Design and performance of an atmospheric pressure ion mobility Fourier transform ion cyclotron resonance mass spectrometer Xiaoting Tang, James E. Bruce and Herbert H. Hill, Jr. * Department of Chemistry, Washington State University, Pullman, WA 99164, USA Received 12 September 2006; Revised 11 January 2007; Accepted 14 January 2007 This manuscript presents our initial results and development of a novel hybrid instrument that combines atmospheric pressure ion mobility spectrometry (AP-IMS) with Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). Our preliminary results obtained from atmos- pheric pressure mobility separation of peptide mixtures combined with high-resolution FTICR mass analysis are demonstrated. The custom IMS system was constructed in-house and was coupled to the commercial FTICR-MS instrument through a flared inlet capillary interface. Dual-gate ion filtration was adapted to allow concurrent measurement of both mobility and m/z values. The feasibility of mobility separation was demonstrated with baseline separation of the peptides bradykinin and angiotensin II and their measured reduced mobility constants which were consistent with those previously reported. Furthermore, the unique size-to-charge separation mechanism of IMS that allows isomer separation was explored and demonstrated with the partial separation of two isomeric phosphopeptides. We feel the combination of IMS and FTICR-MS holds great potential for accurate mass analysis of mobility-selected ions and these results are the first to demonstrate the feasibility of coupling these two techniques. Copyright # 2007 John Wiley & Sons, Ltd. Since its inception in 1970, 1,2 ion mobility spectrometry (IMS) has been combined with a variety of other analytical technologies and employed for detection of a wide range of analytes including both small organic compounds 3–6 and large biological molecules. 7–13 IMS separates ions in the gas phase on the basis of their differential mobility under a uniform weak electric field. It is of great interest in many analytical fields due to the high speed with which this separation technique can be achieved. In its early develop- ments, IMS was termed ‘‘plasma chromatography’’ 1,2 because of its gas-phase separation properties that are analogous to conventional separation techniques such as gas chromatography (GC) and liquid chromatography (LC). In fact, IMS shares similarities to both chromatography and mass spectrometry (MS) but is based on principles different from both, thus it can be coupled to either GC 14 and LC 15 as a detector or MS as a supplementary separation device. While standalone IMS with a Faraday plate detector offers rapid and sensitive detection in real-time with low-cost and field-deployable benefits, coupling IMS with MS has recently attracted increased interest due to additional qualitative and specific fragmentation information that can be gained with MS and MS/MS. One important feature that distinguishes IMS from MS is that IMS separates ions based on their size-to-charge ratio (V/z, where V is cross section) whereas MS measures ions based on their mass-to-charge ratio (m/z). IMS is not only a function of size but also shape and ion-neutral potential interactions. This V/z-based separation mechanism of IMS enables separation of isomers since they can have different conformation in the gas phase even though they have the same m/z. Thus combining IMS with MS produces more comprehensive information than is possible with either technology alone. In addition, as a separation technique, IMS is more advantageous for analysis of biological samples owing to the orders of magnitude faster separation (on ms scale) than typical condensed-phase chromatography such as LC, capillary electrophoresis, or gel electrophoresis. IMS can be operated at either atmospheric pressure or low vacuum pressure (1–10 Torr) and both types of IMS have been coupled with various types of mass spectrometers, 16 such as quadrupole MS, 3,17–21 time-of-flight (TOF)-MS, 7,8,22,23 and ion trap MS. 12,24–26 In addition, low-pressure IMS has been coupled to Fourier transform ion cyclotron resonance (FTICR)-MS. 27 To synchronize the acquisition cycle of IMS and MS, most two-dimensional IMS-MS measurements are achieved by either acquiring IMS spectra for a single m/z value or scanning the m/z window for a selected drift time RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom. 2007; 21: 1115–1122 Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/rcm.2928 *Correspondence to: H. H. Hill, Jr., Department of Chemistry, Washington State University, Pullman, WA 99164, USA. E-mail: hhhill@wsu.edu Contract/grant sponsor: NIH; contract/grant number: R21 DK070274. Contract/grant sponsor: National Science Foundation; contract/ grant number: DBI-0352451. Contract/grant sponsor: US Department of Energy, the Office of Science (BER); contract/grant number: DE-FG02-04ER63924. Contract/grant sponsor: Murdock Charitable Trust. Copyright # 2007 John Wiley & Sons, Ltd.