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High-performance human myocardial two-dimensional electrophoresis database: edition 1996

E C Müller1, B Thiede, U Zimny-Arndt

  • 1Max-Delbrück-Centrum für Molekulare Medizin, Berlin, Germany.

Electrophoresis
|November 1, 1996
PubMed
Summary

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This study presents a high-performance human myocardial two-dimensional electrophoresis (2-DE) gel database, detailing approximately 3300 protein spots. Advanced techniques enabled the identification of 70 proteins, enhancing proteomic analysis of the human heart.

Area of Science:

  • Proteomics
  • Molecular Biology
  • Biochemistry

Background:

  • The human heart proteome is complex and requires advanced techniques for comprehensive analysis.
  • Existing two-dimensional electrophoresis (2-DE) databases may lack detailed protein characterization and accessibility.

Purpose of the Study:

  • To establish a high-performance human myocardial 2-DE gel database.
  • To identify and characterize a significant number of myocardial proteins using advanced proteomic methods.

Main Methods:

  • Utilized large gels (23 x 30 cm) for high-resolution 2-DE, combining isoelectric focusing (IEF) with non-equilibrium pH gradient electrophoresis (NEPHGE) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
  • Employed a combination of mass spectrometry (MALDI-MS), peptide mass fingerprinting, sequencing (N-terminal, internal, ladder), amino acid analysis, and immunoblotting for protein identification.

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  • Developed an integrated workflow including in-gel digestion, peptide purification, and improved computational analysis (FRAGMOD) for sensitive and reliable protein identification.

    • Main Results:

    • Created a master gel database with approximately 3300 characterized protein spots, covering a pH range of 4.5 to 9.6.
    • Successfully identified 70 proteins using a combination of proteomic techniques, with high sensitivity and yield achieved through internal sequencing and MALDI-MS.
    • Developed a clickable, federated database with detailed protein information, including links to SWISS-PROT, other 2-DE databases, and Medline entries.

    Conclusions:

    • The developed high-performance human myocardial 2-DE database provides a valuable resource for cardiac research.
    • The integrated proteomic identification strategies offer high sensitivity and reliability for characterizing complex proteomes.
    • The database design facilitates data sharing and integration within the broader scientific community.