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Toward a better analysis of secreted proteins: the example of the myeloid cells secretome.

ABSTRACT: The analysis of secreted proteins represents a challenge for current proteomics techniques. Proteins are usually secreted at low concentrations in the culture media, which makes their recovery difficult. In addition, culture media are rich in salts and other compounds interfering with most proteomics techniques, which makes selective precipitation of proteins almost mandatory for a correct subsequent proteomics analysis. Last but not least, the non-secreted proteins liberated in the culture medium upon lysis of a few dead cells heavily contaminate the so-called secreted proteins preparations. Several techniques have been used in the past for concentration of proteins secreted in culture media. These techniques present several drawbacks, such as coprecipitation of salts or poor yields at low protein concentrations. Improved techniques based on carrier-assisted TCA precipitation are described and discussed in this report. These techniques have been used to analyze the secretome of myeloid cells (macrophages, dendritic cells) and enabled to analyze proteins secreted at concentrations close to 1 ng/mL, thereby allowing the detection of some of the cytokines (TNF, IL-12) secreted by the myeloid cells upon activation by bacterial products.

SUBMITTER: Chevallet M

PROVIDER: S-EPMC2386146 | biostudies-literature | 2007 Jun

REPOSITORIES: biostudies-literature

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Toward a better analysis of secreted proteins: the example of the myeloid cells secretome.

Chevallet Mireille M Diemer Hélène H Van Dorssealer Alain A Villiers Christian C Villiers Christian C Rabilloud Thierry T

Proteomics 20070601 11

The analysis of secreted proteins represents a challenge for current proteomics techniques. Proteins are usually secreted at low concentrations in the culture media, which makes their recovery difficult. In addition, culture media are rich in salts and other compounds interfering with most proteomics techniques, which makes selective precipitation of proteins almost mandatory for a correct subsequent proteomics analysis. Last but not least, the non-secreted proteins liberated in the culture medi ...[more]

PMID: 17464941

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Project description:Data mining and knowledge discovery techniques have greatly progressed in the last decade. They are now able to handle larger and larger datasets, process heterogeneous information, integrate complex metadata, and extract and visualize new knowledge. Often these advances were driven by new challenges arising from real-world domains, with biology and biotechnology a prime source of diverse and hard (e.g., high volume, high throughput, high variety, and high noise) data analytics problems. The aim of this article is to show the broad spectrum of data mining tasks and challenges present in biological data, and how these challenges have driven us over the years to design new data mining and knowledge discovery procedures for biodata. This is illustrated with the help of two kinds of case studies. The first kind is focused on the field of protein structure prediction, where we have contributed in several areas: by designing, through regression, functions that can distinguish between good and bad models of a protein's predicted structure; by creating new measures to characterize aspects of a protein's structure associated with individual positions in a protein's sequence, measures containing information that might be useful for protein structure prediction; and by creating accurate estimators of these structural aspects. The second kind of case study is focused on omics data analytics, a class of biological data characterized for having extremely high dimensionalities. Our methods were able not only to generate very accurate classification models, but also to discover new biological knowledge that was later ratified by experimentalists. Finally, we describe several strategies to tightly integrate knowledge extraction and data mining in order to create a new class of biodata mining algorithms that can natively embrace the complexity of biological data, efficiently generate accurate information in the form of classification/regression models, and extract valuable new knowledge. Thus, a complete data-to-information-to-knowledge pipeline is presented.

Project description:Background. Bacillus subtilis is a favorable host for the production of industrially relevant proteins because of its capacity of secreting proteins into the medium to high levels, its GRAS (Generally Recognized As Safe) status, its genetic accessibility and its capacity to grow in large fermentations. However, production of heterologous proteins still faces limitations. Results. This study aimed at the identification of bottlenecks in secretory protein production by analyzing the response of B. subtilis at the transcriptome level to overproduction of eight secretory proteins of endogenous and heterologous origin and with different subcellular or extracellular destination: secreted proteins (NprE and XynA of B. subtilis, Usp45 of Lactococcus lactis, TEM-1beta -lactamase of Escherichia coli), membrane proteins (LmrA of L. lactis and XylP of Lactobacillus pentosus) and lipoproteins (MntA and YcdH of B. subtilis). Responses specific for proteins with a common localization as well as more general stress responses were observed. The latter include upregulation of genes encoding intracellular stress proteins (groES/EL, CtsR regulated genes). Specific responses include upregulation of the liaIHGFSR operon under Usp45 and TEM-1 beta-lactamase overproduction; cssRS, htrA and htrB under all secreted proteins overproduction; sigW and SigW-regulated genes mainly under membrane proteins overproduction; and ykrL (encoding an HtpX homologue) specifically under membrane proteins overproduction. Conclusions. The results give better insights into B. subtilis responses to protein overproduction stress and provide potential targets for genetic engineering in order to further improve B. subtilis as a protein production host. Samples for transcriptome analyses were induced at the exponential-growth phase (OD600 = 0.7) with 0.1% subtilin (subtilin containing supernatant of subtilin producing B. subtilis strain ATCC 6633). Cells were harvested 30 min after induction. Three or four independent cultures of each strain (target strains and controls) were used, and cells were sampled for microarray experiment.

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Toward a better analysis of secreted proteins: the example of the myeloid cells secretome.

Publications

Toward a better analysis of secreted proteins: the example of the myeloid cells secretome.

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