Analysis of tryptic peptides using desorption electrospray ionisation combined with ion mobility spectrometry/mass spectrometry Gushinder Kaur-Atwal 1y , Daniel J. Weston 1y , Philip S. Green 2 , Susan Crosland 2 , Philip L. R. Bonner 1 and Colin S. Creaser 1 * 1 School of Biomedical and Natural Sciences, Interdisciplinary Biomedical Research Centre, Nottingham Trent University, Nottingham NG11 8NS, UK 2 Syngenta, Jealott’s Hill International Research Centre, Bracknell RG42 6EY, UK Received 1 December 2006; Revised 19 January 2007; Accepted 19 January 2007 A novel method is reported for rapid protein identification by the analysis of tryptic peptides using desorption electrospray ionisation (DESI) coupled with hyphenated ion mobility spectrometry and quadrupole time-of-flight mass spectrometry (IMS/Q-ToF-MS). Confident protein identification is demonstrated for the analysis of tryptically digested bovine serum albumin (BSA), with no sample pre-treatment or clean-up. Electrophoretic ion mobility separation of ions generated by DESI allowed examination of charge-state and mobility distributions for tryptic peptide mixtures. Selective interrogation of singly charged ions allowed isobaric peptide responses to be distinguished, along with a reduction in spectral noise. The mobility-selected singly charged peptide responses were presented as a pseudo-peptide mass fingerprint ( p-PMF) for protein database searching. Comparative data are shown for electrospray ionisation (ESI) of the BSA digest, without sample clean-up, from which confident protein identification could not be made. Implications for the robustness of the DESI method, together with potential insights into mechanisms for DESI of proteolytic digests, are discussed. Copyright # 2007 John Wiley & Sons, Ltd. Desorption electrospray ionisation (DESI) was introduced by Cooks and co-workers 1 and has been used successfully for the rapid mass spectrometric (MS) analysis of condensed- phase analytes under ambient conditions, without the need for a matrix or sample pre-treatment. DESI relies upon the production of charged droplets from a pneumatically assisted electrospray, which is directed onto analyte molecules in the condensed phase. The impact of the charged droplets on the analyte surface produces, as a result of pneumatic and electrostatic forces, secondary gas-phase ions, 2 which are extracted into the vacuum of the mass spectrometer for analysis. A number of ion formation mechanisms by DESI have been postulated, 3 including chemical sputtering, in which secondary gas-phase ions are generated by the impact of charged solvent droplets and subsequent proton or ion charge transfer, the droplet pick-up mechanism, in which sample molecules become dissolved or adhere to the impacting charged solvent droplets resulting in the formation of secondary gas-phase sample ions, and the volatilisation of neutral species from the analyte surface and subsequent ionisation by gas-phase charge transfer. 1,4 Because DESI is performed under ambient conditions, often requiring little modification of existing mass spectro- meter hardware, the number and diversity of DESI applications have grown rapidly. The technique shows promise for the analysis of chemical warfare agents 5 and the detection of explosives (TNT, RDX) 6,7 in forensic and public safety applications, with analytes desorbed and detected from a number of everyday surfaces such as paper, plastic, metal and leather. Perhaps the most extensive application of DESI has been the rapid analysis of pharmaceutical preparations, such as active drugs from pharmaceutical tablets, patches and creams, 8–11 at levels as low as 0.14% (w/w), and the screening and characterisation of illicit Ecstasy tablets, 12 with high sensitivity and high-throughput capability. The advent of in vivo sampling by DESI of living tissue surfaces, including the detection of pharmaceutical products or metabolites directly from skin, saliva and body fluids (urine or blood), 1,4 provides a non-invasive alternative for high-throughput bioanalysis or screening with little, if any, sample pre-treatment. The application of DESI to biological imaging, to identify potential biomarkers for disease in tissue, 13 and to detect alkaloids in plant tissues, 14 has demonstrated the possibility of obtaining spatially resolved data by DESI. RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom. 2007; 21: 1131–1138 Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/rcm.2941 *Correspondence to: C. S. Creaser, School of Biomedical and Natural Sciences, Interdisciplinary Biomedical Research Centre, Nottingham Trent University, Nottingham NG11 8NS, UK. E-mail: colin.creaser@ntu.ac.uk y Equal experimental contribution was made by these authors. Contract/grant sponsor: Biotechnology and Biological Sciences Research Council (BBSRC) and Syngenta. Copyright # 2007 John Wiley & Sons, Ltd.