Use of Gas-Phase Fractionation to Increase Protein Identifications: Application to the Peroxisome

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.