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1.
Proc Natl Acad Sci U S A ; 118(35)2021 08 31.
Artículo en Inglés | MEDLINE | ID: mdl-34433667

RESUMEN

The fidelity of protein transport in the secretory pathway relies on the accurate sorting of proteins to their correct destinations. To deepen our understanding of the underlying molecular mechanisms, it is important to develop a robust approach to systematically reveal cargo proteins that depend on specific sorting machinery to be enriched into transport vesicles. Here, we used an in vitro assay that reconstitutes packaging of human cargo proteins into vesicles to quantify cargo capture. Quantitative mass spectrometry (MS) analyses of the isolated vesicles revealed cytosolic proteins that are associated with vesicle membranes in a GTP-dependent manner. We found that two of them, FAM84B (also known as LRAT domain containing 2 or LRATD2) and PRRC1, contain proline-rich domains and regulate anterograde trafficking. Further analyses revealed that PRRC1 is recruited to endoplasmic reticulum (ER) exit sites, interacts with the inner COPII coat, and its absence increases membrane association of COPII. In addition, we uncovered cargo proteins that depend on GTP hydrolysis to be captured into vesicles. Comparing control cells with cells depleted of the cargo receptors, SURF4 or ERGIC53, we revealed specific clients of each of these two export adaptors. Our results indicate that the vesicle formation assay in combination with quantitative MS analysis is a robust and powerful tool to uncover novel factors that mediate vesicular trafficking and to uncover cargo clients of specific cellular factors.


Asunto(s)
Proteínas Portadoras/metabolismo , Transporte de Proteínas , Vesículas Transportadoras/metabolismo , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Citosol/metabolismo , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Guanosina Trifosfato/metabolismo , Células HEK293 , Humanos , Técnicas In Vitro , Membranas Intracelulares/metabolismo , Espectrometría de Masas , Proteínas de la Membrana/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteínas de Neoplasias/metabolismo , Vías Secretoras
2.
Anal Chem ; 92(12): 8201-8208, 2020 06 16.
Artículo en Inglés | MEDLINE | ID: mdl-32426967

RESUMEN

The change in glycosylation of serum proteins is often associated with the development of various diseases and thus can be used for diagnosis. In this study, a liquid chromatography-tandem mass spectrometry-based method is used for accurate structural analysis and quantification of site-specific glycoforms of serum α-1-antitrypsin (A1AT) in early-stage HCC and cirrhosis patients. Serum protein A1AT was purified from patient sera by immunoprecipitation with anti-A1AT antibody conjugated agarose beads, and the isolated A1AT protein was digested and analyzed by LC-MS/MS. Two tandem mass spectrometry strategies are integrated in this study: a nontargeted stepped HCD strategy for structural analysis of A1AT glycopeptides and a targeted parallel reaction monitoring (PRM) strategy for quantification of site-specific glycoforms of A1AT in HCC and cirrhosis patient sera. Accordingly, pGlyco2.0 software was used for glycopeptide identification, and Skyline software was used for glycoform quantification using the Y1 ion (peptide+GlcNAc) in MS/MS spectra. Ten site-specific glycopeptides of A1AT were identified with stepped HCD-MS/MS in patient samples, 7 of which were further quantified using HCD-PRM-MS among patient samples. We found that our strategy was able to distinguish isomers of glycopeptides where several isomers showed distinctly different patterns between cirrhosis and HCC patients. We also found that the ratio of different charge states (2+/3+) of one glycopeptide of A1AT can significantly discriminate early-stage HCC from cirrhosis with the area under the receiver operating characteristic curve AUC of 0.9. Further analysis showed that the difference may be related to the sialic acid/galactose linkage of the glycan motif.


Asunto(s)
Carcinoma Hepatocelular/sangre , Neoplasias Hepáticas/sangre , alfa 1-Antitripsina/sangre , Carcinoma Hepatocelular/diagnóstico , Cromatografía Liquida , Femenino , Glicosilación , Humanos , Neoplasias Hepáticas/diagnóstico , Masculino , Persona de Mediana Edad , Isoformas de Proteínas/sangre , Espectrometría de Masas en Tándem
3.
J Proteome Res ; 14(11): 4932-9, 2015 Nov 06.
Artículo en Inglés | MEDLINE | ID: mdl-26448449

RESUMEN

Alterations in glycosylation of serum glycoproteins can provide unique and highly specific fingerprints of malignancy. Our previous mass spectrometric study revealed that the bifucosylation level of serum haptoglobin was distinctly increased in hepatocellular carcinoma (HCC) patients versus liver cirrhosis of all three major etiologies. We have thus developed a method for the analysis of large numbers of serum samples based on a 96-well plate platform for the evaluation of fucosylation changes of serum haptoglobin between HCC versus cirrhosis. Haptoglobin was isolated from the serum of individual patient samples based on an HPLC column immobilized with antihaptoglobin antibody via hydrazide immobilization chemistry. Only 10 µL of serum was required for glycan extraction and processing for MALDI-QIT mass spectrometry analysis using the 96-well plate format. The bifucosylation degrees of haptoglobin in individuals were calculated using a quantitative glycomics method. The MS data confirmed that the bifucosylated tetra-anntenary glycan was upregulated in HCC samples of all etiologies. This study provides a parallel method for processing glycan content for haptoglobin and evaluating detailed changes in glycan structures for a potentially large cohort of clinical serum samples.


Asunto(s)
Alcoholismo/sangre , Carcinoma Hepatocelular/sangre , Hepatitis B/sangre , Hepatitis C/sangre , Cirrosis Hepática/sangre , Neoplasias Hepáticas/sangre , Procesamiento Proteico-Postraduccional , Anciano , Alcoholismo/complicaciones , Alcoholismo/genética , Alcoholismo/patología , Biomarcadores/sangre , Secuencia de Carbohidratos , Carcinoma Hepatocelular/etiología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Cromatografía Líquida de Alta Presión , Femenino , Fucosa/metabolismo , Glicosilación , Haptoglobinas/genética , Haptoglobinas/metabolismo , Hepatitis B/complicaciones , Hepatitis B/genética , Hepatitis B/patología , Hepatitis C/complicaciones , Hepatitis C/genética , Hepatitis C/patología , Humanos , Cirrosis Hepática/complicaciones , Cirrosis Hepática/genética , Cirrosis Hepática/patología , Neoplasias Hepáticas/etiología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Estadificación de Neoplasias , Mapeo Peptídico , Proteolisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Tripsina/química
4.
J Proteome Res ; 14(12): 5388-95, 2015 Dec 04.
Artículo en Inglés | MEDLINE | ID: mdl-26503433

RESUMEN

A method for the detection of fucosylated glycans from haptoglobin in patient serum has been developed that provides enhanced sensitivity. The workflow involves isolation of the haptoglobin using an HPLC-based affinity column followed by glycan removal, extraction, and desialylation. The fucosylated glycans are then derivatized by Meladrazine, which significantly enhances the detection of the glycans in electrospray ionization. The separation of the derivatized glycans in a HILIC column shows that eight glycans from haptoglobin can be detected using less than 1 µL of a serum sample, with excellent reproducibility and quantitation, where without derivatization the glycans could not be detected. The ratio of the fucosylated peaks to their corresponding nonfucosylated forms shows that the fucosylated glycans are upregulated in the case of hepatocellular carcinoma (HCC) samples versus cirrhosis samples, where the relatively low abundance bifucosylated tetra-antennary form can be detected and may be a particularly good marker for HCC.


Asunto(s)
Carcinoma Hepatocelular/sangre , Haptoglobinas/química , Cirrosis Hepática/sangre , Neoplasias Hepáticas/sangre , Espectrometría de Masa por Ionización de Electrospray/métodos , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/química , Cromatografía Líquida de Alta Presión/métodos , Fucosa/sangre , Glicosilación , Humanos , Límite de Detección , Estructura Molecular , Polisacáridos/sangre , Polisacáridos/química , Reproducibilidad de los Resultados , Espectrometría de Masa por Ionización de Electrospray/estadística & datos numéricos
5.
J Proteome Res ; 14(11): 4876-84, 2015 Nov 06.
Artículo en Inglés | MEDLINE | ID: mdl-26403951

RESUMEN

A mass spectrometry-based methodology has been developed to screen for changes in site-specific core-fucosylation (CF) of serum proteins in early stage HCC with different etiologies. The methods involve depletion of high-abundance proteins, trypsin digestion of medium-to-low-abundance proteins into peptides, iTRAQ labeling, and Lens culinaris Agglutinin (LCA) enrichment of CF peptides, followed by endoglycosidase F3 digestion before mass spectrometry analysis. 1300 CF peptides from 613 CF proteins were identified from patients sera, where 20 CF peptides were differentially expressed in alcohol (ALC)-related HCC samples compared with ALC-related cirrhosis samples and 26 CF peptides changed in hepatitis C virus (HCV)-related HCC samples compared with HCV-related cirrhosis samples. Among these, we found three CF peptides from fibronectin upregulated in ALC-related HCC samples compared with ALC-related cirrhosis samples with an AUC (area under the curve) value of 0.89 at site 1007 with a specificity of 85.7% at a sensitivity of 92.9% (generated with 10-fold cross-validation). When combined with the AFP index, the AUC value reached to 0.92 with a specificity of 92.9% at a sensitivity of 100%, significantly improved compared to that with AFP alone (LR test p < 0.001). In HCV-related samples, the CF level of cadherin-5 at site 61 showed the best AUC value of 0.75 but was not as promising as that of fibronectin site 1007 for ALC-related samples. The CF peptides of fibronectin may serve as potential biomarkers for early stage HCC screening in ALC-related cirrhosis patients.


Asunto(s)
Alcoholismo/sangre , Proteínas Sanguíneas/metabolismo , Carcinoma Hepatocelular/sangre , Hepatitis B/sangre , Hepatitis C/sangre , Cirrosis Hepática/sangre , Neoplasias Hepáticas/sangre , Procesamiento Proteico-Postraduccional , Anciano , Anciano de 80 o más Años , Alcoholismo/complicaciones , Alcoholismo/genética , Alcoholismo/patología , Secuencia de Aminoácidos , Antígenos CD/sangre , Antígenos CD/genética , Biomarcadores/sangre , Proteínas Sanguíneas/genética , Cadherinas/sangre , Cadherinas/genética , Carcinoma Hepatocelular/etiología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Femenino , Fibronectinas/sangre , Fibronectinas/genética , Fucosa/metabolismo , Hepatitis B/complicaciones , Hepatitis B/genética , Hepatitis B/patología , Hepatitis C/complicaciones , Hepatitis C/genética , Hepatitis C/patología , Humanos , Cirrosis Hepática/complicaciones , Cirrosis Hepática/genética , Cirrosis Hepática/patología , Neoplasias Hepáticas/etiología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Masculino , Persona de Mediana Edad , Anotación de Secuencia Molecular , Datos de Secuencia Molecular , Estadificación de Neoplasias , Mapeo Peptídico , Proteolisis , Tripsina/química
6.
PLoS One ; 10(3): e0121112, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25799488

RESUMEN

BACKGROUND: Leucine-rich alpha-2-glycoprotein (LRG1) was found to be differentially expressed in sera from patients with Epithelial Ovarian Cancer (EOC). The aim of this study is to investigate the performance of LRG1 for detection of EOC, including early stage EOC, and to evaluate if LRG1 can complement CA125 in order to improve EOC detection using two independent blinded sample sets. METHODS AND RESULTS: Serum LRG1 and CA125 were measured by immunoassays. All assays were performed blinded to clinical data. Using the two independent sample sets (156 participants for sample set 1, and 233 for sample set 2), LRG1 was differentially expressed in EOC cases as compared to healthy, surgical, and benign controls, and its performance was not affected by the conditions of blood collection. The areas under the ROC curve (AUC) for LRG1 in differentiating EOC cases from non-cases were 0.797 and 0.786 for sample set 1 and 2. For differentiating EOC cases from healthy controls, the AUC values for LRG1 were 0.792 and 0.794. At a fixed specificity of 95%, LRG1 detects 52%, and 53.5% of EOC cases from healthy controls for sample set 1 and 2. When combining LRG1 and CA125, the AUC value increased to 0.927, which was improved compared to CA125 (AUC=0.916) (p=0.008) alone in distinguishing EOC cases from non-cases. More importantly, LRG1 also showed potential performance in differentiating early stage EOC from non-cases with an AUC of 0.715 for sample set 1, and 0.690 for sample set 2. The combination of LRG1 and CA125 resulted in an AUC of 0.838, which outperforms CA125 (AUC=0.785) (p=0.018) in detecting early stage EOC cases from non-cases using the larger sample set. CONCLUSIONS: LRG1 could be a useful biomarker alone or in combination with CA125 for the diagnosis of ovarian cancer.


Asunto(s)
Biomarcadores de Tumor/sangre , Antígeno Ca-125/sangre , Glicoproteínas/sangre , Proteínas de la Membrana/sangre , Neoplasias Glandulares y Epiteliales/diagnóstico , Neoplasias Ováricas/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Área Bajo la Curva , Carcinoma Epitelial de Ovario , Femenino , Humanos , Persona de Mediana Edad , Neoplasias Glandulares y Epiteliales/sangre , Neoplasias Glandulares y Epiteliales/patología , Neoplasias Ováricas/sangre , Neoplasias Ováricas/patología , Adulto Joven
7.
J Proteome Res ; 14(4): 1968-78, 2015 Apr 03.
Artículo en Inglés | MEDLINE | ID: mdl-25732060

RESUMEN

Glycosylation has significant effects on protein function and cell metastasis, which are important in cancer progression. It is of great interest to identify site-specific glycosylation in search of potential cancer biomarkers. However, the abundance of glycopeptides is low compared to that of nonglycopeptides after trypsin digestion of serum samples, and the mass spectrometric signals of glycopeptides are often masked by coeluting nonglycopeptides due to low ionization efficiency. Selective enrichment of glycopeptides from complex serum samples is essential for mass spectrometry (MS)-based analysis. Herein, a strategy has been optimized using LCA enrichment to improve the identification of core-fucosylation (CF) sites in serum of pancreatic cancer patients. The optimized strategy was then applied to analyze CF glycopeptide sites in 13 sets of serum samples from pancreatic cancer, chronic pancreatitis, healthy controls, and a standard reference. In total, 630 core-fucosylation sites were identified from 322 CF proteins in pancreatic cancer patient serum using an Orbitrap Elite mass spectrometer. Further data analysis revealed that 8 CF peptides exhibited a significant difference between pancreatic cancer and other controls, which may be potential diagnostic biomarkers for pancreatic cancer.


Asunto(s)
Biomarcadores de Tumor/sangre , Fucosa/metabolismo , Glicopéptidos/sangre , Glicopéptidos/aislamiento & purificación , Espectrometría de Masas/métodos , Neoplasias Pancreáticas/sangre , Análisis de Varianza , Cromatografía Liquida , Glicopéptidos/metabolismo , Humanos , Lectinas de Plantas , Espectrometría de Masas en Tándem , Tripsina
8.
J Proteome Res ; 13(12): 6058-66, 2014 Dec 05.
Artículo en Inglés | MEDLINE | ID: mdl-25393578

RESUMEN

Single amino acid variations are highly associated with many human diseases. The direct detection of peptides containing single amino acid variants (SAAVs) derived from nonsynonymous single nucleotide polymorphisms (SNPs) in serum can provide unique opportunities for SAAV associated biomarker discovery. In the present study, an isobaric labeling quantitative strategy was applied to identify and quantify variant peptides in serum samples of pancreatic cancer patients and other benign controls. The largest number of SAAV peptides to date in serum including 96 unique variant peptides were quantified in this quantitative analysis, of which five variant peptides showed a statistically significant difference between pancreatic cancer and other controls (p-value < 0.05). Significant differences in the variant peptide SDNCEDTPEAGYFAVAVVK from serotransferrin were detected between pancreatic cancer and controls, which was further validated by selected reaction monitoring (SRM) analysis. The novel biomarker panel obtained by combining α-1-antichymotrypsin (AACT), Thrombospondin-1 (THBS1) and this variant peptide showed an excellent diagnostic performance in discriminating pancreatic cancer from healthy controls (AUC = 0.98) and chronic pancreatitis (AUC = 0.90). These results suggest that large-scale analysis of SAAV peptides in serum may provide a new direction for biomarker discovery research.


Asunto(s)
Marcaje Isotópico/métodos , Mutación Missense , Neoplasias Pancreáticas/genética , Péptidos/genética , Polimorfismo de Nucleótido Simple , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Aminoácidos , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Cromatografía Liquida , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Neoplasias Pancreáticas/sangre , Neoplasias Pancreáticas/metabolismo , Péptidos/sangre , Péptidos/metabolismo , Proteoma/análisis , Proteoma/genética , Proteómica/métodos , Curva ROC , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem , Adulto Joven
9.
J Proteome Res ; 13(6): 2887-96, 2014 Jun 06.
Artículo en Inglés | MEDLINE | ID: mdl-24799124

RESUMEN

A mass spectrometry-based methodology has been developed to study changes in core-fucosylation of serum ceruloplasmin that are site-specific between cirrhosis and hepatocellular carcinoma (HCC). The serum samples studied for these changes were from patients affected by cirrhosis or HCC with different etiologies, including alcohol, hepatitis B virus, or hepatitis C virus. The methods involved trypsin digestion of ceruloplasmin into peptides followed by Endo F3 digestion, which removed most of the glycan structure while retaining the innermost N-acetylglucosamine (GlcNAc) and/or core-fucose bound to the peptide. This procedure simplified the structures for further analysis by mass spectrometry, where four core-fucosylated sites (sites 138, 358, 397, and 762) were detected in ceruloplasmin. The core-fucosylation ratio of three of these sites increased significantly in alcohol-related HCC samples (sample size = 24) compared to that in alcohol-related cirrhosis samples (sample size = 18), with the highest AUC value of 0.838 at site 138. When combining the core-fucosylation ratio of site 138 in ceruloplasmin and the alpha-fetoprotein (AFP) value, the AUC value increased to 0.954 (ORsite138 = 12.26, p = 0.017; ORAFP = 3.64, p = 0.022), which was markedly improved compared to that of AFP (AUC = 0.867) (LR test p = 0.0002) alone. However, in HBV- or HCV-related liver diseases, no significant site-specific change in core-fucosylation of ceruloplasmin was observed between HCC and cirrhosis.


Asunto(s)
Consumo de Bebidas Alcohólicas/efectos adversos , Carcinoma Hepatocelular/sangre , Ceruloplasmina/metabolismo , Hepatopatías Alcohólicas/sangre , Neoplasias Hepáticas/sangre , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Aminoácidos , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/química , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/etiología , Estudios de Casos y Controles , Ceruloplasmina/química , Femenino , Glicosilación , Hepatitis B/sangre , Hepatitis B/complicaciones , Hepatitis C/sangre , Hepatitis C/complicaciones , Humanos , Hepatopatías Alcohólicas/diagnóstico , Hepatopatías Alcohólicas/etiología , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/etiología , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Peso Molecular , Procesamiento Proteico-Postraduccional , Curva ROC , Espectrometría de Masas en Tándem
10.
J Proteome Res ; 13(6): 2986-97, 2014 Jun 06.
Artículo en Inglés | MEDLINE | ID: mdl-24807840

RESUMEN

We have developed herein a quantitative mass spectrometry-based approach to analyze the etiology-related alterations in fucosylation degree of serum haptoglobin in patients with liver cirrhosis and hepatocellular carcinoma (HCC). The three most common etiologies, including infection with hepatitis B virus (HBV), infection with hepatitis C virus (HCV), and heavy alcohol consumption (ALC), were investigated. Only 10 µL of serum was used in this assay in which haptoglobin was immunoprecipitated using a monoclonal antibody. The N-glycans of haptoglobin were released with PNGase F, desialylated, and permethylated prior to MALDI-QIT-TOF MS analysis. In total, N-glycan profiles derived from 104 individual patient samples were quantified (14 healthy controls, 40 cirrhosis, and 50 HCCs). A unique pattern of bifucosylated tetra-antennary glycan, with both core and antennary fucosylation, was identified in HCC patients. Quantitative analysis indicated that the increased fucosylation degree was highly associated with HBV- and ALC-related HCC patients compared to that of the corresponding cirrhosis patients. Notably, the bifucosylation degree was distinctly increased in HCC patients versus that in cirrhosis of all etiologies. The elevated bifucosylation degree of haptoglobin can discriminate early stage HCC patients from cirrhosis in each etiologic category, which may be used to provide a potential marker for early detection and to predict HCC in patients with cirrhosis.


Asunto(s)
Biomarcadores de Tumor/sangre , Carcinoma Hepatocelular/sangre , Fucosa/metabolismo , Haptoglobinas/metabolismo , Cirrosis Hepática/sangre , Neoplasias Hepáticas/sangre , Anciano , Consumo de Bebidas Alcohólicas/efectos adversos , Conformación de Carbohidratos , Secuencia de Carbohidratos , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/etiología , Estudios de Casos y Controles , Diagnóstico Diferencial , Detección Precoz del Cáncer , Femenino , Glicosilación , Hepatitis B/sangre , Hepatitis B/complicaciones , Hepatitis C/sangre , Hepatitis C/complicaciones , Humanos , Cirrosis Hepática/diagnóstico , Cirrosis Hepática/etiología , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/etiología , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Procesamiento Proteico-Postraduccional
11.
J Proteome Res ; 13(4): 2197-204, 2014 Apr 04.
Artículo en Inglés | MEDLINE | ID: mdl-24575722

RESUMEN

Altered glycosylation in glycoproteins is associated with carcinogenesis, and certain glycan structures and glycoproteins are well-known markers for tumor progression. To identify potential diagnostic candidate markers, we have developed a novel method for analysis of glycosylation changes of glycoproteins from crude serum samples using lectin-based glycoprotein capture followed by detection with biotin/HRP-conjugated antibodies. The amount of lectin coated on the microplate well was optimized to achieve low background and improved S/N compared with current lectin ELISA methods. In the presence of competing sugars of lectin AAL or with sialic acid removed from the glycoproteins, we confirmed that this method specifically detects glycosylation changes of proteins rather than protein abundance variation. Using our reverse lectin-based ELISA assay, increased fucosylated haptoglobin was observed in sera of patients with ovarian cancer, while the protein level of haptoglobin remained the same between cancers and noncases. The combination of fucosylated haptoglobin and CA125 (AUC = 0.88) showed improved performance for distinguishing stage-III ovarian cancer from noncases compared with CA125 alone (AUC = 0.86). In differentiating early-stage ovarian cancer from noncases, fucosylated haptoglobin showed comparable performance to CA125. The combination of CA125 and fucosylated haptoglobin resulted in an AUC of 0.855, which outperforms CA125 to distinguish early-stage cancer from noncases. Our study provides an alternative method to quantify glycosylation changes of proteins from serum samples, which will be essential for biomarker discovery and validation studies.


Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Glicoproteínas/química , Glicoproteínas/aislamiento & purificación , Lectinas/química , Polisacáridos/química , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/química , Biomarcadores de Tumor/aislamiento & purificación , Biomarcadores de Tumor/metabolismo , Femenino , Glicoproteínas/sangre , Glicoproteínas/metabolismo , Glicosilación , Humanos , Lectinas/metabolismo , Persona de Mediana Edad , Neoplasias Ováricas , Polisacáridos/sangre , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Adulto Joven
12.
Electrophoresis ; 35(15): 2108-15, 2014 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-24285556

RESUMEN

We describe a label-free relative quantification LC-MS/MS method for core-fucosylation in alpha-2-macroglobulin (A2MG) immunoprecipitated from human sera. The method utilizes endoglycosidase F partial deglycosylation to reduce glycosylation microheterogeneity, while retaining the innermost N-acetylglucosamine (GlcNAc) and core fucose. Precursor ion peak areas of partially deglycosylated peptides were obtained and site-specific core-fucosylation ratios based on the peak areas of core-fucosylated and nonfucosylated counterparts were calculated and evaluated for assay development. This assay was applied in a preliminary study of sera samples from normal controls and patients with pancreatic diseases, including pancreatic cancer and chronic pancreatitis. A2MG fucosylation levels at sites N396 and N1424 were found to decrease in both chronic pancreatitis and pancreatic cancer compared to normal controls. The two sites were identified by two peptides and their core-fucosylation ratios were found to be internally consistent. This method provides a platform to quantify fucosylation levels and can be used to study site-specific core-fucosylation aberrations in other glycoproteins for other diseases.


Asunto(s)
Cromatografía Liquida/métodos , Fucosa/análisis , Neoplasias Pancreáticas/metabolismo , Espectrometría de Masas en Tándem/métodos , alfa-Macroglobulinas/análisis , alfa-Macroglobulinas/química , Anciano , Análisis de Varianza , Femenino , Fucosa/química , Fucosa/metabolismo , Glicosilación , Humanos , Masculino , Persona de Mediana Edad , Neoplasias Pancreáticas/sangre , Reproducibilidad de los Resultados , alfa-Macroglobulinas/metabolismo
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