Protocol

Organelle Proteomics

Volume 432 of the series Methods in Molecular Biology™ pp 217-228

Use of Gas-Phase Fractionation to Increase Protein Identifications

Application to the Peroxisome
  • Jacob KennedyAffiliated withZymoGenetics, Inc
  • , Eugene C. YiAffiliated withZymoGenetics, Inc

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Summary

Gas-phase fractionation (GPF), defined as iterative mass spectrometric interrogations of a sample over multiple smaller mass-to-charge (m/z) ranges, enables the ions selected for collision-induced dissociation to come from a greater number of unique peptides compared to the ions selected from the wide mass range scan in automated liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. GPF is described as a means to achieve higher proteome coverage than multiple LC-MS/MS analyses of unfractionated complex peptide mixtures. It is applied to organellar proteomics through analysis of yeast peroxisomal proteins obtained from a discontinuous Nycodenz gradient fraction known to be enriched with yeast peroxisomal membrane proteins.

Key Words

Liquid chromatography-tandem mass spectrometry mass spectrometry gas-phase fractionation yeast peroxisome organelles data-dependent duty cycle subcellular Nycodenz gradient fraction