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1.
J Proteome Res ; 14(6): 2425-36, 2015 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-25855029

RESUMO

Cancer-related alterations in protein glycosylation may serve as diagnostic or prognostic biomarkers or may be used for monitoring disease progression. Clusterin is a medium abundance, yet heavily glycosylated, glycoprotein that is upregulated in clear cell renal cell carcinoma (ccRCC) tumors. We recently reported that the N-glycan profile of clusterin is altered in the plasma of ccRCC patients. Here, we characterized the occupancy and the degree of heterogeneity of individual N-glycosylation sites of clusterin in the plasma of patients diagnosed with localized ccRCC, before and after curative nephrectomy (n = 40). To this end, we used tandem mass spectrometry of immunoaffinity-enriched plasma samples to analyze the individual glycosylation sites in clusterin. We determined the levels of targeted clusterin glycoforms containing either a biantennary digalactosylated disialylated (A2G2S2) glycan or a core fucosylated biantennary digalactosylated disialylated (FA2G2S2) glycan at N-glycosite N374. We showed that the presence of these two clusterin glycoforms differed significantly in the plasma of patients prior to and after curative nephrectomy for localized ccRCC. Removal of ccRCC led to a significant increase in the levels of both FA2G2S2 and A2G2S2 glycans in plasma clusterin. These changes were further confirmed by lectin blotting of plasma clusterin. It is envisioned that these identified glycan alterations may provide an additional level of therapeutic or biomarker sensitivity than levels currently achievable by monitoring expression differences alone.


Assuntos
Carcinoma de Células Renais/metabolismo , Clusterina/metabolismo , Glicopeptídeos/metabolismo , Neoplasias Renais/metabolismo , Polissacarídeos/metabolismo , Carcinoma de Células Renais/patologia , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , Clusterina/química , Glicopeptídeos/química , Humanos , Neoplasias Renais/patologia , Polissacarídeos/química , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem
2.
J Proteome Res ; 12(1): 45-57, 2013 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-23259914

RESUMO

We report progress assembling the parts list for chromosome 17 and illustrate the various processes that we have developed to integrate available data from diverse genomic and proteomic knowledge bases. As primary resources, we have used GPMDB, neXtProt, PeptideAtlas, Human Protein Atlas (HPA), and GeneCards. All sites share the common resource of Ensembl for the genome modeling information. We have defined the chromosome 17 parts list with the following information: 1169 protein-coding genes, the numbers of proteins confidently identified by various experimental approaches as documented in GPMDB, neXtProt, PeptideAtlas, and HPA, examples of typical data sets obtained by RNASeq and proteomic studies of epithelial derived tumor cell lines (disease proteome) and a normal proteome (peripheral mononuclear cells), reported evidence of post-translational modifications, and examples of alternative splice variants (ASVs). We have constructed a list of the 59 "missing" proteins as well as 201 proteins that have inconclusive mass spectrometric (MS) identifications. In this report we have defined a process to establish a baseline for the incorporation of new evidence on protein identification and characterization as well as related information from transcriptome analyses. This initial list of "missing" proteins that will guide the selection of appropriate samples for discovery studies as well as antibody reagents. Also we have illustrated the significant diversity of protein variants (including post-translational modifications, PTMs) using regions on chromosome 17 that contain important oncogenes. We emphasize the need for mandated deposition of proteomics data in public databases, the further development of improved PTM, ASV, and single nucleotide variant (SNV) databases, and the construction of Web sites that can integrate and regularly update such information. In addition, we describe the distribution of both clustered and scattered sets of protein families on the chromosome. Since chromosome 17 is rich in cancer-associated genes, we have focused the clustering of cancer-associated genes in such genomic regions and have used the ERBB2 amplicon as an example of the value of a proteogenomic approach in which one integrates transcriptomic with proteomic information and captures evidence of coexpression through coordinated regulation.


Assuntos
Cromossomos Humanos Par 17 , Genoma Humano , Proteínas , Proteômica , Sequência de Aminoácidos , Cromossomos Humanos Par 17/genética , Cromossomos Humanos Par 17/metabolismo , Bases de Dados de Proteínas , Expressão Gênica , Projeto Genoma Humano , Humanos , Proteínas/classificação , Proteínas/genética , Proteínas/metabolismo
3.
Anal Chem ; 85(17): 8421-8, 2013 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-23901877

RESUMO

MS analysis of sialylated glycans is challenging due to their low ionization efficiency in positive ion mode as well as the possibility of in-source fragmentation. Chemical derivatization strategies have been developed to address this issue focused on removal of the labile acidic proton prior to MS analysis. Highly sialylated negatively charged glycans also exhibit high retention and unsatisfactory separation efficiency when analyzed by hydrophilic interaction liquid chromatography (HILIC) due to their high polarity. Here, we combined linkage specific derivatization of sialic acids by reaction with the condensation reagent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) in methanol with nanoscale liquid chromatographic separation prior to accurate mass Orbitrap MS analysis. Coupling DMT-MM charge neutralization of sialic acids with nano-HILIC-Orbitrap-MS not only allows for linkage specific characterization of sialylated glycans directly from the precursor mass but also improves the preceding HILIC separation by increasing the hydrophobicity and altering the selectivity of the oligosaccharide analytes. We focused on the trisialylated N-glycan fraction from haptoglobin and human plasma, enriched using weak anion exchange chromatography, as this trisialylated fraction has been linked with cancer associated changes in the serum N-glycome. The developed methodology was applied to investigate whether structural alterations in this oligosaccharide pool, enriched from the sera of pathological stage and sex matched patients bearing lung, breast, ovarian, pancreatic, or gastric cancer, demonstrate any degree of cancer specificity or whether changes in expression levels are purely cancer associated. The results of this pilot study indicate limited degrees of cancer specificity, particularly for pancreatic cancer, based on alterations in the relative abundance of specific trisialylated isomers.


Assuntos
Nanotecnologia/métodos , Polissacarídeos/análise , Polissacarídeos/química , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida/métodos , Feminino , Humanos , Isomerismo
4.
Anal Chem ; 85(3): 1689-98, 2013 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-23286506

RESUMO

Improved methods for studying glycans could spur significant advances in the understanding and application of glycobiology. The use of affinity reagents such as lectins and glycan-binding antibodies is a valuable complement to methods involving mass spectrometry and chromatography. Many lectins, however, are not useful as analytic tools due to low affinity in vitro. As an approach to increasing lectin avidity to targeted glycans, we tested the use of lectin multimerization. Several biotinylated lectins were linked together through streptavidin interactions. The binding of certain lectins for purified glycoproteins and glycoproteins captured directly out of biological solutions was increased using multimerization, resulting in the detection of lower concentrations of glycoprotein than possible using monomeric detection. The analysis of glycoproteins in plasma samples showed that the level of binding enhancement through multimerization was not equivalent across patient samples. Wheat germ agglutinin (WGA) reactive glycans on fibronectin and thrombospondin-5 were preferentially bound by multimers in pancreatic cancer patient samples relative to control samples, suggesting a cancer-associated change in glycan density that could be detected only through lectin multimerization. This strategy could lead to the more sensitive and informative detection of glycans in biological samples and a broader spectrum of lectins that are useful as analytical reagents.


Assuntos
Glicoproteínas/metabolismo , Lectinas/metabolismo , Multimerização Proteica/fisiologia , Humanos , Neoplasias Pancreáticas/metabolismo , Ligação Proteica/fisiologia , Aglutininas do Germe de Trigo/metabolismo
5.
Mol Cell Proteomics ; 10(12): M111.010298, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21947365

RESUMO

A challenge in the treatment of lung cancer is the lack of early diagnostics. Here, we describe the application of monoclonal antibody proteomics for discovery of a panel of biomarkers for early detection (stage I) of non-small cell lung cancer (NSCLC). We produced large monoclonal antibody libraries directed against the natural form of protein antigens present in the plasma of NSCLC patients. Plasma biomarkers associated with the presence of lung cancer were detected via high throughput ELISA. Differential profiling of plasma proteomes of four clinical cohorts, totaling 301 patients with lung cancer and 235 healthy controls, identified 13 lung cancer-associated (p < 0.05) monoclonal antibodies. The monoclonal antibodies recognize five different cognate proteins identified using immunoprecipitation followed by mass spectrometry. Four of the five antigens were present in non-small cell lung cancer cells in situ. The approach is capable of generating independent antibodies against different epitopes of the same proteins, allowing fast translation to multiplexed sandwich assays. Based on these results, we have verified in two independent clinical collections a panel of five biomarkers for classifying patient disease status with a diagnostics performance of 77% sensitivity and 87% specificity. Combining CYFRA, an established cancer marker, with the panel resulted in a performance of 83% sensitivity at 95% specificity for stage I NSCLC.


Assuntos
Anticorpos Monoclonais/metabolismo , Biomarcadores Tumorais/sangue , Carcinoma Pulmonar de Células não Pequenas/sangue , Detecção Precoce de Câncer/métodos , Neoplasias Pulmonares/sangue , Proteoma/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Anticorpos Monoclonais/química , Especificidade de Anticorpos , Área Sob a Curva , Biomarcadores Tumorais/imunologia , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Estudos de Casos e Controles , Fator H do Complemento/imunologia , Fator H do Complemento/metabolismo , Feminino , Glicoproteínas/sangue , Glicoproteínas/imunologia , Haptoglobinas/imunologia , Haptoglobinas/metabolismo , Humanos , Imunoensaio/métodos , Neoplasias Pulmonares/diagnóstico , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Proteômica , Curva ROC , Adulto Jovem , alfa 1-Antiquimotripsina/sangue , alfa 1-Antiquimotripsina/imunologia
6.
J Proteome Res ; 11(2): 808-17, 2012 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-22098410

RESUMO

Protein biomarkers are critical for diagnosis, prognosis, and treatment of disease. The transition from protein biomarker discovery to verification can be a rate limiting step in clinical development of new diagnostics. Liquid chromatography-selected reaction monitoring mass spectrometry (LC-SRM MS) is becoming an important tool for biomarker verification studies in highly complex biological samples. Analyte enrichment or sample fractionation is often necessary to reduce sample complexity and improve sensitivity of SRM for quantitation of clinically relevant biomarker candidates present at the low ng/mL range in blood. In this paper, we describe an alternative method for sample preparation for LC-SRM MS, which does not rely on availability of antibodies. This new platform is based on selective enrichment of proteotypic peptides from complex biological peptide mixtures via isoelectric focusing (IEF) on a digital ProteomeChip (dPC) for SRM quantitation using a triple quadrupole (QQQ) instrument with an LC-Chip (Chip/Chip/SRM). To demonstrate the value of this approach, the optimization of the Chip/Chip/SRM platform was performed using prostate specific antigen (PSA) added to female plasma as a model system. The combination of immunodepletion of albumin and IgG with peptide fractionation on the dPC, followed by SRM analysis, resulted in a limit of quantitation of PSA added to female plasma at the level of ∼1-2.5 ng/mL with a CV of ∼13%. The optimized platform was applied to measure levels of PSA in plasma of a small cohort of male patients with prostate cancer (PCa) and healthy matched controls with concentrations ranging from 1.5 to 25 ng/mL. A good correlation (r(2) = 0.9459) was observed between standard clinical ELISA tests and the SRM-based assay. Our data demonstrate that the combination of IEF on the dPC and SRM (Chip/Chip/SRM) can be successfully applied for verification of low abundance protein biomarkers in complex samples.


Assuntos
Proteínas Sanguíneas/análise , Cromatografia Líquida/instrumentação , Focalização Isoelétrica/instrumentação , Espectrometria de Massas/instrumentação , Análise Serial de Proteínas/instrumentação , Sequência de Aminoácidos , Animais , Biomarcadores/sangue , Proteínas Sanguíneas/isolamento & purificação , Bovinos , Cromatografia Líquida/métodos , Feminino , Humanos , Técnicas de Imunoadsorção , Focalização Isoelétrica/métodos , Limite de Detecção , Modelos Lineares , Masculino , Espectrometria de Massas/métodos , Dados de Sequência Molecular , Fragmentos de Peptídeos/análise , Antígeno Prostático Específico/análise , Antígeno Prostático Específico/isolamento & purificação , Proteômica/instrumentação , Proteômica/métodos , Reprodutibilidade dos Testes , Tripsina/química
7.
Electrophoresis ; 33(12): 1746-54, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22740463

RESUMO

Aberrant protein glycosylation has been shown to be associated with disease processes and identification of disease-specific glycoproteins and glycosylation changes may serve as potential diagnostic and therapeutic biomarkers. However despite recent advances in proteomic-based biomarker discovery, this knowledge has not yet translated into an extensive mining of the glycoproteome for potential biomarkers. The major challenge for a comprehensive glycoproteomics analysis arises primarily from the enormous complexity and the large dynamic range in protein constituent in biological samples. Methods that specifically target glycoproteins are therefore necessary to facilitate their selective enrichment prior to their identification by MS-based analysis. The use of lectins, with selective affinities for specific carbohydrate epitopes, to enrich glycoprotein fractions coupled with modern MS, have greatly enhanced the identification of the glycoproteome. On account of their ability to specifically bind cell surface carbohydrates lectins have, during the recent past, found extensive applications in elucidation of the architecture and dynamics of cell surface carbohydrates, glycoconjugate purification, and structural characterization. Combined with complementary depletion and MS technologies, lectin affinity chromatography is becoming the most widely employed method of choice for biomarker discovery in cancer and other diseases.


Assuntos
Cromatografia de Afinidade/métodos , Glicoproteínas/química , Lectinas/química , Proteômica/métodos , Glicoproteínas/sangue , Glicoproteínas/isolamento & purificação , Humanos , Proteoma/química
8.
Biotechnol Bioeng ; 109(12): 3007-17, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22752974

RESUMO

Within the biotechnology industry there is a continuous drive to better and more fully understand the biopharmaceutical process in order to achieve better process control. A method to monitor and quantitate glycomic changes that occur in CHO cells during a bioreactor campaign could help to address this. The goal of the method presented here is to provide data that may help to understand the changes in glycosylation that are occurring, within the cell, to proteins other than the expressed biotherapeutic. The method involves the lysing of cells to gain access to intracellular proteins. The expressed biotherapeutic is specifically removed by affinity chromatography, while the remaining proteins are subjected to deglycosylation by treatment with PNGase F. The released glycans are derivatized with isotopic tags, and quantitative analysis by MALDI-TOF MS is performed. The MALDI-TOF MS method allows for the simultaneous analysis of both neutral and sialylated glycans, displays a linear dynamic range over two orders of magnitude for both neutral and sialylated glycans and achieves sub-picomolar sensitivity. This method may yield valuable information that gives further insight into the inner-workings of CHO cells, potentially taking another step towards fully understanding and controlling the biopharmaceutical process.


Assuntos
Reatores Biológicos , Glicômica/métodos , Glicoproteínas/metabolismo , Polissacarídeos/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Eletroforese em Gel de Poliacrilamida , Glicoproteínas/análise , Glicosilação , Marcação por Isótopo , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/análise , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Polissacarídeos/análise , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Proteína Estafilocócica A/análise , Proteína Estafilocócica A/metabolismo , ortoaminobenzoatos/química , ortoaminobenzoatos/metabolismo
9.
Anal Bioanal Chem ; 402(9): 2687-700, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22274286

RESUMO

The development of a general method for the purification and quantitative glycomic analysis of human plasma samples to characterize global glycosylation changes shall be presented. The method involves multiple steps, including the depletion of plasma via multi-affinity chromatography to remove high abundant proteins, the enrichment of the lower abundant glycoproteins via multi-lectin affinity chromatography, the isotopic derivatization of released glycans, and quantitative analysis by MALDI-TOF MS. Isotopic derivatization of glycans is accomplished using the well-established chemistry of reductive amination to derivatize glycans with either a light analog ((12)C anthranilic acid) or a heavy analog ((13)C(7) anthranilic acid), which allows for the direct comparison of the alternately labeled glycans by MALDI-TOF MS. The method displays a tenfold linear dynamic range for both neutral and sialylated glycans with sub-picomolar sensitivity. Additionally, by using anthranilic acid, a very sensitive fluorophore, as the derivatization reagent, the glycans can be analyzed by chromatography with fluorescence detection. The utility of this methodology is highlighted by the many diseases and disorders that are known to either show or be the result of changes in glycosylation. A method that provides a generic approach for sample preparation and quantitative data will help to further advance the field of glycomics.


Assuntos
Proteínas Sanguíneas/análise , Proteínas Sanguíneas/isolamento & purificação , Cromatografia/métodos , Glicômica/métodos , Glicoproteínas/sangue , Glicoproteínas/isolamento & purificação , Espectrometria de Massas/métodos , Proteínas Sanguíneas/metabolismo , Glicoproteínas/metabolismo , Glicosilação , Humanos , Polissacarídeos/análise , Polissacarídeos/metabolismo
10.
J Sep Sci ; 35(18): 2445-52, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22997032

RESUMO

Lectins are capable of recognizing specific glycan structures and serve as invaluable tools for the separation of glycosylated proteins from nonglycosylated proteins in biological samples. We report on the optimization of native multi-lectin affinity chromatography, combining three lectins, namely, concanavalin A, jacalin, and wheat germ agglutinin for fractionation of cellular glycoproteins from MCF-7 breast cancer lysate. We evaluated several conditions for optimum recovery of total proteins and glycoproteins such as low pH and saccharide elution buffers, and the inclusion of detergents in binding and elution buffers. Optimum recovery was observed with overnight incubation of cell lysate with lectins at 4°C, and inclusion of detergent in binding and saccharide elution buffers. Total protein and bound recoveries were 80 and 9%, respectively. Importantly, we found that high saccharide strength elution buffers were not necessary to release bound glycoproteins. This study demonstrates that multi-lectin affinity chromatography can be extended to total cell lysate to investigate the cellular glycoproteome.


Assuntos
Neoplasias da Mama/química , Técnicas de Cultura de Células , Glicoproteínas/análise , Lectinas/química , Neoplasias da Mama/metabolismo , Cromatografia de Afinidade , Cromatografia Líquida , Imunofluorescência , Humanos , Concentração de Íons de Hidrogênio , Células MCF-7 , Espectrometria de Massas em Tandem , Células Tumorais Cultivadas
11.
Anal Chem ; 83(6): 2029-37, 2011 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-21338062

RESUMO

Site-specific analysis of protein glycosylation is important for biochemical and clinical research efforts. Glycopeptide analysis using liquid chromatography-collision-induced dissociation/electron transfer dissociation mass spectrometry (LC-CID/ETD-MS) allows simultaneous characterization of the glycan structure and attached peptide site. However, due to the low ionization efficiency of glycopeptides during electrospray ionization, 200-500 fmol of sample per injection is needed for a single LC-MS run, which makes it challenging for the analysis of limited amounts of glycoprotein purified from biological matrixes. To improve the sensitivity of LC-MS analysis for glycopeptides, an ultranarrow porous layer open tubular (PLOT) LC column (2.5 m × 10 µm i.d.) was coupled to a linear ion trap (LTQ) collision-induced dissociation/electron transfer dissociation mass spectrometer to provide sensitive analysis of N-linked protein glycosylation heterogeneity. The potential of the developed method is demonstrated by the characterization of site-specific glycosylation using haptoglobin (Hpt) as a model protein. To limit the amount of haptoglobin to low picomole amounts of protein, we affinity purified it from 1 µL of pooled lung cancer patient plasma. A total of 26 glycoforms/glycan compositions on three Hpt tryptic glycopeptides were identified and quantified from 10 LC-MS runs with a consumption of 100 fmol of Hpt digest (13 ng of protein, 10 fmol per injection). Included in this analysis was the determination of the glycan occupancy level. At this sample consumption level, the high sensitivity of the PLOT LC-LTQ-CID/ETD-MS system allowed glycopeptide identification and structure determination, along with relative quantitation of glycans presented on the same peptide backbone, even for low abundant glycopeptides at the ∼100 amol level. The PLOT LC-MS system is shown to have sufficient sensitivity to allow characterization of site-specific protein glycosylation from trace levels of glycosylated proteins.


Assuntos
Cromatografia Líquida/métodos , Haptoglobinas/isolamento & purificação , Haptoglobinas/metabolismo , Neoplasias Pulmonares/sangue , Espectrometria de Massas/métodos , Sequência de Aminoácidos , Sítios de Ligação , Glicosilação , Haptoglobinas/química , Humanos , Imunoprecipitação , Isomerismo , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Porosidade , Especificidade por Substrato
12.
Anal Chem ; 83(12): 4845-54, 2011 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-21513341

RESUMO

The discovery of breast cancer associated plasma/serum biomarkers is important for early diagnosis, disease mechanism elucidation, and determination of treatment strategy for the disease. In this study of serum samples, a multidimensional fractionation platform combined with mass spectrometric analysis were used to achieve the identification of medium to lower abundance proteins, as well as to simultaneously detect glycan and abundance changes. Immuno-affinity depletion and multi-lectin chromatography (M-LAC) were integrated into an automated HPLC platform to remove high abundance protein and fractionate glycoproteins. The collected glycoproteomes were then subjected to isoelectric focusing (IEF) separation by a digital ProteomeChip (dPC), followed by in-gel digestion and LC-MS analysis using an Orbitrap mass spectrometer. As a result, the total number of identified proteins increased significantly when the IEF fractionation step was included as part of the platform. Relevant proteins with biological and disease significance were observed and the dynamic range of the serum proteome measurement was extended. In addition, potential glycan changes were indicated by comparing proteins in control and cancer samples in terms of their affinity to the multi-lectin column (M-LAC) and the pI profiles in IEF separation. In conclusion, a proteomics platform including high abundance protein depletion, lectin affinity fractionation, IEF separation, and LC-MS analysis has been applied to discover breast cancer-associated proteins. The following candidates, thrombospondin-1 and 5, alpha-1B-glycoprotein, serum amyloid P-component, and tenascin-X, were selected as promising examples of the use of this platform. They show potential abundance and glycan changes and will be further investigated in future studies.


Assuntos
Neoplasias da Mama/metabolismo , Cromatografia de Afinidade/métodos , Focalização Isoelétrica/métodos , Lectinas/química , Proteoma/análise , Biomarcadores Tumorais/sangue , Proteína de Matriz Oligomérica de Cartilagem , Cromatografia Líquida de Alta Pressão/métodos , Proteínas da Matriz Extracelular/sangue , Feminino , Glicoproteínas/sangue , Humanos , Espectrometria de Massas/métodos , Proteínas Matrilinas , Componente Amiloide P Sérico/análise , Tenascina/sangue , Trombospondina 1/sangue
13.
Biomed Microdevices ; 13(3): 565-71, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21455756

RESUMO

Lectins are a group of proteins that bind specifically and reversibly to mono- and oligosaccharide carbohydrate structures that are present on the surfaces of mammalian cells. The use of lectins as capture agents in microfluidic channels was examined with a focus on cells associated with T and B lymphocytic leukemia. In addition to examining the adhesion of Jurkat T and Raji B lymphocytes to a broad panel of lectins, this work also examined the capture of these cells from whole blood. Captured T and B lymphocytes were eluted from the microfluidic devices with a solution of the lectin's inhibiting sugar. The capture and release steps were accomplished in under 1 h. The significance of this work lies within the realm of low-cost capture of abundant target cells with non-stimulatory elution capability.


Assuntos
Linfócitos B/citologia , Separação Celular/instrumentação , Leucemia/sangue , Técnicas Analíticas Microfluídicas/instrumentação , Lectinas de Plantas/metabolismo , Linfócitos T/citologia , Linfócitos B/metabolismo , Adesão Celular , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Células Jurkat , Lectinas de Plantas/química , Linfócitos T/metabolismo
14.
J Proteome Res ; 9(4): 1834-42, 2010 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-20146545

RESUMO

A mass spectrometric (MS)-based strategy for antigen (Ag) identification and characterization of globally produced monoclonal antibodies (mAbs) is described. Mice were immunized with a mixture of native glycoproteins, isolated from the pooled plasma of patients with nonsmall cell lung cancer (NSCLC), to generate a library of IgG-secreting hybridomas. Prior to immunization, the pooled NSCLC plasma was subjected to 3-sequential steps of affinity fractionation, including high abundant plasma protein depletion, glycoprotein enrichment, and polyclonal antibody affinity chromatography normalization. In this paper, to demonstrate the high quality of the globally produced mAbs, we selected 3 mAbs of high differentiating power against a matched, pooled normal plasma sample. After production of large quantities of the mAbs from ascites fluids, Ag identification was achieved by immunoaffinity purification, SDS-PAGE, Western blotting, and MS analysis of in-gel digest products. One antigen was found to be complement factor H, and the other two were mapped to different subunits of haptoglobin (Hpt). The 2 Hpt mAbs were characterized in detail to assess the quality of the mAbs produced by the global strategy. The affinity of one of the mAbs to the Hpt native tetramer form was found to have a K(D) of roughly 10(-9) M and to be 2 orders of magnitude lower than the reduced form, demonstrating the power of the mAb proteomics technology in generating mAbs to the natural form of the proteins in blood. The binding of this mAb to the beta-chain of haptoglobin was also dependent on glycosylation on this chain. The characterization of mAbs in this work reveals that the global mAb proteomics process can generate high-quality lung cancer specific mAbs capable of recognizing proteins in their native state.


Assuntos
Anticorpos Monoclonais/biossíntese , Antígenos/química , Neoplasias Pulmonares/imunologia , Proteômica/métodos , Adulto , Animais , Anticorpos Monoclonais/imunologia , Antígenos/imunologia , Antígenos/metabolismo , Biomarcadores Tumorais/química , Biomarcadores Tumorais/metabolismo , Western Blotting , Eletroforese em Gel de Poliacrilamida , Glicoproteínas/sangue , Glicoproteínas/imunologia , Glicosilação , Haptoglobinas/química , Haptoglobinas/metabolismo , Humanos , Neoplasias Pulmonares/sangue , Masculino , Espectrometria de Massas , Camundongos , Pessoa de Meia-Idade
15.
J Proteome Res ; 9(9): 4337-45, 2010 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-20690678

RESUMO

The California poppy, Eschscholzia californica, produces benzophenanthridine alkaloids (BPAs), an important class of biologically active compounds. Cell cultures of E. californica were investigated as an alternative and scalable method for producing these valuable compounds; treatment with yeast extract increased production from low levels to 23 mg/g dry weight (DW) of BPAs. A shotgun proteomic analysis of E. californica cell cultures was undertaken to explore changes in metabolism associated with enhanced BPA production. We implemented differential centrifugation and then shotgun proteomics based on nanoliquid chromatography/mass spectrometry (nano-LC-MS/MS) for peptide separation and analysis. A unigene database available for E. californica was translated and utilized for protein identification. Approximately 646 proteins (3% false discovery rate at the protein level) were identified. Differentially abundant proteins observed with elicitation included enzymes involved in (S)-adenosyl methionine (SAM) biosynthesis and BPA biosynthesis. These results demonstrate (1) the identification of proteins from a medicinal plant using shotgun proteomics combined with a well-annotated, translated unigene database and (2) the potential utility of proteomics for exploring changes in metabolism associated with enhanced secondary metabolite production.


Assuntos
Benzofenantridinas/biossíntese , Eschscholzia/metabolismo , Proteínas de Plantas/química , Proteoma/química , Saccharomyces cerevisiae/química , Cromatografia Líquida , Eschscholzia/microbiologia , Redes e Vias Metabólicas , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Mapeamento de Peptídeos/métodos , Proteínas de Plantas/metabolismo , Proteoma/metabolismo , Espectrometria de Massas em Tandem , Tripsina/metabolismo
16.
Anal Chem ; 82(1): 115-20, 2010 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-19957969

RESUMO

This publication describes the development of an automated platform for the study of the plasma glycoproteome. The method consists of targeted depletion in-line with glycoprotein fractionation. A key element of this platform is the enabling of high throughput sample processing in a manner that minimizes analytical bias in a clinical sample set. The system, named High Performance Multi-Lectin Affinity Chromatography (HP-MLAC), is composed of a serial configuration of depletion columns containing anti-albumin antibody and protein A with in-line multilectin affinity chromatography (M-LAC) which consists of three mixtures of lectins concanavalin A (ConA), jacalin (JAC), and wheat germ agglutinin (WGA). We have demonstrated that this platform gives high recoveries for the fractionation of the plasma proteome (> or = 95%) and excellent stability (over 200 runs). In addition, glycoproteomes isolated using the HP-MLAC platform were shown to be highly reproducible and glycan specific as demonstrated by rechromatography of selected fractions and proteomic analysis of the unbound (glycoproteome 1) and bound (glycoproteome 2) fractions.


Assuntos
Proteínas Sanguíneas/química , Glicoproteínas/sangue , Proteômica/instrumentação , Proteômica/métodos , Automação , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
17.
Anal Chem ; 82(21): 8998-9005, 2010 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-20936840

RESUMO

Liquid chromatography-selected reaction monitoring (LC-SRM) is a highly specific and sensitive mass spectrometry (MS) technique that is widely being applied to selectively qualify and validate candidate markers within complex biological samples. However, in order for LC-SRM methods to take on these attributes, target-specific optimization of sample processing is required, in order to reduce analyte complexity, prior to LC-SRM. In this study, we have developed a targeted platform consisting of protein immunoaffinity enrichment on magnetic beads and LC-SRM for measuring carbonic anhydrase 12 (CA12) protein in a renal cell carcinoma (RCC) cell line (PRC3), a candidate biomarker for RCC whose expression at the protein level has not been previously reported. Sample processing and LC-SRM assay were optimized for signature peptides selected as surrogate markers of CA12 protein. Using LC-SRM coupled with stable isotope dilution, we achieved limits of quantitation in the low fmol range sufficient for measuring clinically relevant biomarkers with good intra- and interassay accuracy and precision (≤17%). Our results show that using a quantitative immunoaffinity capture approach provides specific, accurate, and robust assays amenable to high-throughput verification of potential biomarkers.


Assuntos
Anidrases Carbônicas/análise , Anidrases Carbônicas/metabolismo , Carcinoma de Células Renais/enzimologia , Sequência de Aminoácidos , Linhagem Celular Tumoral , Cromatografia Líquida/métodos , Humanos , Imunoprecipitação/métodos , Limite de Detecção , Espectrometria de Massas/métodos , Dados de Sequência Molecular , Peptídeos/análise , Peptídeos/metabolismo , Reprodutibilidade dos Testes
18.
J Proteome Res ; 8(10): 4732-42, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19725534

RESUMO

Liquid Chromatography Mass Spectrometry (LC-MS) based proteomics is an important tool in detecting changes in peptide/protein abundances in samples potentially leading to the discovery of disease biomarker candidates. We present CLUE-TIPS (Clustering Using Euclidean distance in Tanimoto Inter-Point Space), an approach that compares complex proteomic samples for similarity/dissimilarity analysis. In CLUE-TIPS, an intersample distance feature map is generated from filtered, aligned and binarized raw LC-MS data by applying the Tanimoto distance metric to obtain normalized similarity scores between all sample pairs for each m/z value. We developed clustering and visualization methods for the intersample distance map to analyze various samples for differences at the sample level as well as the individual m/z level. An approach to query for specific m/z values that are associated with similarity/dissimilarity patterns in a set of samples was also briefly described. CLUE-TIPS can also be used as a tool in assessing the quality of LC-MS runs. The presented approach does not rely on tandem mass-spectrometry (MS/MS), isotopic labels or gels and also does not rely on feature extraction methods. CLUE-TIPS suite was applied to LC-MS data obtained from plasma samples collected at various time points and treatment conditions from immunosuppressed mice implanted with MCF-7 human breast cancer cells. The generated raw LC-MS data was used for pattern analysis and similarity/dissimilarity detection. CLUE-TIPS successfully detected the differences/similarities in samples at various time points taken during the progression of tumor, and also recognized differences/similarities in samples representing various treatment conditions.


Assuntos
Cromatografia Líquida/métodos , Análise por Conglomerados , Espectrometria de Massas/métodos , Proteômica/métodos , Algoritmos , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Camundongos , Transplante de Neoplasias , Proteínas/metabolismo
19.
Anal Chem ; 81(17): 7357-62, 2009 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-19663468

RESUMO

The productivity of mammalian cell culture expression systems is critically important to the production of biopharmaceuticals. In this study, a high-producing Chinese hamster ovary cell culture which was transfected with the apoptosis inhibitor Bcl-X(L) gene was compared to a low-producing control that was not transfected. Shotgun proteomics was used to compare the high and low-producing fed-batch cell cultures at different growth time points. The goals of this study were twofold; it would be of value to find a biomarker that could predict cell lines with higher growth efficiency and to gain mechanistic insights into the effects of the introduction of a foreign gene that is known to have growth regulating properties in human cells. A total of 392 proteins were identified in this study, and 32 of these proteins were determined to be differentially expressed. In the high-producing cell culture, several proteins related to protein metabolism were upregulated, such as eukaryotic translation initiation factor 3 and ribosome 40S. In addition, several intermediate filament proteins such as vimentin and annexin, as well as histone H1.2 and H2A, were downregulated in the high producer. The expression of these proteins may be indicative of cellular productivity. A growth inhibitor, galectin-1, was downregulated in the high producer, which may be linked to the expression of Bcl-X(L). The molecular chaperone BiP was upregulated significantly in the high producer and may indicate an unfolded protein response due to endoplasmic reticulum (ER) stress. Several proteins involved in regulation of the cell cycle such as RACK1 and GTPase Ran were found to be differentially expressed, which may be due to a differentially controlled cell cycle between low- and high-producing cell cultures.


Assuntos
Ovário/citologia , Ovário/metabolismo , Proteoma/análise , Proteoma/genética , Proteômica , Proteína bcl-X/genética , Animais , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Feminino , Expressão Gênica , Transfecção
20.
Clin Chem ; 54(11): 1805-14, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18801941

RESUMO

BACKGROUND: The mechanisms underlying psoriatic pathogenesis are not fully understood and might be elucidated by identifying novel disease-related molecular markers, including autoantigens. METHODS: We used 2 proteomic methods to analyze plasma samples from 20 psoriasis patients and 20 matched healthy donors. The first method focused on evaluating changes in glycoprotein concentrations and the plasma proteome, and the second method assessed endogenous proteolytic activity by analyzing the low molecular weight component of plasma. RESULTS: The integrated proteomic and peptidomic analysis identified a number of proteins and their fragments present at different concentrations in the plasma of psoriasis patients and healthy donors. We used ELISA to independently verify the changes in the concentrations of several of these proteins. One intriguing finding, increased concentrations of cytoskeletal and actin-binding proteins and their peptides in psoriatic plasma, suggested disease-related cell leakage of these proteins and their increased proteolysis. Among the increased proteins and peptides were thymosin beta 4, talin 1, actin gamma, filamin, and profilin. Increased concentrations of Ca(2+)-binding proteins calgranulins A and B in psoriatic plasma were also observed, confirming previous reports, and appeared to be relevant to the increase of cytoskeletal components. Another notable change in psoriatic plasma was a striking decrease in fibrinogen fragments. CONCLUSIONS: The identified increased concentrations of cytoskeletal proteins, their peptides, and calgranulins in psoriatic plasma, as well as the underlying altered protease activity, are proposed to be related to psoriasis pathogenesis.


Assuntos
Proteínas de Ligação ao Cálcio/sangue , Proteínas do Citoesqueleto/sangue , Peptídeos/sangue , Psoríase/sangue , Adulto , Idoso , Sequência de Aminoácidos , Proteínas de Ligação ao Cálcio/química , Estudos de Casos e Controles , Cromatografia Líquida , Proteínas do Citoesqueleto/química , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Hidrólise , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Proteômica , Espectrometria de Massas em Tandem
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