RESUMO
Hepatocellular cancer is a serious human disease with an unfortunately low survival rate. It further poses a significant epidemic threat to our society through its viral vectors associated with cirrhosis conditions preceding the cancer. A search for biomarkers of these diseases enlists analytical glycobiology, in general, and quantitative biomolecular mass spectrometry (MS), in particular, as valuable approaches to cancer research. The recent advances in quantitative glycan permethylation prior to MALDI-MS oligosaccharide profiling has enabled us to compare the glycan quantitative proportions in the small serum samples of cancer and cirrhotic patients against control individuals. We were further able to fractionate the major serum proteins from the minor components and compare statistically their differential glycosylation, elucidating some causes of quantitatively unusual glycosylation events. Numerous glycan structures were tentatively identified and connected with the origin proteins, with a particular emphasis on sialylated and fucosylated glycans.
RESUMO
Heavy metal exposure in children has been associated with a variety of physiological and neurological problems. The goal of this study was to utilize proteomics to enhance the understanding of biochemical interactions responsible for the health problems related to lead and mercury exposure at concentrations well below CDC guidelines. Blood plasma and serum samples from 34 children were depleted of their most abundant proteins using antibody-based affinity columns and analyzed using two different methods, LC-MS/MS and 2-D electrophoresis coupled with MALDI-TOF/MS and tandem mass spectrometry. Apolipoprotein E demonstrated an inverse significant association with lead concentrations (average being one microgram/deciliter) as deduced from LC-MS/MS and 2-D electrophoresis and confirmed by Western blot analysis. This coincides with prior findings that Apolipoprotein E genotype moderates neurobehavioral effects in individuals exposed to lead. Fifteen other proteins were identified by LC-MS/MS as proteins of interest exhibiting expressional differences in the presence of environmental lead and mercury.
Assuntos
Proteínas Sanguíneas/química , Intoxicação por Chumbo/sangue , Intoxicação por Mercúrio/sangue , Proteoma/efeitos dos fármacos , Proteômica/métodos , Sequência de Aminoácidos , Apolipoproteínas E/sangue , Apolipoproteínas E/química , Proteínas Sanguíneas/metabolismo , Western Blotting , Criança , Cromatografia Líquida , Estudos Transversais , Eletroforese em Gel Bidimensional , Feminino , Humanos , Masculino , Fragmentos de Peptídeos/sangue , Fragmentos de Peptídeos/química , Proteoma/química , Proteoma/metabolismo , Reprodutibilidade dos Testes , Fatores Sexuais , Fatores Socioeconômicos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem , Tripsina/sangue , Tripsina/metabolismoRESUMO
The study of protein glycosylation in biological fluids and tissues has substantial medical importance, as changes in glycan structures have now been associated with a number of diseases. Quantification of glycomic-profile changes is becoming increasingly important in the search for disease biomarkers. Here, we report a highly reproducible combination of a glycomic sample preparation/solid-phase derivatization of glycoprotein-derived N-linked glycans with their subsequent microchip-based separation and mass-spectrometric (MS) measurements. Following our previously described reductive beta-elimination for O-linked glycans with ammonia-borane complex to reduce N-linked structures, the N-linked alditol structures are effectively methylated in dimethylformamide medium to avoid artefacts in MS measurements. Reversed-phase microfluidic liquid chromatography (LC) of methylated N-linked oligosaccharide alditols resolved some closely related structures into regular retention increments, aiding in their structural assignments. Optimized LC gradients, together with nanospray MS, have been applied here in the quantitative measurements of N-linked glycans in blood serum, distinguishing breast cancer patients from control individuals.
Assuntos
Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/química , Cromatografia de Fase Reversa/métodos , Espectrometria de Massas/métodos , Polissacarídeos/sangue , Polissacarídeos/química , Amônia/química , Biomarcadores Tumorais/isolamento & purificação , Boranos/química , Neoplasias da Mama/diagnóstico , Feminino , Glicômica/métodos , Glicoproteínas/sangue , Glicoproteínas/química , Humanos , Metilação , Polissacarídeos/isolamento & purificação , Reprodutibilidade dos TestesRESUMO
Although glycoproteomic studies provide unique opportunities for cancer research, it has been necessary to develop specific methods for analysis of oncologically interesting glycoproteins. We describe a general, multimethodological approach for quantitative glycoproteomic analysis of fucosylated glycoproteins in human blood serum. A total of 136 putative fucosylated glycoproteins were identified with very high confidence in three clinically relevant sample pools (N=5 for each), with a mean CV of 3.1% observed for replicate analyses. Two samples were collected from subjects diagnosed with esophagus disease states, high-grade dysplasia plus esophageal adenocarcinoma, while the third sample was representative of a disease-free condition. Some glycoproteins, observed to be significantly upregulated in esophageal adenocarcinoma, i.e. more than twofold higher than in the disease-free condition, are briefly discussed. Further investigation will be necessary to validate these findings; however, the method itself is demonstrated to be an effective tool for quantitative glycoproteomics of clinical samples.
Assuntos
Adenocarcinoma/sangue , Neoplasias Esofágicas/sangue , Fucose/sangue , Glicoproteínas/sangue , Proteoma/análise , Colágeno Tipo I/análise , Colágeno Tipo I/sangue , Cadeia alfa 1 do Colágeno Tipo I , Fetuína-B , Glicoproteínas/análise , Humanos , Lectinas/química , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/metabolismo , Tripsina/metabolismo , alfa-Fetoproteínas/análise , alfa-Fetoproteínas/metabolismoRESUMO
Glycosylation of proteins is one of the most common posttranslational modifications which has its bearing on function and biological activity. Assigning the glycosylation sites and their inherent microheterogeneities are key structural issues addressing various glycoprotein functions. This chapter describes three different approaches all based on liquid chromatography/tandem mass spectrometry (LC/MS-MS), which are commonly employed for the assignment of protein glycosylation sites and their microheterogeneities. Comparing the LC/MS-MS analysis of a native glycoprotein tryptic digest to that of a deglycosylated tryptic digest can be accomplished through a routine LC/MS instrument. The use of a scanning mass spectrometer capable of switching between high-voltage and low-voltage scans, combined with monitoring carbohydrate-characteristic oxonium ions, is yet another analytical approach utilized for characterization of the glycosylation sites of glycoproteins. These two approaches do not address the problem originating from the ion suppression associated with coeluting peptides. The use of on-line glycopeptide enrichment in conjunction with LC/MS-MS is a third approach, which reduces ion suppression, thus offering a more sensitive approach to the characterization of protein glycosylation sites.
Assuntos
Cromatografia Líquida/métodos , Glicoproteínas/análise , Glicoproteínas/metabolismo , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bovinos , Cromatografia de Afinidade , Glicopeptídeos/análise , Glicopeptídeos/química , Glicopeptídeos/metabolismo , Glicoproteínas/química , Glicosilação , Lectinas/química , Dados de Sequência Molecular , Dióxido de Silício/química , Tripsina/metabolismo , alfa-Fetoproteínas/análise , alfa-Fetoproteínas/química , alfa-Fetoproteínas/metabolismoRESUMO
Posttranslational modifications (PTM) of proteins are among the key biological regulators of function, activity, localization, and interaction. The fact that no more than 30,000-50,000 proteins are encoded by the human genome underlines the importance of posttranslational modifications in modulating the activities and functions of proteins in health and disease. With approximately 50% of all proteins now considered to be glycosylated, its physiological importance in mammalian systems is imperative. Aberrant glycosylation has now been recognized as an attribute of many mammalian diseases, including hereditary disorders, immune deficiencies, neurodegenerative diseases, cardiovascular conditions, and cancer. As many potential disease biomarkers may be glycoproteins present in only minute quantities in tissue extracts and physiological fluids, glycoprotein isolation and enrichment may be critical in a search for such biomarkers. For decades, efforts have been focused on the development of glycoprotein enrichment from complex biological samples. Logically, the great majority of these enrichment methodologies rely on the use of immobilized lectins, which permit selective enrichment of the pools of glycoproteins for proteomic/glycomic studies. In this chapter, lectin affinity chromatography in different formats are described, including tubes; packed columns, and microfluidic channels.
Assuntos
Cromatografia de Afinidade/métodos , Genoma Humano , Glicoproteínas/análise , Glicoproteínas/isolamento & purificação , Lectinas/química , Técnicas Analíticas Microfluídicas/métodos , Biomarcadores/análise , Biomarcadores/química , Doenças Cardiovasculares/metabolismo , Doenças Genéticas Inatas/metabolismo , Glicoproteínas/química , Glicoproteínas/metabolismo , Glicosilação , Humanos , Doenças do Sistema Imunitário/metabolismo , Neoplasias/metabolismo , Doenças Neurodegenerativas/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
Reproducible and efficient affinity enrichment is increasingly viewed as an essential step in many investigations leading to the discovery of new biomarkers. In this work, we have evaluated the repeatability of lectin enrichment of glycoproteins from human blood serum through both qualitative and quantitative proteomic approaches. In a comprehensive evaluation of lectin binding, we have performed 30 separate microscale lectin affinity chromatography experiments, followed by a conventional sample purification, and LC-MS/MS analysis of the enriched glycoproteins. Two lectin affinity matrixes, both with Con A lectin, immobilized to the same solid support but differing in the amount of immobilized lectin, were investigated to characterize their binding properties. Both qualitative and quantitative data indicate acceptable repeatability and binding efficiency for the lectin materials received from two different commercial sources.
Assuntos
Cromatografia de Afinidade/métodos , Concanavalina A/química , Glicoproteínas/sangue , Lectinas/química , Cromatografia de Afinidade/instrumentação , Cromatografia Líquida/instrumentação , Cromatografia Líquida/métodos , Feminino , Glicoproteínas/química , Humanos , Nanotecnologia/instrumentação , Nanotecnologia/métodos , Ligação Proteica , Proteoma , Valores de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização por Electrospray/instrumentação , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/instrumentação , Espectrometria de Massas em Tandem/métodosRESUMO
We report here the use of high-performance lectin affinity enrichment of glycoproteins at microscale levels using a series of silica-bound lectins. The potential of this approach is being demonstrated for the glycoprotein enrichment from microliter volumes of human blood serum. Individual injections of sample to the affinity microcolumns packed with four lectin materials with different glycan specificities (Con A, SNA-I, UEA-I, PHA-L), followed by off-line reversed-phase pre-fractionation and nano-LC/MS/MS, permitted identification of 108 proteins in the lectin-bound fractions spanning a concentration dynamic range of 7-10 orders of magnitude. In contrast, multi-lectin microcolumn affinity chromatography, an alternative enrichment approach allowed identification of only 67 proteins. An attractive feature of high-performance lectin affinity chromatography at microscale levels is the substantial reduction of sample losses that are commonly experienced with extensive sample preparation needed for larger sample volumes.
Assuntos
Cromatografia de Afinidade/métodos , Cromatografia Líquida/métodos , Glicoproteínas/sangue , Espectrometria de Massas em Tandem/métodos , Glicoproteínas/isolamento & purificação , Humanos , LectinasRESUMO
In simplifying the evaluation and quantification of high-throughput label-free quantitative proteomic data, we introduce ProteinQuant Suite. It comprises three standalone complementary computer utilities, namely ProtParser, ProteinQuant, and Turbo RAW2MGF. ProtParser is a filtering utility designed to evaluate database search results. Filtering is performed according to different criteria that are defined by the end-user. ProteinQuant then utilizes this parsed list of peptides and proteins in conjunction with mzXML or mzData files generated from the raw files for quantification. This quantification is based on the automatic detection and integration of chromatographic peaks representative of the liquid chromatography/mass spectrometry (LC/MS) elution profiles of identified peptides. Turbo RAW2MGF was developed to extend the applicability of ProteinQuant Suite to data collected from different types of mass spectrometers. It directly processes raw data files generated by Xcalibur, a ThermoElectron data acquisition software, and generates a MASCOT generic file (MGF). This file format is needed since the protein identification results generated by the database search employing this file format include information required for the precise identification and quantification of chromatographic peaks. The performance of ProteinQuant Suite was initially validated using LC/MS/MS generated for a mixture of standard proteins as well as standard proteins spiked in a complex biological matrix such as blood serum. Automated quantification of the collected data resulted in calibration curves with R(2) values higher than 0.95 with linearity spanning over more than 2 orders of magnitude with peak quantification reproducibility better than 15% (RSD). ProteinQuant Suite was also applied to confirm the binding preference of standard glycoproteins to Con A lectin using a sample consisting of both standard glycoproteins and proteins.
Assuntos
Biologia Computacional/métodos , Proteômica/métodos , Software , Cromatografia Líquida , Bases de Dados de Proteínas , Glicoproteínas/análise , Humanos , Espectrometria de Massas , Peptídeos/análise , Proteínas/análise , Soro/químicaRESUMO
We describe an effective analytical approach to identify trace glycoproteins in a small volume of human serum. The system is based on automatable affinity enrichment through silica-based lectin microcolumns and a further separation of the retained glycoproteins on a reversed-phase liquid chromatography with superficially porous packing, operating at high temperature. The fractionated sample is further directed into a 96-well plate for trypsinization and LC-MS/MS analysis. Using a major-component depleted blood serum (16 microg total protein), we were able to identify 271 glycoproteins through this analytical system.
Assuntos
Glicoproteínas/sangue , Proteômica/métodos , Soro/química , Cromatografia de Afinidade/métodos , Humanos , Lectinas , Espectrometria de Massas/métodosRESUMO
Interactions of boar, bull, and human seminal plasma proteins with heparin and phosphorylcholine were studied by affinity LC using heparin immobilized to a Toyopearl support. A step gradient elution from 0.15 to 1.50 M NaCl was employed to elute the seminal plasma proteins. Relative amounts of the heparin-binding fraction of seminal plasma proteins (H+) in seminal plasma of three species were determined. Further on, the fraction of seminal plasma proteins interacting with phosphorylcholine-binding proteins (P+) was evaluated. P+ proteins were not found in human seminal plasma and their highest amount was present in bull seminal plasma. A CE method was developed for separation of seminal plasma proteins. Various capillaries and separation conditions were tested; the best resolution was obtained in a bare-silica capillary, with a micellar system consisting of a 0.02 M borate buffer and 0.05 M SDS pH 10.0. The optimized conditions were applied to the identification of the components in boar plasma.
Assuntos
Cromatografia de Afinidade/métodos , Cromatografia Líquida/métodos , Eletroforese Capilar/métodos , Proteínas de Plasma Seminal/análise , Animais , Heparina/química , Humanos , Masculino , Fosforilcolina/química , Isoformas de Proteínas/análiseRESUMO
Silica-based lectin microcolumns are described in this study together with the chemical procedures necessary for their preparation. The analytical merits of Canavalia ensiformis and Sambucus nigra lectins, [immobilized on activated macroporous silica], such as binding capacity, trapping reproducibility, and substrate selectivity, have been evaluated using model glycoproteins. The described microcolumns are applicable to high-pressure analytical schemes utilizing microvalving procedures, washing steps, and quantitative desorption for LC/MS analysis. The described analytical systems are amenable to the applications aiming at fractionation of complex glycopeptide mixtures and determination of the sites of glycosylation.