Proteomics on an Orbitrap benchtop mass spectrometer using all-ion fragmentation

Area of Science:

• Proteomics
• Mass Spectrometry
• Analytical Chemistry

Background:

• Orbitrap mass analyzers offer high sensitivity, resolution, and mass accuracy.
• Hybrid LTQ-Orbitrap instruments are common in proteomics.
• Stand-alone Exactive Orbitrap lacks ion isolation but has higher energy collisional dissociation.

Purpose of the Study:

• To evaluate the Exactive Orbitrap's capability for protein mixture characterization using all-ion fragmentation (AIF) and MS/MS scans.
• To adapt and apply existing peak recognition algorithms for AIF data analysis.
• To demonstrate the utility of AIF on the Exactive for complex proteomics applications.

Main Methods:

• Alternating MS and all-ion fragmentation (AIF) MS/MS scans on the Exactive Orbitrap.
• Utilizing MaxQuant software for peak recognition at precursor and product ion levels.
• Implementing stepped collision energy with cumulative MS readout for broad precursor fragmentation.

Main Results:

• AIF identified 45 of 48 proteins in an equimolar standard mixture, and all with a small database.
• The method detected proteins with over 100-fold abundance differences in a high dynamic range standard.
• Unambiguous identification and relative quantification of a low-abundance immunoprecipitated protein in gel slices.

Conclusions:

• All-ion fragmentation (AIF) on the Exactive Orbitrap is an effective technique for characterizing protein mixtures.
• This approach significantly enhances protein identification and quantification capabilities in proteomics.
• AIF on benchtop Orbitrap instruments presents an attractive technology for diverse proteomics analyses.