Resonance excitation and dynamic collision-induced dissociation in quadrupole ion traps using higher-order excitation frequencies U ¨ nige A. Laskay and Glen P. Jackson * Center for Intelligent Chemical Instrumentation, Department of Chemistry and Biochemistry, Ohio University, Athens, OH 45701-2979, USA Received 15 January 2008; Revised 25 April 2008; Accepted 27 May 2008 Fragmentation of the pentapeptide leucine enkephalin (YGGFL) is accomplished via higher-order resonances combined with simultaneous analysis of low-mass product ions. Two methods of achieving excitation are explored: (1) 0.5 ms resonant excitation at the v and at V-v secular frequencies of ion motion (where V is the radio-frequency (rf) drive frequency) in a manner similar to both pulsed q collision-induced dissociation (PQD) and high amplitude short time excitation (HASTE), and (2) 0.5 ms pulse of the v or at V-v excitation frequencies when the secular frequency of the ions is quickly swept across resonance conditions (pulsed q dynamic CID, PqDCID). In both methods of excitation, the rf amplitude on the ring electrode is rapidly decreased after excitation, therefore enabling analysis of low- mass product ions. Maximum fragmentation efficiencies of 20% can be obtained with pulsed CID with both regular and high-order frequency excitation, while pulsed DCID offers maximum efficien- cies of 12%. All the excitation methods studied offer increased internal energy depositions when compared to conventional CID, as measured by the a 4 /b 4 product ion ratios of leucine enkephalin. These ratios were as high as 13:1 for pulsed CID and 8:1 for PqDCID. Successful mass analysis of the low-mass ions is observed with both pulsed CID and PqDCID. The combined benefit of high internal energy deposition and wider dynamic mass range offers the possibility of increased sequence coverage and the identification of unique internal fragments or high-energy product ions which may provide complementary information to biological applications of conventional CID. This is the first report on deliberate fragmentation of precursor ions at a higher-order component of the ion secular frequency combined with a successful mass analysis of the low-mass ions through pulsed CID and PqDCID. Copyright # 2008 John Wiley & Sons, Ltd. The employment of the quadrupole ion trap (QIT) as a platform for tandem mass spectrometry (MS/MS) arises from the great flexibility and sensitivity of this instrument in analysis of volatile organic compounds. Through the combination of ion traps with atmospheric ionization sources such as electrospray ionization (ESI), fragmentation of biological molecules can be tackled in both the top-down 1 and bottom-up 2 approaches. Increasing the size of precursor ions of interest, such as proteins, lipids or polysaccharides, increases the number of degrees of freedom such that probing the fragmentation of such large species is proble- matic. Due to the low internal energy depositions in the high collision-frequency environment of ion traps, tandem mass spectra in ion traps tend to lack fragments arising from pathways with high activation barriers. While this is often not a problem, per se, fundamental chemistry and appli- cations such as de novo peptide sequencing could benefit from the ability to probe higher energy pathways. Increasing the collection efficiency and collision energy is a problem well known to researchers developing QITs, and one can find extensive efforts towards optimization of all stages of the mass analysis. For example, an improved injection and more efficient isolation may increase the number of ions successfully mass-analyzed; 3 investigating different scanning parameters such as scanning rate, 4,5 scan direction 6,7 or ejection q z leads to a better sensitivity and, to a certain degree, mass resolution may be enhanced. 8 As for the aspect of fragmentation, increasing the number and energy of collisions utilizing different bath gases with variable pressures have been successfully accomplished. 9,10 A more selective fragmentation of precursor ions can be achieved by application of customized waveforms to achieve on- resonance conditions for the ions of interest. 11,12 Ions trapped in the quadrupolar field of the ion trap have relatively stable trajectories and modest kinetic energies. Perturbation of the motion of the ions using supplementary alternating current (ac) waveforms leads to a modification of their kinetic energies and may lead to their fragmentation via collisions with the bath gas, or to ion ejection, if their excursion path exceeds the physical dimensions of the trap or if their trajectories become unstable. Mathematically, the RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom. 2008; 22: 2342–2348 Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/rcm.3618 *Correspondence to: G. P. Jackson, Department of Chemistry and Biochemistry, Ohio University, 136 Clippinger Laboratories, Athens, OH 45701-2979, USA. E-mail: jacksong@ohio.edu Contract/grant sponsor: NSF; contract/grant number: 064757. Copyright # 2008 John Wiley & Sons, Ltd.