheimer's or Parkinson's disease.
Assuntos
Proteínas do Líquido Cefalorraquidiano/isolamento & purificação , Metabolômica/métodos , Proteômica/métodos , Encéfalo/metabolismo , Líquido Cefalorraquidiano/metabolismo , Proteínas do Líquido Cefalorraquidiano/metabolismo , Cromatografia Gasosa/métodos , Cromatografia Líquida de Alta Pressão/métodos , Humanos , Espectrometria de Massas/métodos , Metanol/química , Proteólise , Tripsina , Fluxo de TrabalhoRESUMO
In the field of proteomics, the emerging "top-down" MS-based proteomics approach can be used to obtain a bird's eye view of all intact proteoforms present in a sample. This alternative to the "bottom-up" approach based on tryptic protein digestion processes has some unique advantages for assessing PTMs and sequence variations. However, it requires some dedicated tools for sample preparation and LC-MS analysis, which makes it more complex to handle than the bottom-up approach. In this study, a simple methodology is presented for characterizing intact proteins in biological fluid. This method yields quantitative information using an MS1 profiling approach and makes it possible to identify the proteins regulated under various clinical conditions.
Assuntos
Biomarcadores , Proteínas do Líquido Cefalorraquidiano , Cromatografia Líquida , Espectrometria de Massas , Proteoma , Proteômica , Proteínas do Líquido Cefalorraquidiano/isolamento & purificação , Proteínas do Líquido Cefalorraquidiano/metabolismo , Interpretação Estatística de Dados , Humanos , Proteômica/métodos , Extração em Fase SólidaRESUMO
The cerebrospinal fluid (CSF) proteome data set presented herein was obtained after immunodepletion of abundant proteins and off-gel electrophoresis fractionation of a commercial pool of normal human CSF; liquid chromatography tandem mass spectrometry analysis was performed with a linear ion trap-Orbitrap Elite. We report the identification of 12â¯344 peptides mapping on 2281 proteins. In the context of the Chromosome-centric Human Proteome Project (C-HPP), the existence of seven missing proteins is proposed to be validated. This data set is available to the ProteomeXchange Consortium ( http://www.proteomexchange.org/ ) with the data set identifier PXD008029.
Assuntos
Proteínas do Líquido Cefalorraquidiano/análise , Proteoma/análise , Proteínas do Líquido Cefalorraquidiano/isolamento & purificação , Cromatografia Líquida , Mapeamento Cromossômico , Cromossomos Humanos , Humanos , Espectrometria de Massas em TandemRESUMO
Proteomics offers the simultaneous detection of a large number of proteins in a single experiment and can provide important information regarding crucial aspects of specific proteins, particularly post-translational modifications (PTMs). Investigations of oxidative PTMs are currently performed using focused redox proteomics techniques, which rely on gel electrophoresis separations of intact proteins with the final detection of oxidative PTMs being performed by mass spectrometry (MS) analysis. The application of this technique to human biofluids is being subject of increasing investigation and is expected to provide new insights on the oxidative status of the peripheral proteome in neurological diseases such as Alzheimer's disease, towards purposes of early diagnosis and prognosis. This chapter describes all the experimental steps to perform redox proteomics analysis of cerebrospinal fluid and plasma/serum samples.
Assuntos
Métodos Analíticos de Preparação de Amostras/métodos , Proteínas Sanguíneas/análise , Proteínas Sanguíneas/isolamento & purificação , Proteínas do Líquido Cefalorraquidiano/análise , Proteínas do Líquido Cefalorraquidiano/isolamento & purificação , Imunoquímica/métodos , Proteômica/métodos , Proteínas Sanguíneas/química , Proteínas Sanguíneas/metabolismo , Proteínas do Líquido Cefalorraquidiano/química , Proteínas do Líquido Cefalorraquidiano/metabolismo , Colódio/química , Eletroforese em Gel de Poliacrilamida , Humanos , Focalização Isoelétrica , Membranas Artificiais , Oxirredução , Proteólise , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tripsina/metabolismoRESUMO
Analysis of the cerebrospinal fluid (CSF) proteome has proven valuable to the study of neurodegenerative disorders. To identify new protein/pathway alterations and candidate biomarkers for amyotrophic lateral sclerosis (ALS), we performed comparative proteomic profiling of CSF from sporadic ALS (sALS), healthy control (HC), and other neurological disease (OND) subjects using label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 1712 CSF proteins were detected and relatively quantified by spectral counting. Levels of several proteins with diverse biological functions were significantly altered in sALS samples. Enrichment analysis was used to link these alterations to biological pathways, which were predominantly related to inflammation, neuronal activity, and extracellular matrix regulation. We then used our CSF proteomic profiles to create a support vector machines classifier capable of discriminating training set ALS from non-ALS (HC and OND) samples. Four classifier proteins, WD repeat-containing protein 63, amyloid-like protein 1, SPARC-like protein 1, and cell adhesion molecule 3, were identified by feature selection and externally validated. The resultant classifier distinguished ALS from non-ALS samples with 83% sensitivity and 100% specificity in an independent test set. Collectively, our results illustrate the utility of CSF proteomic profiling for identifying ALS protein/pathway alterations and candidate disease biomarkers.
Assuntos
Doença de Alzheimer/diagnóstico , Esclerose Lateral Amiotrófica/diagnóstico , Proteínas do Líquido Cefalorraquidiano/isolamento & purificação , Doença dos Neurônios Motores/diagnóstico , Proteoma/isolamento & purificação , Proteínas Adaptadoras de Transdução de Sinal/líquido cefalorraquidiano , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/isolamento & purificação , Adulto , Doença de Alzheimer/líquido cefalorraquidiano , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/líquido cefalorraquidiano , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/isolamento & purificação , Esclerose Lateral Amiotrófica/líquido cefalorraquidiano , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Biomarcadores/líquido cefalorraquidiano , Proteínas de Ligação ao Cálcio/líquido cefalorraquidiano , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/isolamento & purificação , Estudos de Casos e Controles , Moléculas de Adesão Celular/líquido cefalorraquidiano , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/isolamento & purificação , Proteínas do Líquido Cefalorraquidiano/líquido cefalorraquidiano , Proteínas do Líquido Cefalorraquidiano/genética , Cromatografia Líquida/métodos , Diagnóstico Diferencial , Matriz Extracelular/química , Proteínas da Matriz Extracelular/líquido cefalorraquidiano , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/isolamento & purificação , Humanos , Imunoglobulinas/líquido cefalorraquidiano , Imunoglobulinas/genética , Imunoglobulinas/isolamento & purificação , Inflamação , Pessoa de Meia-Idade , Doença dos Neurônios Motores/líquido cefalorraquidiano , Doença dos Neurônios Motores/genética , Doença dos Neurônios Motores/patologia , Proteoma/genética , Proteoma/metabolismo , Proteômica/métodos , Sensibilidade e Especificidade , Máquina de Vetores de Suporte , Sinapses/genética , Sinapses/metabolismo , Transmissão Sináptica , Espectrometria de Massas em Tandem/métodosRESUMO
The progressive neurodegeneration in Alzheimer's disease is believed to be linked to the presence of prefibrillar aggregates of the amyloid-ß (Aß) peptide in the brain. The exact role of these aggregates in the disease pathology is, however, still an open question. Any mechanism by which oligomeric Aß may cause damage to neuronal cells must, in one way or another, involve interactions with other molecules. Here, we identify proteins in human serum and cerebrospinal fluid that bind to stable protofibrils formed by an engineered variant of Aß42 (Aß42CC). We find that the protofibrils attract a substantial number of protein binding partners. Many of the 101 identified proteins are involved in lipid transport and metabolism, the complement system, or in hemostasis. Binding of representative proteins from all of these groups with micromolar affinity was confirmed using surface plasmon resonance. In addition, binding of apolipoprotein E to the protofibrils with nanomolar affinity was demonstrated. We also find that aggregation of Aß enhances protein binding, as lower amounts of proteins bind monomeric Aß. Proteins that bind to Aß protofibrils might contribute to biological effects in which these aggregates are involved. Our results therefore suggest that an improved understanding of the mechanisms by which Aß causes cytotoxicity and neurodegeneration might be gained from studies carried out in biologically relevant matrices in which Aß-binding proteins are present.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Apolipoproteínas E/química , Proteínas Sanguíneas/química , Proteínas do Líquido Cefalorraquidiano/química , Fragmentos de Peptídeos/química , Idoso , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Apolipoproteínas E/isolamento & purificação , Proteínas Sanguíneas/isolamento & purificação , Proteínas do Líquido Cefalorraquidiano/isolamento & purificação , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Expressão Gênica , Humanos , Cinética , Masculino , Pessoa de Meia-Idade , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Agregados Proteicos , Ligação Proteica , Engenharia de Proteínas , Mapeamento de Interação de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ressonância de Plasmônio de SuperfícieRESUMO
The cerebrospinal fluid (CSF) proteome is of great interest for investigation of diseases and conditions involving the CNS. However, the presence of high-abundance proteins (HAPs) can interfere with the detection of low-abundance proteins, potentially hindering the discovery of new biomarkers. Therefore, an assessment of the CSF subproteome composition requires depletion strategies. Existing methods are time consuming, often involving multistep protocols. Here, we present a rapid, accurate, and reproducible method for preparing the CSF proteome, which allows the identification of a high number of proteins. This method involves acetonitrile (ACN) precipitation for depleting HAPs, followed by immediate trypsination. As an example, we demonstrate that this method allows discrimination between multiple sclerosis patients and healthy subjects.
Assuntos
Proteínas do Líquido Cefalorraquidiano/isolamento & purificação , Proteoma/isolamento & purificação , Esclerose/líquido cefalorraquidiano , Acetonitrilas/química , Proteínas do Líquido Cefalorraquidiano/análise , Proteínas do Líquido Cefalorraquidiano/metabolismo , Precipitação Química , Cromatografia Líquida , Humanos , Proteoma/análise , Proteoma/metabolismo , Espectrometria de Massas em Tandem , Tripsina/metabolismoRESUMO
Over the last years there has been an increased focus on the importance of knowing the effect of pre-analytical influence on the proteomes under study, particularly in the field of biomarker discovery. We present three proteomics studies examining the effect of blood contamination and the rostro-caudal gradient (RCG) on the cerebrospinal fluid (CSF) proteome, in addition to plasma/CSF protein ratios. The studies showed that the central nervous system (CNS) derived proteins appeared to be unaffected by the RCG, while the plasma-derived proteins showed an increase in concentration towards the lumbar area. This implies that the concentration of the plasma-derived proteins in CSF will vary depending on the volume of CSF that is collected. In the CSF samples spiked with blood, 262 of 814 quantified proteins showed an abundance increase of more than 1.5 fold, while 403 proteins had a fold change of less than 1.2 and appeared to be unaffected by blood contamination. Proteins with a high plasma/CSF ratio appeared to give the largest effect on the CSF proteome upon blood contamination. The results give important background information on how factors like blood contamination, RCG and blood-CNS-barrier influences the CSF proteome. This information is particularly important in the field of biomarker discovery, but also for routine clinical measurements. The data from the blood contamination and RCG discovery studies have been deposited to the ProteomeXchange with identifier PXD000401.
Assuntos
Proteínas Sanguíneas/líquido cefalorraquidiano , Proteínas do Líquido Cefalorraquidiano/metabolismo , Paralisia Supranuclear Progressiva/líquido cefalorraquidiano , Artefatos , Proteínas Sanguíneas/isolamento & purificação , Proteínas do Líquido Cefalorraquidiano/isolamento & purificação , Cromatografia por Troca Iônica , Cromatografia de Fase Reversa , Humanos , Proteoma/isolamento & purificação , Proteoma/metabolismo , Punção EspinalRESUMO
Cerebrospinal fluid (CSF) is produced in the brain by cells in the choroid plexus at a rate of 500 mL/day. It is the only body fluid in direct contact with the brain. Thus, any changes in the CSF composition will reflect pathological processes and make CSF a potential source of biomarkers for different disease states. Proteomics offers a comprehensive view of the proteins found in CSF. In this study, we use a recently developed nongel based method of sample preparation of CSF followed by liquid chromatography-high accuracy mass spectrometry (LC-MS) for MS and MS/MS analyses, allowing unambiguous identification of peptides/proteins. Gel-eluted liquid fraction entrapment electrophoresis (Gelfree) is used to separate a CSF complex protein mixture in 12 user-selectable liquid-phase molecular weight fractions. Using this high throughput workflow, we have been able to separate CSF intact proteins over a broad mass range (3.5-100 kDa) with high resolution (between 15 and 100 kDa) in 2 h and 40 min. We have completely eliminated albumin and were able to interrogate the low abundance CSF proteins in a highly reproducible manner from different CSF samples at the same time. Using LC-MS as a downstream analysis, we identified 368 proteins using MidiTrap G-10 desalting columns and 166 proteins (including 57 unique proteins) using Zeba spin columns with a 5% false discovery rate (FDR). Prostaglandin D2 synthase, Chromogranin A, Apolipoprotein E, Chromogranin B, Secretogranin III, Cystatin C, VGF nerve growth factor, and Cadherin 2 are a few of the proteins that were characterized. Gelfree-LC-MS is a robust method for the analysis of the human proteome that we will use to develop biomarkers for several neurodegenerative diseases and to quantitate these markers using multiple reaction monitoring.
Assuntos
Proteínas do Líquido Cefalorraquidiano/isolamento & purificação , Proteoma/isolamento & purificação , Biomarcadores/metabolismo , Proteínas do Líquido Cefalorraquidiano/química , Proteínas do Líquido Cefalorraquidiano/metabolismo , Cromatografia de Fase Reversa , Humanos , Peso Molecular , Proteoma/química , Proteoma/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
Cerebrospinal fluid (CSF) can be considered the most promising biosample for the discovery and analysis of biomarkers in neuroscience, an area of great medical need. CSF is a body fluid that surrounds the brain and provides a rich pool of biochemical markers, both proteomic and metabolomic, that reflect the state of neurological processes. Such biomarkers can either serve as diagnostic or prognostic biomarkers to improve the characterization of patients and preclinical disease models, or can be used to demonstrate drug-related exposure and efficacy. Here, we describe the proteomic toolbox for studying CSF from a drug-discovery perspective, and the trends and challenges that lie ahead.
Assuntos
Proteínas do Líquido Cefalorraquidiano/metabolismo , Proteoma/metabolismo , Biomarcadores/química , Biomarcadores/metabolismo , Proteínas do Líquido Cefalorraquidiano/química , Proteínas do Líquido Cefalorraquidiano/isolamento & purificação , Cromatografia Líquida , Descoberta de Drogas , Eletroforese em Gel Bidimensional , Humanos , Imunoensaio , Espectrometria de Massas/métodos , Doenças do Sistema Nervoso/líquido cefalorraquidiano , Doenças do Sistema Nervoso/tratamento farmacológico , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Análise Serial de Proteínas , Proteoma/química , Proteoma/isolamento & purificação , ProteômicaRESUMO
The cerebrospinal fluid (CSF) perfuses the brain and spinal cord. CSF contains proteins and peptides important for brain physiology and potentially also relevant for brain pathology. Hence, CSF is the perfect source to search for new biomarkers to improve diagnosis of neurological diseases as well as to monitor the performance of disease-modifying drugs. This chapter presents methods for SELDI-TOF profiling of CSF as well as useful advice regarding pre-analytical factors to be considered.