Approaching complete peroxisome characterization by gas-phase fractionation

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Direct microliquid chromatography/electrospray ionization-tandem mass spectrometry (microLC/ESI-MS/MS) is crucial for proteome analysis.
• Improving proteome coverage and reproducibility in peptide ion selection remains a challenge.

Purpose of the Study:

• To evaluate the utility of gas-phase fractionation (GPF) for enhancing proteome coverage and reproducibility.
• To compare GPF in the m/z dimension (GPF(m/z)) and relative ion intensity dimension (GPF(RI)) with standard methods.

Main Methods:

• Proteolytic digestion of yeast whole-cell lysate and peroxisomal membrane protein fractions.
• Analysis using direct microLC/ESI-MS/MS with and without GPF.
• GPF applied in the m/z dimension across specific ranges and in the relative ion intensity dimension.

Main Results:

• GPF(m/z) identified 1.3x more proteins than triplicate standard analysis for yeast whole-cell lysate, with lower average codon bias.
• GPF(RI) identified more proteins per m/z unit scanned compared to GPF(m/z) and standard analysis.
• GPF(m/z) detected 93% of known peroxisomal proteins, significantly higher than the 73% detected by standard scans.

Conclusions:

• GPF, particularly GPF(m/z), significantly improves proteome coverage and reproducibility in microLC/ESI-MS/MS.
• GPF is effective for analyzing complex proteomes, including specific fractions like peroxisomal membrane proteins.