TopSpot

TopSpot analyzes 2-dimensional electrophoretic (2DE) gel images to support large-scale proteomic identification and database construction.

Key Features:

• Data Acquisition and Format Compatibility: Processes 2DE gel images in BMP format or scanned versions for qualitative and quantitative analysis of protein spots.
• High-Performance Techniques Integration: Handles data from large-format gels (23 x 30 cm) using isoelectric focusing with anodic sample preparation and nonequilibrium running conditions (NEPHGE) followed by SDS-PAGE in the second dimension.
• Protein Identification Capabilities: Supports integration of amino acid analysis, N-terminal and internal sequencing, immunostaining, matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS), peptide mass fingerprinting, post-source decay MALDI-MS, ladder sequencing by carboxypeptidase P, in-gel digestion, and peptide purification.
• Database Construction: Facilitates building proteomic databases exemplified by a human myocardial 2-DE gel database with approximately 3300 characterized protein spots annotated with isoelectric point (pI) and molecular mass across pH 4.5–9.6.
• Federated Database Compliance: Adheres to four rules for a federated database to support interoperability and comprehensive data integration.

Scientific Applications:

• Human myocardial proteome mapping: Enables analysis and cataloguing of complex protein mixtures from human myocardial tissue using 2DE gel data.
• Protein identification and characterization: Supports determination of protein identity, isoelectric point, and molecular mass by integrating sequencing and mass spectrometry data.
• Post-translational modification and biomarker studies: Facilitates analysis of protein expression patterns, post-translational modifications, and disease biomarker discovery by combining 2DE gel results with peptide and sequencing data.

Methodology:

Processes BMP or scanned 2DE gel images for qualitative and quantitative spot analysis and integrates identification data from amino acid analysis, N-terminal and internal sequencing, immunostaining, MALDI-MS (including peptide mass fingerprinting and post-source decay), ladder sequencing by carboxypeptidase P, in-gel digestion, and peptide purification; accepts data derived from large-format isoelectric focusing (anodic sample preparation, NEPHGE) followed by SDS-PAGE.