Development of top-down proteomics (TDP) is progressing at a rapid rate. As with well-established bottom-up proteomic methods, analysis requires the extraction of proteins from raw samples and often uses chromatography for separation of complex mixtures to simplify the identification and/or quantification of a proteome.
Unlike in bottom-up proteomics where proteins are digested and resultant peptides analysed; proteins remain intact allowing for the characterisation of individual proteoforms (1). The proteoform describes a protein with specific, localised modifications that result from biological events such as genetic variation, post-translational modifications and sequence cleavage events. By analysing intact proteins, this avoids problems that exist in bottom-up methods such as the introduction of artefacts caused by enzymatic digestion, and information loss, which requires the inference of protein structure and function from a subset of peptides.
Due to the high dynamic range of protein concentrations in a sample, top-down proteomics presents further challenges for separation and identification of proteoforms. Chromatographic separation of intact proteins is essential; however, this added complexity can benefit from the employment of orthogonal separation methods, such as gas-phase fractionation using high field asymmetric waveform ion mobility spectrometry (FAIMS).
The FAIMS Pro device attached to a Fusion Lumos provides an orthogonal separation method to chromatographic separation of intact proteins via gas-phase fractionation. FAIMS applies dispersion (DV) and compensation voltages (CV) to separate species in the gas phase by different sizes, conformations (i.e., collisional cross sections) and charge states.
Here, we demonstrate the use of FAIMS to determine its effectiveness as a gas phase fractionation method by applying different compensation voltages for top-down proteomics application of proteins from an E. coli lysate. We anticipate that using FAIMS in TDP workflows should result in the identification of unique proteoforms. FAIMS in TDP workflows should aid in characterisation of intact proteins from cell lysates.