Proteins play a key role in cell function regulation. Understanding how a protein modulates cell activity may give new insight into disease mechanisms and elucidate biomarkers. A protein’s structure can affect its biological activity; therefore, recent research has focused on determining a protein’s structure, especially where modifications occur, such as phosphorylation. Identifying alterations in a protein’s phosphorylated state can be used as therapeutic and preventative biomarkers. Mass spectrometry (MS) has been a growing technique for identifying and sequencing proteins. Proteins are digested into their respective peptides for analysis by MS. In order to identify and sequence numerous proteins, data-dependent analysis is often employed. The computer selects a predefined number of the highest abundance peaks for further analysis by tandem MS, regardless of their relevance. Often, analytes of interest are in low abundance in a complicated biological matrix, making them difficult to analyze. Sample clean-up methods have been utilized, including immobilized metal affinity chromatography (IMAC) and high performance liquid chromatography (HPLC), but they are time-consuming. Here is presented high-field asymmetric-waveform ion mobility spectrometry (FAIMS) for the rapid separation of tryptic peptide ions.

FAIMS separates ions in milliseconds based on their difference in mobility at high and low fields. The FAIMS device can be used as a stand-alone device or can be coupled to a mass spectrometer in between the ionization source and the mass spectrometer entrance. Here FAIMS is explored as a separation technique for tryptic peptides ions, particularly phosphopeptides. Variation in FAIMS parameters, such as gas composition and flow rate, dispersion voltages, electrode temperature and geometries are explored for their effect on tryptic peptide ion separation. The addition of FAIMS to a data-dependent HPLC-MS analysis is addressed.