RESUMEN
BACKGROUND: Mouse allergy is an important cause of indoor asthma and allergic rhinoconjunctivitis. The major mouse allergen, Mus m 1, is a complex of homologous pheromone-binding lipocalins called major urinary proteins (MUPs). METHODS: We analyzed the proteome of MUPs in mouse urine, commercial mouse epithelial extracts, and environmental samples using several approaches. These include as follows: two-dimensional electrophoresis and immunoblotting; liquid chromatography-high-resolution mass spectrometry (LC/HRMS); multiple reaction monitoring (MRM) mass spectrometry; and LC/HRMS analysis of glycans at the N-66 residue of MUP3. RESULTS: Albumin is predominant in the extracts, while MUPs are predominant in urine. LC/HRMS of 4 mouse allergen extracts revealed surprising heterogeneity. Of 22 known mouse MUPs, only 6 (MUP3, MUP4, MUP5, MUP13, MUP20, and MUP21) could be identified with MRM using unique peptides. Assessment of MUP content in urine, extracts, and dust samples showed good correlation between MRM and other methods working with different detection principles. All 6 identifiable MUPs were found in electrophoretically separated urine bands, but only MUP3 and MUP20 were above LOQ in unseparated mouse urine, and only MUP3, MUP4, and MUP20 were found in mouse epithelial extracts. Glycan heterogeneity was noted among 4 individual inbred mice: of 13 glycan structures detected, 8 were unique to one mouse, and only 2 glycan modifications were present in all 4 mice. CONCLUSIONS: Using mass spectrometry and MRM, mouse allergen extracts and urine samples are shown to be complex and heterogeneous. The efficacy and safety of commercial mouse allergen extracts will be improved with better controls of allergen content.
Asunto(s)
Alérgenos , Asma , Alérgenos/química , Animales , Asma/etiología , Polvo , Ratones , Proteoma , OrinaRESUMEN
BACKGROUND: Allergen extracts are the primary tool for diagnosis and treatment of allergic diseases. In the United States, most allergen extracts are non-standardized. More sophisticated analytical approaches are needed to characterize these products and enable manufacturers and regulators to better determine potency. OBJECTIVE: To expand the multiple reaction monitoring (MRM) assay for an in-depth characterization of German cockroach (GCr; Blattella germanica) allergen extracts. METHODS: We applied advanced liquid chromatography (LC) and mass spectrometry (MS) techniques including MRM. The expanded LC/MRM-MS method was optimized to measure known GCr allergens and their isoforms/variants in commercial extracts and environmental samples. We performed isoform-specific allergen measurements in multiple extracts from four commercial sources and extracts prepared using environmental samples from urban homes. To investigate causes of heterogeneity, we examined over 30 extraction process variables. RESULTS: Evaluation of the commercial extracts confirmed the variability of production lots and commercial sources. Commonly used defatting and extraction protocols yielded extracts with comparable allergen profiles and content. However, the identity and quality of source materials was a major contributor to variability. In comparing commercial GCr extracts to environmental samples, relative quantities of Bla g 1, Bla g 2, Bla g 3, Bla g 4 and Bla g 11 were similar, while Bla g 5, Bla g 6, Bla g 7 and Bla g 8 were present in the environmental samples but largely absent for the commercial extracts. CONCLUSIONS AND CLINICAL RELEVANCE: LC/MRM-MS can be used to measure all known GCr allergens in commercial allergen extracts and environmental samples. Significant differences exist between allergen profiles of commercial extracts and the profiles of environmental samples from dwellings. This analytical platform can serve as a template to achieve better product characterization of similarly complex products.
Asunto(s)
Alérgenos/química , Blattellidae/química , Mezclas Complejas/química , Proteínas de Insectos/química , Animales , Cromatografía Liquida , Espectrometría de MasasRESUMEN
Susceptibility to influenza A virus is determined by a balance of viral and host factors. The genetic background of the host contributes to the severity of disease, but the influenza-related proteomes of cells from different individuals have not been compared. We used high-resolution mass spectrometry to identify proteins in normal human bronchial epithelial (NHBE) cells isolated from three different donors. Infection of each NHBE cell culture with influenza A/California/07/2009 (H1N1) resulted in expression of viral proteins and a variety of host proteins, including interferons, interferon-stimulated genes, and secreted chemokines/cytokines. The expression level of viral proteins corresponded to the level of host proteins that support influenza infection (i.e., pro-viral proteins); however, production of infectious virus was inversely related to the levels of antiviral proteins, suggesting that a balance of pro-viral proteins and the antiviral response controls virus replication. In summary, our results demonstrate that expression levels of pro-viral as well as antiviral factors are different for each donor and suggest that relative quantitation of these factors may provide a way to identify individuals or population groups who are susceptible to severe influenza disease.
Asunto(s)
Células Epiteliales/metabolismo , Interacciones Huésped-Patógeno , Subtipo H1N1 del Virus de la Influenza A/genética , Proteoma/genética , Proteínas Virales/genética , Citocinas/genética , Citocinas/inmunología , Células Epiteliales/virología , Regulación de la Expresión Génica , Humanos , Subtipo H1N1 del Virus de la Influenza A/metabolismo , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/inmunología , Interferones/genética , Interferones/inmunología , Metalotioneína/genética , Metalotioneína/inmunología , Anotación de Secuencia Molecular , Cultivo Primario de Células , Proteoma/inmunología , Proteómica/instrumentación , Proteómica/métodos , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/virología , Transducción de Señal , Superóxido Dismutasa/genética , Superóxido Dismutasa/inmunología , Ubiquitina/genética , Ubiquitina/inmunología , Carga Viral , Proteínas Virales/metabolismoRESUMEN
The major objective of personalized medicine is to select optimized drug therapies and to a large degree such mission is determined by the expression profiles of cytochrome(s) P450 (CYP). Accordingly, a proteomic case study in personalized medicine is provided by the superfamily of cytochromes P450. Our knowledge about CYP isozyme expression on a protein level is very limited and based exclusively on DNA/mRNA derived data. Such information is not sufficient because transcription and translation events do not lead to correlated levels of expressed proteins. Here we report expression profiles of CYPs in human liver obtained by mass spectrometry (MS)-based proteomic approach. We analyzed 32 samples of human liver microsomes (HLM) of different sexes, ages and ethnicity along with samples of recombinant human CYPs. We have experimentally confirmed that each CYP isozyme can be effectively differentiated by their unique isozyme-specific tryptic peptide(s). Trypsin digestion patterns for almost 30 human CYP isozymes were established. Those findings should assist in selecting tryptic peptides suitable for MS-based quantitation. The data obtained demonstrate remarkable differences in CYP expression profiles. CYP2E1, CYP2C8 and CYP4A11 were the only isozymes found in all HLM samples. Female and pediatric HLM samples revealed much more diverse spectrum of expressed CYPs isozymes compared to male HLM. We have confirmed expression of a number of "rare" CYP (CYP2J2, CYP4B1, CYP4V2, CYP4F3, CYP4F11, CYP8B1, CYP19A1, CYP24A1 and CYP27A1) and obtained first direct experimental data showing expression of such CYPs as CYP2F1, CYP2S1, CYP2W1, CYP4A22, CYP4X1, and CYP26A1 on a protein level.
Asunto(s)
Sistema Enzimático del Citocromo P-450/análisis , Sistema Enzimático del Citocromo P-450/genética , Espectrometría de Masas/métodos , Microsomas Hepáticos/metabolismo , Proteómica/métodos , Secuencia de Aminoácidos , Femenino , Humanos , Masculino , Microsomas Hepáticos/química , Datos de Secuencia MolecularRESUMEN
Bone-marrow derived mesenchymal stromal cells (BMSCs) have been in clinical trials for therapy. One major bottleneck in the advancement of BMSC-based products is the challenge associated with cell isolation, characterization, and ensuring cell fitness over the course of in vitro cell propagation steps. The data in this report is part of publications that explored the proteomic changes following in vitro passaging of BMSCs [4] and the molecular heterogeneity in cultures obtained from different human donors [5], [6].The methodological details involving cell manufacturing, proteome harvesting, protein identification and quantification as well as the bioinformatic analyses were described to ensure reproducibility of the results.
RESUMEN
Human bone marrow stromal cells (hBMSCs, also loosely called bone marrow-derived mesenchymal stem cells) are the subject of increasing numbers of clinical trials and laboratory research. Our group recently reported on the optimization of a workflow for a sensitive proteomic study of hBMSCs. Here, we couple this workflow with a label-free protein quantitation method to investigate the molecular responses of hBMSCs to long-term in vitro passaging. We explored the proteomic responses of hBMSCs by assessing the expression levels of proteins at early passage (passage 3, P3) and late passage (P7). We used multiple biological as well as technical replicates to ensure that the detected proteomic changes are repeatable between cultures and thus likely to be biologically relevant. Over 1700 proteins were quantified at three passages and a list of differentially expressed proteins was compiled. Bioinformatics-based network analysis and term enrichment revealed that metabolic pathways are largely altered, where many proteins in the glycolytic, pentose phosphate, and TCA pathways were shown to be largely upregulated in late passages. We also observed significant proteomic alterations in functional categories including apoptosis, and ER-based protein processing and sorting following in vitro cell aging. We posit that the comprehensive map outlined in this report of affected phenotypes as well as the underpinning molecular factors tremendously benefit the effort to uncovering targets that are not just used only to monitor cell fitness but can be employed to slowdown the in vitro aging process in hBMSCs and hence ensure manufacturing of cells with known quality, efficacy and stability.
Asunto(s)
Técnicas In Vitro/métodos , Células Madre Mesenquimatosas/metabolismo , Proteómica/métodos , Diferenciación Celular , Células Cultivadas , Humanos , Células Madre Mesenquimatosas/citologíaRESUMEN
The clinical application of human bone marrow stromal cells (hBMSCs) largely depends on their capacity to expand in vitro. We have conducted a comprehensive comparative proteomic analysis of culture-expanded hBMSCs obtained from different human donors. The data reveal extensive donor-to-donor proteomic heterogeneity. Processing and database-searching of the tandem MS data resulted in a most comprehensive to date proteomic dataset for hBMSC. A total of 7753 proteins including 712 transcription and translation regulators, 384 kinases, 248 receptor proteins, and 29 cytokines were confidently identified. The proteins identified are mainly nuclear (43.2%) and the share of proteins assigned to more than one subcellular location constitutes 10% of the identified proteome. Bioinformatics tools (IPA, DAVID, and PANTHER) were used to annotate proteins with respect to cellular locations, functions, and other physicochemical characteristics. We also compared the proteomic profile of hBMSCs to recently compiled datasets for human and mouse pluripotent stem cells. The result shows the extent of similarity between the three cell populations and also identified 253 proteins expressed uniformly by all lines of hBMSCs but not reported in the proteomic datasets of the two pluripotent stem cells. Overall, the proteomic database reported in this paper can serve as a reference map for extensive evaluation of hBMSC to explain their biology as well as identify possible marker candidates for further evaluation.
Asunto(s)
Células de la Médula Ósea/metabolismo , Células Madre Mesenquimatosas/metabolismo , Proteoma/metabolismo , Adulto , Animales , Diferenciación Celular/fisiología , Células Cultivadas , Femenino , Humanos , Masculino , Ratones , Proteoma/análisis , Proteoma/genética , Proteómica/métodos , Adulto JovenRESUMEN
A comprehensive analysis of the membrane proteome is essential to explain the biology of multipotent stromal cells and identify reliable protein biomarkers for the isolation as well as tracking of cells during differentiation and maturation. However, proteomic analysis of membrane proteins is challenging and they are noticeably under-represented in numerous proteomic studies. Here we introduce new approach, which includes high pressure-assisted membrane protein extraction, protein fractionation by gel-eluted liquid fraction entrapment electrophoresis (GELFREE), and combined use of liquid chromatography MALDI and ESI tandem mass spectrometry. This report presents the first comprehensive proteomic analysis of membrane proteome of undifferentiated and culture-expanded human bone marrow multipotent stromal cells (hBM-MSC) obtained from different human donors. Gene ontology mapping using the Ingenuity Pathway Analysis and DAVID programs revealed the largest membrane proteomic dataset for hBM-MSC reported to date. Collectively, the new workflow enabled us to identify at least two-fold more membrane proteins compared to published results on hBM-MSC. A total of 84 CDs were identified including 14 CDs identified for the first time. This dataset can serve as a basis for further exploration of self-renewal, differentiation and characterization of hBM-MSC.