Gas -phase separations of synthetic and biopolymers with high -field asymmetric waveform ion mobility spectrometry and Fourier -transform ion cyclotron resonance mass spectrometry

Electrospray ionization (ESI) has enabled proteins and other high molecular weight compounds to be ionized and introduced into the gas-phase. ESI is readily combined with mass spectrometry and this combination is used to determine the molecular weight of biomolecules with unprecedented accuracy. Fourier-transform ion cyclotron resonance mass spectrometry (FT/ICR MS) has a high resolution (108). Using FT/ICR MS, it is possible to identify tens of thousands of unique compounds in a complex mixture, such as crude oil. However, adding a separation method prior to mass spectrometry can enhance analytical capabilities to better unravel complex systems. For example, liquid chromatography has been combined with mass spectrometry in the analysis of complex protein mixtures.;Two methods for separating ions in the gas phase prior to mass spectrometry are drift tube ion mobility spectrometry (IMS) and high-field asymmetric waveform ion mobility spectrometry (FAIMS). These rapid separations have great potential for increasing sample throughput, a critical need in proteomics and other applications. Drift tube ion mobility separates ions based on collisional cross section. FAIMS separates ions based on the difference between the high and low field ion mobility for an ion. Advantages of FAIMS over drift tube IMS are that FAIMS has much higher sensitivity than drift tube IMS and is readily incorporated with a variety of mass spectrometers. This facilitates rapid multidimensional separation of ions in the gas phase. FAIMS has been used to separate tryptic digest ions, improve detection in environmental chemistry, and resolve atomic isotopes and other isomeric ions.;Here, the first results from the combination of FAIMS with FT/ICR MS are reported. FAIMS is used to selectively introduce different gas-phase conformers of proteins into the mass spectrometer. Once introduced into the FT/ICR mass spectrometer, the FAIMS separated conformers can be characterized with additional methods, such as H/D exchange and electron capture dissociation (ECD). By using previously measured collisional cross sections of ions separated by FAIMS, it is shown that H/D exchange and collisional cross section are orthogonal separations of the gas-phase ion populations. ECD of protein ions results in extensive backbone cleavages and is used to characterize proteins in the gas-phase. ECD was originally thought to depend solely on the ion charge state. By using FAIMS FT/ICR ECD MS, it is clearly demonstrated for the first time that the gas-phase ion conformation can have greater impact on electron capture efficiency than charge alone.