JOURNAL OF MASS SPECTROMETRY J. Mass Spectrom. 2007; 42: 117–126 Published online 8 December 2006 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/jms.1151 Do collisions inside the collision cell play a relevant role in CID-LIFT experiments? † Gloriano Moneti, 1 Simona Francese, 1 Guido Mastrobuoni, 1 Giuseppe Pieraccini, 1 Roberta Seraglia, 2 Giovanni Valitutti 2 and Pietro Traldi 2* 1 CISM, viale G.Pieraccini, 6 50139 Firenze, Italy 2 ISTM-CNR, C.so Stati Uniti, 4 35127 Padova, Italy Received 3 July 2006; Accepted 30 October 2006 Collision experiments are a valid approach to characterize the ionic species generated by matrix assisted laser desorption ionization (MALDI). When a time-of-flight analyzer is employed, three different approaches are available for these experiments: the postsource decay (PSD), the LIFT and the MALDI- TOF/TOF. The last two are of particular interest because of the overcoming of the PSD problems related to mass calibration of the product ion spectra. Experiments performed by LIFT on linear or cyclic peptides, in presence or in absence of collision gas in the collision cell, gave evidence of an unexpected behavior: the two spectra were practically superimposable, and in the former case only a few new fragmentation channels were activated with low yield. These results mean that the selected ion exhibits a large amount of internal energy, capable of promoting fragmentation processes in the time window corresponding to the flight time between ion source and the acceleration electrode placed after the collision cell. Experiments performed by varying the plume density show that this internal energy uptake occurs in the expanding plume, through multiple collisions. The LIFT data have been compared with those achieved by collisions of ESI-generated [MH] + ions of angotensin II performed under ‘in-source’ conditions and by triple-quadrupole experiments. The obtained results show a strong similarity among the spectra, indicating that the internal energy uptake in a MALDI source is comparable with that of 40-eV ions colliding with Ar in a triple-quadrupole instrument. Copyright  2006 John Wiley & Sons, Ltd. KEYWORDS: LIFT; MALDI/MS; collisions; TOF/TOF INTRODUCTION Nowadays matrix assisted laser desorption ionization (MALDI) mass spectrometry is one of the most powerful techniques for researches in the biomedical field, in partic- ular, in proteomics. The preferred formation of protonated molecules allows immediate fingerprinting of complex pro- tein mixtures. On the other hand, the analysis of tryptic digests and the use of the data so obtained for database research lead, in the most of cases, to effective and unequiv- ocal protein identification. To achieve more specific and, consequently, more structurally diagnostic data, techniques based on MALDI followed by collisional activation have been developed. It was in 1992 that Spengler et al. 1 showed that in MALDI a large fraction of the desorbed analyte ions undergo delayed fragmentation reactions (occurring during the flight) and that the m/z values of related decomposition ions can be determined by a reflectron time-of-flight (RETOF) analyzer. This technique was called postsource decay (PSD) and, until L Correspondence to: Pietro Traldi, ISTM-CNR, C.so Stati Uniti, 4, I-35100 Padova, Italy. E-mail: pietro.traldi@adr.pd.cnr.it † Paper presented at the 24th Informal Meeting on Mass Spectrometry, Ustro ˜ n, Poland, 14–18 May 2006. few years ago, it was the only way to achieve structural information on MALDI-generated ionic species of interest. It consists in ion selection by a suitable gating potential, which allows the selection and injection in the flight tube only the ion of interest; for the mass analysis of its fragment ions the reflectron voltages have to be reduced stepwise. Typically ten spectral segments are recorded sequentially with enough overlap to lead to the complete product ion spectrum. The main problem related to this approach lies in mass calibration of product ion spectrum. To avoid tedious and time-consuming procedures by model decomposition reactions (for which precursor and product ions are well known), computer software have been developed, based on the geometrical parameters and the electrical fields employed, as well as on the flight times of precursor and product ions. 2 More recently, to improve the power of the MALDI- MS/MS approach, other two approaches have been pro- posed. The first is based on the reduction of the acceleration voltage of the MALDI source, so to achieve an ion beam with a kinetic energy of the order of 8–10 keV. The selection of the ion of interest is performed by an electrostatic gating, by which ions with m/z values lower and higher than that of interest (i.e. with higher and lower speeds) are ejected from Copyright  2006 John Wiley & Sons, Ltd.