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

Proteomics - Tập 7 Số 11 - Trang 1757-1770 - 2007
Mireille Chevallet1,2, Hélène Diemer3, Alain Van Dorssealer3, Christian Villiers1, Thierry Rabilloud1
1CEA-DSV/DRDC/ICH, Laboratoire d'Immunochimie, CEA-Grenoble, Grenoble, France
2INSERM U548, Controle moléculaire de la réponse immune spécifique, CEA-Grenoble, Grenoble, France
3CNRS UMR7178, Institut Pluridisciplinaire Hubert Curien, ECPM, Strasbourg, France

Tóm tắt

AbstractThe 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.

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Tài liệu tham khảo

10.1002/elps.1150190616

10.1074/jbc.M006143200

Liu Y., 1999, Proc. Natl. Acad. Sci. USA, 14694

10.1002/(SICI)1522-2683(20000401)21:6<1054::AID-ELPS1054>3.0.CO;2-8

10.1038/nbt819

10.1002/pmic.200401258

10.1002/pmic.200300691

10.1074/jbc.M211980200

10.1016/j.cellimm.2004.04.002

10.1002/elps.1150090110

10.1006/abio.1997.2248

10.1006/abio.1995.1242

10.1016/S0003-2697(76)80064-4

10.1002/elps.1150101115

10.1002/elps.11501501223

10.1002/elps.1150180303

10.1002/pmic.200300589

10.1002/pmic.200500567

10.1002/(SICI)1522-2683(19990301)20:3<601::AID-ELPS601>3.0.CO;2-6

10.1002/pmic.200300642

10.1002/(SICI)1522-2683(19991201)20:18<3551::AID-ELPS3551>3.0.CO;2-2

10.4049/jimmunol.161.9.4476

10.4049/jimmunol.175.2.839

10.1002/pmic.200600096

10.1006/abio.1998.3018

10.1002/elps.11501301195

10.1002/elps.1150170716

10.1016/S0091-679X(08)60115-6

10.1128/jb.138.2.650-652.1979

10.1006/abio.1999.4149

10.1021/pr025541x

10.1002/pmic.200401178

10.1002/elps.200410387

10.1021/pr050375p