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1.
Biotechnol Bioeng ; 121(4): 1284-1297, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38240126

RESUMO

Product association of host-cell proteins (HCPs) to monoclonal antibodies (mAbs) is widely regarded as a mechanism that can enable HCP persistence through multiple purification steps and even into the final drug substance. Discussion of this mechanism often implies that the existence or extent of persistence is directly related to the strength of binding but actual measurements of the binding affinity of such interactions remain sparse. Two separate avenues of investigation of HCP-mAb binding are reported here. One is the measurement of the affinity of binding of individual, commonly persistent Chinese hamster ovary (CHO) HCPs to each of a set of mAbs, and the other uses quantitative proteomic measurements to assess binding of HCPs in a null CHO harvested cell culture fluid (HCCF) to mAbs produced in the same cell line. The individual HCP measurements show that the binding affinities of individual HCPs to different mAbs can vary appreciably but are rarely very high, with only weak pH dependence. The measurements on the null HCCF allow estimation of individual HCP-mAb affinities; these are typically weaker than those seen in affinity measurements on isolated HCPs. Instead, the extent of binding appears correlated with the initial abundance of individual HCPs in the HCCF and the forms of the HCPs in the solution, i.e., whether HCPs are present as free molecules or as parts of large aggregates. Separate protein A chromatography experiments performed by feeding different fractions of a mAb-containing HCCF obtained by size-exclusion chromatography (SEC) showed clear differences in the number and identity of HCPs found in the protein A eluate. These results indicate a significant role for HCP-mAb association in determining HCP persistence through protein A chromatography, presumably through binding of HCP-mAb complexes to the resin. Overall, the results illustrate the importance of considering more fully the biophysical context of HCP-product association in assessing the factors that may affect the phenomenon and determine its implications. Knowledge of the abundances and the forms of individual or aggregated HCPs in HCCF are particularly significant, emphasizing the integration of upstream and downstream bioprocessing and the importance of understanding the collective properties of HCPs in addition to just the biophysical properties of individual HCPs.


Assuntos
Anticorpos Monoclonais , Proteômica , Cricetinae , Animais , Cricetulus , Proteômica/métodos , Células CHO , Anticorpos Monoclonais/química , Cromatografia em Gel , Proteína Estafilocócica A/química
2.
Biotechnol Bioeng ; 121(1): 291-305, 2024 01.
Artigo em Inglês | MEDLINE | ID: mdl-37877536

RESUMO

Host-cell proteins (HCPs) are the foremost class of process-related impurities to be controlled and removed in downstream processing steps in monoclonal antibody (mAb) manufacturing. However, some HCPs may evade clearance in multiple purification steps and reach the final drug product, potentially threatening drug stability and patient safety. This study extends prior work on HCP characterization and persistence in mAb process streams by using mass spectrometry (MS)-based methods to track HCPs through downstream processing steps for seven mAbs that were generated by five different cell lines. The results show considerable variability in HCP identities in the processing steps but extensive commonality in the identities and quantities of the most abundant HCPs in the harvests for different processes. Analysis of HCP abundance in the harvests shows a likely relationship between abundance and the reproducibility of quantification measurements and suggests that some groups of HCPs may hinder the characterization. Quantitative monitoring of HCPs persisting through purification steps coupled with the findings from the harvest analysis suggest that multiple factors, including HCP abundance and mAb-HCP interactions, can contribute to the persistence of individual HCPs and the identification of groups of common, persistent HCPs in mAb manufacturing.


Assuntos
Anticorpos Monoclonais , Cricetinae , Animais , Humanos , Anticorpos Monoclonais/química , Reprodutibilidade dos Testes , Cricetulus , Espectrometria de Massas , Células CHO
3.
Biotechnol Bioeng ; 120(4): 1068-1080, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36585356

RESUMO

In the production of biopharmaceuticals such as monoclonal antibodies (mAbs) and vaccines, the residual amounts of host-cell proteins (HCPs) are among the critical quality attributes. In addition to overall HCP levels, individual HCPs may elude purification, potentially causing issues in product stability or patient safety. Such HCP persistence has been attributed mainly to biophysical interactions between individual HCPs and the product, resin media, or residual chromatin particles. Based on measurements on process streams from seven mAb processes, we have found that HCPs in aggregates, not necessarily chromatin-derived, may play a significant role in the persistence of many HCPs. Such aggregates may also hinder accurate detection of HCPs using existing proteomics methods. The findings also highlight that certain HCPs may be difficult to remove because of their functional complementarity to the product; specifically, chaperones and other proteins involved in the unfolded protein response (UPR) are disproportionately present in the aggregates. The methods and findings described here expand our understanding of the origins and potential behavior of HCPs in cell-based biopharmaceutical processes and may be instrumental in improving existing techniques for HCP detection and clearance.


Assuntos
Produtos Biológicos , Agregados Proteicos , Cricetinae , Animais , Humanos , Cricetulus , Anticorpos Monoclonais , Proteômica/métodos , Células CHO
4.
Biotechnol Bioeng ; 115(10): 2489-2503, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29896879

RESUMO

Protein precipitates that arise during bioprocessing can cause manufacturing challenges, but they can also aid in clearance of host-cell protein (HCP) and DNA impurities. Such precipitates differ from many protein precipitates that have been studied previously in their heterogeneous composition, particularly in the presence of high concentrations of the product protein. Here, we characterize the precipitates that form after neutralization of protein A purified and viral-inactivated material of an Fc-fusion protein produced in Chinese hamster ovary cells. The physical growth of precipitate particles was observed by optical microscopy, transmission electron microscopy, dynamic light scattering, and small-angle and ultra-small-angle X-ray scattering to characterize the precipitate microstructure and growth mechanism. The precipitate microstructure is well-described as a mass fractal with fractal dimension approximately 2. The growth is governed by a diffusion-limited aggregation mechanism as indicated by a power-law dependence on time of the size of the principal precipitate particles. Optical microscopy shows that these primary particles can further aggregate into larger particles in a manner that appears to be promoted by mixing. Absorbance experiments at varying pH and salt concentrations reveal that the growth is largely driven by attractive electrostatic interactions, as growth is hindered by an increase in ionic strength. The solution conditions that resulted in the most significant particle growth are also correlated with the greatest removal of soluble impurities (DNA and HCPs). Proteomic analysis of the precipitates allows identification of O ( 100 ) unique HCP impurities, depending on the buffer species (acetate or citrate) used for the viral inactivation. Most of these proteins have pI values near the precipitation pH, supporting the likely importance of electrostatic interactions in driving precipitate formation.


Assuntos
Precipitação Fracionada , Fragmentos Fc das Imunoglobulinas , Modelos Químicos , Proteômica , Proteínas Recombinantes de Fusão , Animais , Células CHO , Cricetinae , Cricetulus , Fragmentos Fc das Imunoglobulinas/biossíntese , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/isolamento & purificação , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação
5.
Prostate ; 77(7): 729-742, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28168724

RESUMO

BACKGROUND: Previous studies showed that human bone marrow stromal HS-5 cells secreted unidentified factor(s) inducing PCa cell death. Herein, the HS-5-derived factor (HS-5 DF) was characterized and identified. METHODS: Conditioned media from confluent HS-5 cells were collected and modified for biochemical characteristic testing of HS-5 DF. Cell survival was measured by apoptosis assay and live/dead assay. Fibulin-1 was identified from gel electrophoresis and mass spectrometry. The validation of Fibulin-1 as a HS-5 DF was done by immunoprecipitation (IP) and genetic knockdown by CRISPR/Cas9 system. RESULTS: HS-5 DF was trypsin and heat sensitive, but pH stable. The tentative size of the factor fell between 30 kDa and 100 kDa. TGF-ß1 treatment led to a suppression of HS-5 DF activity, a property consistent with bone metastasis in prostate cancer. Examination of TGF-ß1 down regulated proteins led to identification of fibulin-1 as a candidate for the DF. IP of Fibulin-1 from HS-5 CM and CRISPR knockdown of Fibulin-1 showed a significant reduction of HS-5 CM-derived PCa cell death. These results strongly support a role for fibulin-1 in HS-5 bone marrow stromal cell induction of PCa cell death. CONCLUSION: Our data indicate that Fibulin-1 functions as a HS-5 bone marrow stromal cell-derived factor inducing prostate cancer cell death. Prostate 77:729-742, 2017. © 2016 Wiley Periodicals, Inc.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Células-Tronco Mesenquimais , Neoplasias da Próstata , Apoptose/fisiologia , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/secundário , Morte Celular/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Meios de Cultivo Condicionados/análise , Meios de Cultivo Condicionados/metabolismo , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Fator de Crescimento Transformador beta1/metabolismo
6.
Electrophoresis ; 36(15): 1690-8, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25884272

RESUMO

This work presents improved protease digestion conditions for membrane protein detection. The enzymatic digest of bacteriorhodopsin (BR), a model membrane protein with seven transmembrane domains (TMDs) was investigated. An initial in-gel digestion identified 17% BR sequence coverage, including part of the seventh TMD. To improve sequence coverage, BR digest was tested with different concentrations of RapiGest, methanol (MeOH) and SDS using either trypsin or chymotrypsin. Two improved conditions, 0.01% SDS or the combination of 10% MeOH and 0.01% RapiGest, were chosen. Trypsin digestions in both conditions achieved more than 40% BR sequence coverage compared to 0% using standard digestion conditions. Peptides detected from trypsin and chymotrypsin digestions in the same condition were combined to maximize sequence coverage. The same conditions were applied to a different membrane protein with one TMD, Selenoprotein S, and proteins from Escherichia coli. For Selenoprotein S, a higher sequence coverage, including a peptide from the TMD, was detected from the improved condition compared to the typical condition. The application of both improved conditions to a membrane protein fraction of Escherichia coli resulted in the identification of 309 (SDS) and 329 (MeOH/RapiGest) unique proteins of which 140/309 and 148/329 were membrane proteins.


Assuntos
Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteômica/métodos , Tripsina/metabolismo , Bacteriorodopsinas/análise , Bacteriorodopsinas/química , Bacteriorodopsinas/metabolismo , Proteínas de Escherichia coli/análise , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/análise , Mapeamento de Peptídeos , Estrutura Terciária de Proteína
7.
Electrophoresis ; 35(12-13): 1821-7, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24756957

RESUMO

Intravenous immunoglobulin (IVIg) therapy has shown promise in the treatment of Alzheimer's disease (AD). In this study, serial cerebrospinal fluid (CSF) samples from a group of subjects with AD undergoing IVIg immunotherapy are analyzed to identify IVIg-related changes. CSF samples from eight subjects were collected before therapy, after 6 months of therapy, and after a 3-month drug washout period. Samples were analyzed using a gel-based proteomics strategy and IVIg-related changes were determined by gel spot percent volumes. An initial assessment of the data revealed consistent and considerable change in 69 spots. A statistical analysis revealed 79 protein spots with a significant change after 6 months; furthermore, in a subset of these (25), the percent volume change was either maintained or reversed in the washout samples. The proteins that showed a significant change during IVIg therapy, including Ig molecules, gelsolin, transferrin, and transthyretin, have been previously implicated in AD. This study provides preliminary findings regarding a group of CSF proteins that may be associated with the treatment of AD, as well as the potential use of IVIg as an AD immunotherapy.


Assuntos
Doença de Alzheimer/líquido cefalorraquidiano , Doença de Alzheimer/tratamento farmacológico , Proteínas do Líquido Cefalorraquidiano/líquido cefalorraquidiano , Imunoglobulinas Intravenosas/administração & dosagem , Proteoma/análise , Biomarcadores/líquido cefalorraquidiano , Proteínas do Líquido Cefalorraquidiano/química , Eletroforese em Gel Bidimensional/métodos , Humanos , Proteômica
8.
Biotechnol Bioeng ; 111(5): 904-12, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24254318

RESUMO

Downstream processing of monoclonal antibodies (mAbs) has evolved to allow the specific process for a new product to be developed largely by empirical specialization of a platform process that enables removal of impurities of different kinds. A more complete characterization of impurities and the product itself would provide insights into the rational design of efficient downstream processes. This work identifies and characterizes host cell protein (HCP) product-associated impurities, that is, HCP species carried through the downstream processes via direct interactions with the mAb. Interactions between HCPs and mAbs are characterized using cross-interaction chromatography under solution conditions typical of those used in downstream processing. The interacting species are then identified by two-dimensional gel electrophoresis and mass spectrometry. This methodology has been applied to identify product-associated impurities in one particular purification step, namely protein A affinity chromatography, for four therapeutic mAbs as well as the Fab and Fc domains of one of these mAbs. The results show both the differences in HCP-mAb interactions among different mAbs, and the relative importance of product association compared to co-elution in protein A affinity chromatography.


Assuntos
Anticorpos Monoclonais/isolamento & purificação , Cromatografia de Afinidade/métodos , Proteínas/análise , Proteínas/química , Proteínas Recombinantes/isolamento & purificação , Animais , Células CHO , Cricetinae , Cricetulus , Eletroforese em Gel Bidimensional
9.
Methods ; 61(3): 269-76, 2013 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-23523702

RESUMO

Clostridium acetobutylicum (Cac) is an anaerobic, endospore-forming, Gram-positive bacterium with tremendous promise for use as a biocatalyst for the production of fuels and solvents. Cac proteomic sample preparation for shotgun analysis typically involves a multitude of reagents for harsh lysis conditions and to maintain protein solubility. We describe a protein extraction and preparation method for Cac that is compatible with proteomic shotgun analysis using isobaric labeling approaches. The method is applied to the analysis of Cac grown under butanol stress and labeled using iTRAQ 4-plex reagents. This method relies on the use of calcium carbonate to facilitate lysis by sonication and a commercially available kit to remove detergents prior to labeling. This workflow resulted in the identification and quantitation of 566 unique proteins using ProteinPilot software with a false discovery rate of 0.01% for peptide matches and 0.70% for protein matches. Ninety-five proteins were found to have statistically higher expression levels in butanol-stressed Cac as compared to non-stressed Cac. Sixty-one proteins were found to have statistically lower expression levels in stressed versus non-stressed cells. This method may be applicable to other Gram-positive organisms.


Assuntos
Proteínas de Bactérias/isolamento & purificação , Clostridium acetobutylicum/química , Peptídeos/isolamento & purificação , Proteoma/isolamento & purificação , Proteômica/métodos , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Reatores Biológicos , Butanóis/farmacologia , Carbonato de Cálcio/química , Cromatografia Líquida , Clostridium acetobutylicum/efeitos dos fármacos , Clostridium acetobutylicum/crescimento & desenvolvimento , Clostridium acetobutylicum/metabolismo , Fermentação , Anotação de Sequência Molecular , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/metabolismo , Proteoma/química , Proteoma/metabolismo , Estresse Fisiológico , Espectrometria de Massas em Tandem , Fluxo de Trabalho
10.
J Chromatogr A ; 1702: 464081, 2023 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-37244165

RESUMO

Recent work has shown that aggregates in monoclonal antibody (mAb) solutions may be made up not just of mAb oligomers but can also harbor hundreds of host-cell proteins (HCPs), suggesting that aggregate persistence through downstream purification operations may be related to HCP clearance. We have examined this in a primary analysis of aggregate persistence through processing steps that are typically implemented for HCP reduction, demonstrating that the phenomenon is relevant to depth filtration, protein A chromatography and flow-through anion-exchange (AEX) polishing. Confocal laser scanning microscopy observations show that aggregates compete with the mAb to adsorb specifically in protein A chromatography and that this competitive interaction is integral to the efficacy of protein A washes. Column chromatography reveals that the protein A elution tail can have a relatively high concentration of aggregates, which corroborates analogous observations from recent HCP studies. Similar measurements in flow-through AEX chromatography show that relatively large aggregates that harbor HCPs and that persist into the protein A eluate can be retained to an extent that appears to depend primarily on the resin surface chemistry. The total aggregate mass fraction of both protein A eluate pools (∼ 2.4 - 3.6%) and AEX flow-through fractions (∼ 1.5 - 3.2%) correlates generally with HCP concentrations measured using enzyme-linked immunosorbent assay (ELISA) as well as the number of HCPs that may be identified in proteomic analysis. This suggests that quantification of the aggregate mass fraction may serve as a convenient albeit imperfect surrogate for informing early process development decisions regarding HCP clearance strategies.


Assuntos
Cromatografia , Proteômica , Cricetinae , Animais , Cricetulus , Proteômica/métodos , Células CHO , Anticorpos Monoclonais/química , Proteína Estafilocócica A/química , Ânions
11.
Biotechnol Prog ; 39(4): e3343, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37020359

RESUMO

Host-cell proteins (HCPs) and high molecular weight (HMW) species have historically been treated as independent classes of impurities in the downstream processing of monoclonal antibodies (mAbs), but recent indications suggest that they may be partially linked. We have explored this connection with a shotgun proteomic analysis of HMW impurities that were isolated from harvest cell culture fluid (HCCF) and protein A eluate using size-exclusion chromatography (SEC). As part of the proteomic analysis, a cross-digest study was performed in which samples were analyzed using both the standard and native digest techniques to enable a fair comparison between bioprocess pools. This comparison reveals that the HCP profiles of HCCF and protein A eluate overlap substantially more than previous work has suggested, because hundreds of HCPs are conserved in aggregates that may be up to ~50 nm in hydrodynamic radius and that persist through the protein A capture step. Quantitative SWATH proteomics suggests that the majority of the protein A eluate's HCP mass is found in such aggregates, and this is corroborated by ELISA measurements on SEC fractions. The SWATH data also show that intra-aggregate concentrations of individual HCPs are positively correlated between aggregates that were isolated from HCCF and protein A eluate, and species that have generally been considered difficult to remove tend to be more concentrated than their counterparts. These observations support prior hypotheses regarding aggregate-mediated HCP persistence through protein A chromatography and highlight the importance of this persistence mechanism.


Assuntos
Anticorpos Monoclonais , Proteômica , Cricetinae , Animais , Anticorpos Monoclonais/química , Cricetulus , Proteômica/métodos , Células CHO , Cromatografia Líquida/métodos , Proteína Estafilocócica A/química
12.
Electrophoresis ; 33(13): 1947-57, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22806459

RESUMO

Optimized 2DE sample preparation protocols that maximize total protein recovery are fundamental to improving proteome capture and increasing the utility of 2DE, which is in part limited by inadequate recovery of proteins with diverse physicochemical properties. Maintaining protein solubility is an important factor for protein recovery, but the multitude of solubility-enhancing agents and the relatively low-throughput nature of 2DE limit the systematic study of sample preparation. In this work, design of experiment (DOE) approaches are used to optimize protein recovery by altering the levels of four solubility-enhancing agents (urea, DTT, CHAPS, and SDS) in the initial suspension solution. Protein recovery is quantified by a total protein concentration assay, which is demonstrated to be representative of SDS-PAGE and 2DE recovery. DOE methodologies are presented as relatively high-throughput procedures for optimizing 2DE sample preparation parameters for a variety of sample types. Optimal suspension solution compositions are shown to vary across a model protein solution (no urea or DTT), Chinese hamster ovary (CHO) cell lysate (8 M urea, ≥2% CHAPS, ≥32.5 mM DTT), and Escherichia coli cell lysate (8 M urea, 4% CHAPS, 65 mM DTT), with optimized conditions increasing 2DE protein recovery at least 50% compared to suboptimal conditions.


Assuntos
Eletroforese em Gel Bidimensional/métodos , Proteoma/química , Proteômica/métodos , Animais , Células CHO , Bovinos , Cricetinae , Cricetulus , Proteínas de Escherichia coli , Ensaios de Triagem em Larga Escala , Limite de Detecção , Proteoma/análise , Projetos de Pesquisa
13.
Expert Rev Proteomics ; 7(6): 919-29, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21142892

RESUMO

Alzheimer's disease is a progressive neurodegenerative disorder and the most common form of dementia. The disease is confirmed by the presence of neuritic plaques and neurofibrillary tangles in the cerebral cortex at autopsy, but the accuracy of antemortem diagnosis, especially at the early stages of the disease, is not ideal. Thus, there is a substantial need for the discovery and validation of diagnostic biomarkers. Many Alzheimer's disease biomarker discovery studies emphasize the analysis of cerebrospinal fluid (CSF) because of its close association with the brain. Here, we review recent mass spectrometry-based studies of Alzheimer's disease CSF, and additionally discuss issues associated with CSF in proteomics studies.


Assuntos
Doença de Alzheimer/diagnóstico , Doença de Alzheimer/líquido cefalorraquidiano , Animais , Biomarcadores/líquido cefalorraquidiano , Humanos , Proteômica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem , Eletroforese em Gel Diferencial Bidimensional
14.
Dela J Public Health ; 6(2): 10-15, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34467100

RESUMO

OBJECTIVES: To increase testing capability for SARS-CoV-2 during a rapidly evolving public health emergency, we aimed to deploy a validated laboratory-developed real-time reverse transcription polymerase chain reaction (RT-PCR) diagnostic test for SARS-CoV-2 on an accelerated timeline and using reagent supply chains that were not constrained. METHODS: A real-time RT-PCR assay that detects the structural envelope (E) gene of SARS-CoV-2 was developed and validated on the Roche cobas 6800 instrument platform with the omni Utility channel reagents, which performs automated nucleic acid extraction and purification, PCR amplification, and detection. In silico analysis was performed for both inclusivity of all SARS-CoV-2 variants and cross reactivity with other pathogenic organisms. Positive control material was used to determine the Limit of Detection (LOD) and patient samples (positive and negative) confirmed by another authorized assay were used for clinical validation. Experiments were carried out at the Christiana Care Health System's Molecular Diagnostics Laboratory (Newark, DE) between April 1 and April 4, 2020. RESULTS: A real-time RT-PCR assay for SARS-Cov-2 was developed and validated in just two weeks. For all oligonucleotides, 100% homology to the available SARS-CoV-2 sequences was observed. Greater than 80% homology between one or more oligonucleotides was observed for SARS-Cov (Urbani strain) and Influenza A, however risk of cross reactivity was deemed to be low. The limit of detection (LOD) of the assay was 250 copies/mL. The assay identified 100% of positive patient samples (30/30) and 100% of negative patient samples (29/29 patient negatives and 1/1 saline). Up to 92 samples can be run on a single plate and analysis takes approximately 3.5 hours. CONCLUSIONS: In this work, we demonstrate the development and validation of a single target laboratory-developed test for SARS-CoV-2 in two weeks. Key considerations for complementary supply chains enabled development on an accelerated timeline and an increase in testing capability.

15.
Biomaterials ; 29(26): 3514-20, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18514307

RESUMO

Bovine pericardium is an important biomaterial with current application in glutaraldehyde-fixed bioprosthetic heart valves and possible future application as an unfixed biological scaffold for tissue engineering. The importance of both humoral and cell-mediated rejection responses toward fixed and unfixed xenogeneic tissues has become increasingly apparent. However, the full scope and specific identities of bovine pericardium proteins that can elicit an immune response remain largely unknown. In this study, an immunoproteomic approach was used to survey bovine pericardium proteins for their ability to elicit a humoral immune response in rabbits. A two-stage protein extraction protocol was used to separate bovine pericardium proteins into water- and lipid-soluble fractions. Two-dimensional (2-D) gel electrophoresis was performed to separate the proteins from each fraction. Western blots were generated from 2-D gels of both bovine pericardium protein fractions. These blots were probed with serum from rabbits immunized with bovine pericardium and a secondary antibody was used to assess for IgG positivity. Western blots were compared to duplicate 2-D gels and proteins in matched spots were identified by tandem mass spectrometry. Thirty-one putative protein antigens were identified, eight of which are known to be antigenic from previous studies. All of the putative antigens demonstrated progressive staining intensity with increasing days of post-exposure serum. Identified antigenic proteins represented a variety of functional and structural protein types, and included both cellular and matrix proteins. The results of this study have implications for the use of bovine pericardium as a biomaterial in bioprostheses and tissue engineering applications, as well as xenotransplantation in general.


Assuntos
Antígenos Heterófilos/metabolismo , Pericárdio , Proteoma/imunologia , Animais , Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Bovinos , Dados de Sequência Molecular , Pericárdio/química , Pericárdio/imunologia , Coelhos
16.
ACS Biomater Sci Eng ; 4(3): 836-845, 2018 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-29552635

RESUMO

Cells interact with and remodel their microenvironment, degrading large extracellular matrix (ECM) proteins (e.g., fibronectin, collagens) and secreting new ECM proteins and small soluble factors (e.g., growth factors, cytokines). Synthetic mimics of the ECM have been developed as controlled cell culture platforms for use in both fundamental and applied studies. However, how cells broadly remodel these initially well-defined matrices remains poorly understood and difficult to probe. In this work, we have established methods for widely examining both large and small proteins that are secreted by cells within synthetic matrices. Specifically, human mesenchymal stem cells (hMSCs), a model primary cell type, were cultured within well-defined poly(ethylene glycol) (PEG)-peptide hydrogels, and these cell-matrix constructs were decellularized and degraded for subsequent isolation and analysis of deposited proteins. Shotgun proteomics using liquid chromatography and mass spectrometry identified a variety of proteins, including the large ECM proteins fibronectin and collagen VI. Immunostaining and confocal imaging confirmed these results and provided visualization of protein organization within the synthetic matrices. Additionally, culture medium was collected from the encapsulated hMSCs, and a Luminex assay was performed to identify secreted soluble factors, including vascular endothelial growth factor (VEGF), endothelial growth factor (EGF), basic fibroblast growth factor (FGF-2), interleukin 8 (IL-8), and tumor necrosis factor alpha (TNF-α). Together, these methods provide a unique approach for studying dynamic reciprocity between cells and synthetic microenvironments and have the potential to provide new biological insights into cell responses during three-dimensional (3D) controlled cell culture.

17.
Artigo em Inglês | MEDLINE | ID: mdl-23735279

RESUMO

There is significant interest in the development of methods to validate novel biomarkers for Alzheimer's disease (AD) diagnosis. Previously, a proteomic panel of cerebrospinal fluid (CSF) biomarker candidates that differentiated AD and non-AD CSF with accuracy higher than 90% was found; information about these CSF proteins can be used to develop multiple reaction monitoring (MRM) based analytical assays, which offer the possibility of quantifying protein expression level changes in samples, as well as, validation among multiple laboratories. Here we report an MRM assay that demonstrates good linearity (average R(2)=0.969) and reproducibility (average coefficient of variance of 6.93%) for the proposed AD CSF biomarkers. MRM quantification results of Aß1-40, Aß1-42, retinol-binding protein and cystatin C correlated well with those from ELISA (average R(2)=0.974). Analysis shows that 12 out of 16 selected targets exhibit the same trend in protein expression as that in literature.


Assuntos
Doença de Alzheimer/líquido cefalorraquidiano , Proteínas do Líquido Cefalorraquidiano/líquido cefalorraquidiano , Proteômica/métodos , Sequência de Aminoácidos , Biomarcadores/líquido cefalorraquidiano , Cromatografia Líquida , Humanos , Espectrometria de Massas , Dados de Sequência Molecular , Reprodutibilidade dos Testes
18.
J Proteome Res ; 6(3): 1145-52, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17284062

RESUMO

Microbial communities are of great environmental, medical, and industrial significance. To date, biomolecular methods to study communities have focused on identifying species, with limited capabilities to reveal functions. Proteomics has the potential to yield functional information about these communities, but the application of proteomic methods to complex mixtures of unsequenced organisms is in its infancy. In this study, 2DE, MALDI-TOF/TOF MS, and de novo peptide sequencing were used for the separation and identification of proteins differentially expressed over time following exposure of a bacterial community to an inhibitory level of cadmium. Significant community proteome responses after 0.25, 1, 2, and 3 h of exposure to cadmium were observed, with more than 100 protein expression changes detected at each time point. Several temporal responses were observed, and the most common expression pattern was immediate up- or down-regulation within 15 min of shock followed by maintenance of that level. More than 100 unique differentially expressed proteins were identified through database searching and de novo sequencing. Proteins of importance in the cadmium shock included ATPases, oxidoreductases, and transport proteins. The ability of proteomics to detect the differential regulation of these proteins even during short cadmium exposures shows that it is a powerful tool in explaining cellular mechanisms for a mixed culture. This is the first report of the large-scale identification of proteins involved in the dynamic response of a community of unsequenced bacteria using de novo sequencing.


Assuntos
Proteínas de Bactérias/análise , Cádmio/farmacologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Proteômica/métodos , Microbiologia da Água , Adenosina Trifosfatases/análise , Adenosina Trifosfatases/genética , Proteínas de Bactérias/genética , Proteínas de Transporte/análise , Proteínas de Transporte/genética , Eletroforese em Gel Bidimensional , Cinética , Oxirredutases/análise , Oxirredutases/genética , Análise de Sequência de Proteína , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem
19.
Ann Neurol ; 61(2): 120-9, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17167789

RESUMO

OBJECTIVE: To find a panel of proteins in antemortem cerebrospinal fluid (CSF) that could be used to differentiate between samples from Alzheimer's disease (AD) patients and samples from healthy and neurological control subjects. METHODS: For a test cohort, antemortem CSF proteins from 34 AD and 34 non-AD patients were separated using two-dimensional gel electrophoresis. The resulting protein patterns were analyzed using the random forest multivariate statistical method. Protein spots of interest were identified using tandem time-of-flight mass spectrometry. A validation cohort consisting of CSF from 18 AD patients and 10 non-AD subjects was analyzed in a similar way. RESULTS: Using the test cohort, a panel of 23 spots was identified that could be used to differentiate AD and non-AD gels with a sensitivity of 94%, a specificity of 94%, and a predicted classification error rate of only 5.9%. These proteins are related to the transport of beta-amyloid, the inflammatory response, proteolytic inhibition, and neuronal membrane proteins. The 23 spots separately classified the validation cohort with 90% sensitivity (probable AD subjects), 83% specificity, and a predicted classification error rate of 14% in a blinded analysis. The total observed sensitivity is 93%, the total observed specificity is 90%, and the predicted classification error rate is 8.3%. INTERPRETATION: A panel of possible CSF biomarkers for AD has been identified using proteomic methods.


Assuntos
Doença de Alzheimer/líquido cefalorraquidiano , Doença de Alzheimer/diagnóstico , Biomarcadores/líquido cefalorraquidiano , Proteômica , Estudos de Coortes , Eletroforese em Gel Bidimensional , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Método Simples-Cego , Espectrometria de Massas em Tandem
20.
NeuroRx ; 3(3): 327-35, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16815216

RESUMO

Two-dimensional protein electrophoresis (2-DE) has undergone many technical improvements in the past 30 years, resulting in an analytical method that is unparalleled in the resolution of complex protein mixtures and capable of quantifying changes in protein expression from a wide variety of tissues and samples. The technique has been applied in many studies of neurologic disease to identify changes in spot patterns that correlate with disease. The true power of the technique emerges when it is coupled to state-of-the-art methods in mass spectrometry, which enable identification of the protein or proteins contained within a spot of interest on a 2-DE map. Investigators have successfully applied the technique to gain improved understanding of neurologic disease mechanisms in humans and in animal models and to discover biomarkers that are useful in the clinical setting. An important extension to these efforts that has not been realized thus far is the desire to profile changes in protein expression that result from therapy to help relate disease-modifying effects at the molecular level with clinical outcomes. Here we review the major advances in 2-DE methods and discuss specific examples of its application in the study of neurologic diseases.


Assuntos
Biomarcadores , Eletroforese em Gel Bidimensional/métodos , Doenças do Sistema Nervoso , Proteômica , Animais , Humanos , Doenças do Sistema Nervoso/diagnóstico , Doenças do Sistema Nervoso/genética , Doenças do Sistema Nervoso/metabolismo , Análise Serial de Proteínas/métodos
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