2D DIGE analysis of the bursa of Fabricius reveals characteristic proteome profiles for different stages of chicken B‐cell development

Antibody producing B‐cells are an essential component of the immune system. In contrast to human and mice where B‐cells develop in the bone marrow, chicken B‐cells develop in defined stages in the bursa of Fabricius, a gut associated lymphoid tissue. In order to gain a better understanding of critical biological processes like immigration of B‐cell precursors into the bursa anlage, their differentiation and final emigration from the bursa we analyzed the proteome dynamics of this organ during embryonic and posthatch development. Samples were taken from four representative developmental stages (embryonic day (ED) 10, ED18, day 2, and day 28) and compared in an extensive 2D DIGE approach comprising six biological replicates per time point. Cluster analysis and PCA demonstrated high reliability and reproducibility of the obtained data set and revealed distinctive proteome profiles for the selected time points, which precisely reflect the differentiation processes. One hundred fifty three protein spots with significantly different intensities were identified by MS. We detected alterations in the abundance of several proteins assigned to retinoic acid metabolism (e.g. retinal‐binding protein 5) and the actin‐cytoskeleton (e.g. vinculin and gelsolin). By immunohistochemistry, desmin was identified as stromal cell protein associated with the maturation of B‐cell follicles. Strongest protein expression difference (10.8‐fold) was observed for chloride intracellular channel 2. This protein was thus far not associated with B‐cell biology but our data suggest an important function in bursa B‐cell development.     

Differential cytoplasmic and whole cellular expression of histone H1 within the avian bursa of Fabricius

Bursal anti-steroidogenic peptide (BASP), purified from the chicken bursa of Fabricius (BF), has been previously demonstrated to be a potent and efficacious inhibitor of steroid hormone biosynthesis from chicken ovarian, and both mammalian and avian adrenal cells in vitro. Other studies have demonstrated that BASP can markedly reduce avian and mammalian mitogen-stimulated lymphocyte proliferation. Recent studies have indicated that BASP has a structural and functional relationship with histone H1. Immunohistochemical studies using a monoclonal antibody, which is known to recognize a common histone H1 epitope from several plant and animal species identified the protein within the cytoplasm and nucleus of distinct cells within both the cortex and medulla of all BF follicles. Additionally, epithelial cells within the BF expressed the protein strongly in the cytoplasm with reduced nuclear staining. In contrast, the same antibody did not recognize the protein in thymus of the same animals. The differential expression of histone H1 immunoreactivity within selected cells of the BF may support a previous proposed role of histone H1 in extranuclear and extracellular signaling in chickens and possibly other species.     

The bursa of Fabricius: a central organ providing for contact between the lymphoid system and intestinal content

Small inert particles, after oral or intracloacal administration to chickens, are readily taken up and deposited throughout the medullary portion of bursal follicles. Epithelial tuft cells transporting this material are distinctively equipped with lytic enzymes. No entry of living bacteria into the lymphoid tissue was observed. These findings are indicative of an antigen-driven development of the bursal system protected by local bactericidal mechanisms.     

Expanding the organismal scope of proteomics: cross-species protein identification by mass spectrometry and its implications

Due to the limited applicability of conventional protein identification methods to the proteomes of organisms with unsequenced genomes, researchers have developed approaches to identify proteins using mass spectrometry and sequence similarity database searches. Both the integration of mass spectrometry with bioinformatics and genomic sequencing drive the expanding organismal scope of proteomics.     

The hard cell: from proteomics to a whole cell model

Proteomics has provided a wealth of data related to the nature of the proteome, including subcellular location, copy number, interaction partners and protein complexes. This raises the question of whether it is feasible to combine these data, together with other data related to overall cellular structure, to construct a static picture of the cell. In this minireview, we discuss these data, and the issues of turning them into whole cell models.     

Tissue imaging using MALDI-MS: a new frontier of histopathology proteomics

Modern pathology is an amalgam of many disciplines, such as microbiology, biochemistry and immunology, which historically have been intermingled with the practice of clinical medicine. For centuries, the pre-eminent pathological tool, at least in the context of patients, was a post-mortem examination. With the advent of optical microscopes, morphology became a predominant means of developing tissue classification. A further paradigm shift occurred in the attempt to understand the nature and origin of disease; the recognition that, ultimately, it is the derangement in the structure and function of genes and proteins that causes human disease. More recent progress in pathology has led to the use of genomics and molecular technologies, including DNA sequencing, microarray analysis, PCR, in situ hybridization and proteomics. Today, the newest frontier appears to be histopathology proteomics, which adds the mass spectrometer to the arsenal of tools for the direct analysis of tissue biopsies and molecular diagnosis. Typically called MALDI imaging, this technique takes mass spectral snapshots of intact tissue slices, revealing how proteins and peptides are spatially distributed within a given sample. In this review, MALDI imaging technology is presented as well as applications of such technology in cancer or neurodegenerative diseases.     

Towards cracking the epigenetic code using a combination of high-throughput epigenomics and quantitative mass spectrometry-based proteomics

High-throughput genomic sequencing and quantitative mass spectrometry (MS)-based proteomics technology have recently emerged as powerful tools, increasing our understanding of chromatin structure and function. Both of these approaches require substantial investments and expertise in terms of instrumentation, experimental methodology, bioinformatics, and data interpretation and are, therefore, usually applied independently from each other by dedicated research groups. However, when applied reiteratively in the context of epigenetics research these approaches are strongly synergistic in nature.     

Subcellular proteomics revealed the epithelial-mesenchymal transition phenotype in lung cancer

Subcellular proteomics was used to compare the protein profiles between human lung adenocarcinoma A549 cells and human bronchial epithelial (HBE) cells. In total, 106 differential proteins were identified and the altered expression levels of partial identified proteins were confirmed by Western blot analysis. Importantly, pathway analysis and biological validation revealed epithelial-mesenchymal transition (EMT) phenotype shift in A549 cells as compared with HBE cells. The EMT phenotype of A549 cells can be increased by self-producing TGF-β1 and significantly decreased by silencing heterogeneous nuclear ribonucleoprotein (hnRNPK) expression. As EMT has been considered as an important event during malignant tumor progression and metastasis, investigating EMT and deciphering the related pathways may lead to more efficient strategies to fight lung cancer progression. By integrating the subcellular proteomic data with EMT-related functional studies, we revealed new insights into the EMT progress of lung carcinogenesis, providing clues for further investigations on the discovery of potential therapeutic targets.     

Isolation, modulatory functions on murine B cell development and antigen-specific immune responses of BP11, a novel peptide from the chicken bursa of Fabricius

The bursa of Fabricius (BF) is the central humoral immune organ unique to birds which plays important roles in B lymphocyte differentiation. Here, a new bursal peptide (BP11) with the amino acid sequence DVAGKLPDNRT was identified and characterized from BF. It was proved that BP11 promoted CFU pre-B formation, and regulated B cell differentiation, including increase the percentage of immature and mature B cells in BM cells co-cultured with IL-7. BP11 also exerted immunomodulatory function on antigen-specific immune responses in BALB/c mice immunized with inactivated influence virus (AIV, H9N2 subtype) vaccine, including enhancing AIV-specific antibody and cytokine production. Furthermore, it was noteworthy that BP11 stimulated antibody productions and potentiates the Th1 and Th2-type immune responses in dose-dependent manner in chicken. These results suggested that BP11 might be highly relevant for the development of avian immune system.     

Trypsin digest coupled with two-dimensional shotgun proteomics reveals the involvement of multiple signaling pathways in functional remodeling of late-gestation uteri in rats

Pregnant uteri become quiescent after functional remodeling but details are not fully known. Here we revealed uterine proteins of late-gestation rats by 2-D shotgun proteomic analysis and correlated protein expression with uterine functions. After duplication, 239 proteins were identified. About 190 proteins commonly found in duplicate analyses were subjected to functional annotation. The proteins associated with signal transduction fell into three known pathways. Western blotting and functional data indicated that: (i) a reduction of Na(+)/K(+)-ATPase-related proteins was associated with the decrease of contraction rate, (ii) a reduction of tyrosine hydroxylase and cyclic AMP-dependent protein kinase type II-alpha regulatory chain (PKARII alpha) was associated with an increase in the relaxation response to 8-bromo-cAMP, and (iii) in the presence of Ras, an increased expression of nucleolin was associated with the elevation of Bcl-xL, an antiapoptotic protein. In conclusion, 2-D shotgun proteomic analysis provides a global database of uterine proteins for hypothesis-driven studies. Our data suggest that in late-gestation uteri down-regulation of PKARII alpha and Na(+)/K(+)-ATPase may cause functional remodeling and lead to uterine quiescent. Up-regulation of antiapoptotic proteins (nucleolin and Bcl-xL) in the Ras-mediated pathway may maintain cell survival and counteract cell loss during remodeling.     

Systemic sclerosis biomarkers discovered using mass-spectrometry-based proteomics: a systematic review

Abstract

Context: Systemic sclerosis (SSc) is an autoimmune disease with incompletely known physiopathology. There is a great challenge to predict its course and therapeutic response using biomarkers.

Objective: To critically review proteomic biomarkers discovered from biological specimens from systemic sclerosis patients using mass spectrometry technologies.

Methods: Medline and Embase databases were searched in February 2014.

Results: Out of the 199 records retrieved, a total of 20 records were included, identifying 116 candidate proteomic biomarkers.

Conclusion: Research in SSc proteomic biomarkers should focus on biomarker validation, as there are valuable mass-spectrometry proteomics studies in the literature.     

MSDA,a proteomics software suite for in‐depth Mass Spectrometry Data Analysis using grid computing

One of the major bottlenecks in the proteomics field today resides in the computational interpretation of the massive data generated by the latest generation of high‐throughput MS instruments. MS/MS datasets are constantly increasing in size and complexity and it becomes challenging to comprehensively process such huge datasets and afterwards deduce most relevant biological information. The Mass Spectrometry Data Analysis (MSDA, https://msda.unistra.fr ) online software suite provides a series of modules for in‐depth MS/MS data analysis. It includes a custom databases generation toolbox, modules for filtering and extracting high‐quality spectra, for running high‐performance database and de novo searches, and for extracting modified peptides spectra and functional annotations. Additionally, MSDA enables running the most computationally intensive steps, namely database and de novo searches, on a computer grid thus providing a net time gain of up to 99% for data processing.     

Improved metabolites of pharmaceutical ingredient grade Ginkgo biloba and the correlated proteomics analysis

Ginkgo biloba is an attractive and traditional medicinal plant, and has been widely used as a phytomedicine in the prevention and treatment of cardiovascular and cerebrovascular diseases. Flavonoids and terpene lactones are the major bioactive components of Ginkgo, whereas the ginkgolic acids (GAs) with strong allergenic properties are strictly controlled. In this study, we tested the content of flavonoids and GAs under ultraviolet‐B (UV‐B) treatment and performed comparative proteomic analyses to determine the differential proteins that occur upon UV‐B radiation. That might play a crucial role in producing flavonoids and GAs. Our phytochemical analyses demonstrated that UV‐B irradiation significantly increased the content of active flavonoids, and decreased the content of toxic GAs. We conducted comparative proteomic analysis of both whole leaf and chloroplasts proteins. In total, 27 differential proteins in the whole leaf and 43 differential proteins in the chloroplast were positively identified and functionally annotated. The proteomic data suggested that enhanced UV‐B radiation exposure activated antioxidants and stress‐responsive proteins as well as reduced the rate of photosynthesis. We demonstrate that UV‐B irradiation pharmaceutically improved the metabolic ingredients of Ginkgo, particularly in terms of reducing GAs. With high UV absorption properties, and antioxidant activities, the flavonoids were likely highly induced as protective molecules following UV‐B irradiation.     

Global identification of miR-373-regulated genes in breast cancer by quantitative proteomics

Although microRNAs (miRNAs) have been reported to play an important role in carcinogenesis, their molecular mechanism remains largely unknown because of our limited understanding of miRNA target genes. miR-373 was found to be capable of promoting breast cancer invasion and metastasis, but only a target gene was experimentally identified on the basis of mRNA expression analysis. In this study, we used SILAC-based quantitative proteomics to globally identify the genes regulated by miR-373. Totally, 3666 proteins were identified, and 335 proteins were found to be regulated by miR-373. Among the 192 proteins that were downregulated by miR-373, 27 (14.1%) were predicted to have at least one potential match site at their 3'-UTR for miR-373 seed sequence. However, miR-373 did not affect the mRNA level of the five selected candidate targets, TXNIP, TRPS1, RABEP1, GRHL2 and HIP1, suggesting that the protein expressions were regulated by miR-373 via translational inhibition instead of mRNA degradation. Luciferase and mutation assays validated that TXNIP and RABEP1 were the direct target genes of miR-373. More than 30 proteins reported to be involved in cancer invasion and metastasis were found to be regulated by miR-373 in breast cancer for the first time.     

Identification of estrogen-responsive proteins in MCF-7 human breast cancer cells using label-free quantitative proteomics

17beta-Estradiol (E(2)) is a key regulatory steroid hormone that is involved in the control of a number of developmental and other functions. The aim of the present work was to identify estrogen-dependent proteomic changes by determining the levels of expressed proteins in MCF-7 human breast cancer cells following treatment with E(2). A number of methods exist for differential analysis of complex proteomic mixtures. Here, a label-free mass spectrometric approach comparing the ion intensities of tryptic peptides was adopted, which was combined with prefractionation of whole cell lysate proteins by 1-D SDS-PAGE. Using this approach, 60 proteins were found to be affected by E(2). These comprised 55 up-regulated and five down-regulated proteins. These proteins varied widely in their physiochemical properties with pIs of 4-12 and molecular weights of 9-500 kDa. Pathway analysis revealed that the majority of changes were related and together describe an up-regulated pathway consistent with the events of cell proliferation. The quantitative approach used here is relatively straightforward, avoids the use of costly labelling reagents, was reproducible within acceptable limits and has a linear response over a useful concentration range.     

Identification of membrane-associated proteins from Campylobacter jejuni strains using complementary proteomics technologies

Campylobacter jejuni is the leading cause of food- and water-borne illness world-wide. The membrane-associated proteome of a recent C. jejuni gastrointestinal isolate (JHH1) was generated by sodium carbonate precipitation and ultracentrifugation followed by 2-DE and MALDI-TOF MS as well as 2-DLC (strong cation exchange followed by RP chromatography) of trypsin digests coupled to MS/MS (2-DLC/MS/MS). 2-DE/MS identified 77 proteins, 44 of which were predicted membrane proteins, while 2-DLC/MS/MS identified 432 proteins, of which 206 were predicted to be membrane associated. A total of 453 unique proteins (27.4% of the C. jejuni theoretical proteome), including 187 bona fide membrane proteins were identified in this study. Membrane proteins were also compared between C. jejuni JHH1 and ATCC 700297 to identify factors potentially associated with increased gastrointestinal virulence. We identified 28 proteins that were significantly (>two-fold) more abundant in, or unique to, JHH1, including eight proteins involved in chemotaxis signal transduction and flagellar motility, the amino acid-binding surface antigens CjaA and CjaC, and four outer membrane proteins (OMPs) of unknown function (Cj0129c, Cj1031, Cj1279c, and Cj1721c). Immunoblotting using convalescent patient sera generated post-gastrointestinal infection revealed 13 (JHH1) and 12 (ATCC 700297) immunoreactive proteins. These included flagellin (FlaA) and CadF as well as Omp18, Omp50, Cj1721c, PEB1A, PEB2, and PEB4A. This study provides a comprehensive analysis of membrane-associated proteins from C. jejuni.     

基于生物质谱的蛋白质C末端研究进展

蛋白质的C末端在蛋白质进行各项生命活动过程中都起着极其重要的作用。它不仅标志着DNA转录翻译成蛋白质过程的初步完成,更是参与和调控了蛋白质的各种生理功能。研究蛋白质的C末端不仅有利于完整蛋白质的鉴定,对于在分子水平理解蛋白质的信号传导和生化功能是十分必要的。文中结合我们的研究工作,综述了近年来基于生物质谱的蛋白质C末端研究的相关进展,包括了C末端的识别、鉴定以及蛋白质C末端肽段富集的新方法和新技术。