Proteomics of Vibrio cholerae
Combining high-throughput mass spectrometry with isobaric tags for relative and absolute quantification (iTRAQ) allows for the identification and relative quantification of proteins from multiple samples. Furthermore, low-abundance proteins that are usually not detected can be enriched by using only the relevant fraction of the proteome, e.g., cytoplasmic, membrane proteins, or secreted proteins. Described here is a workflow for isolation and enrichment of secreted and membrane proteins that is compatible with mass spectrometry. Isolated proteins are reduced, alkylated, and digested with trypsin, and obtained peptides are labeled with iTRAQ reagent and separated by strong cation exchange to reduce the complexity. Finally, the peptides are separated by reverse-phase chromatography, spotted on a MALDI target plate, and analyzed by MALDI TOF-TOF.
Key wordsProteomics iTRAQ Secreted proteins Cell envelope 2D-LC-MS/MS
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