This paper reports detailed studies on the internal energy of ions formed in matrix-assisted laser desorption/ionization (MALDI) using delayed extraction MALDI-time-of-flight (TOF) and atmospheric pressure (AP) MALDI mass spectrometric (MS) methods. We use benzylpyridinium cations as internal energy probes. Our study reveals three distinct contributions to internal energy build-up in vacuum-MALDI (classical MALDI-TOF), each having different effects on ion fragmentation. Some fragments are formed before ion extraction (i.e. no more than 100 ns after the laser impact), and they are therefore well resolved and recorded as sharp signals in the MALDI-TOFMS scan. This prompt fragmentation can have two origins: (i) in-plume thermal activation, presumably always present, and (ii) in-plume chemical activation, in the course of reactions with hydrogen radicals. In addition to early internal energy build-up associated with these well-resolved promptly formed fragments, a broad peak slightly offset to higher masses could be detected corresponding to fragments formed after the extraction has started. This second signal corresponds to a third source of internal energy in MALDI ions, (iii) the extraction-induced collisional activation of the ions with the neutral components of the plume. These three contributions are difficult to quantify in vacuum-MALDI, because of the combined influence of several parameters (nature of the matrix, spot-to-spot variability, total laser exposure, delay time, acceleration voltage) on extraction-induced fragmentation. AP-MALDI, on the other hand, has two advantages for comparative studies of analyte fragmentation. First, extraction-induced fragmentation is absent, and only the contributions of early plume activation remain. Second, the reproducibility is far better than in vacuum-MALDI. AP-MALDI is therefore expected to shed new light on the early steps of the MALDI process.

In this paper, we report the measurement of the degree of analyte fragmentation in AP-MALDI as a function of the matrix and of the laser fluence. The analytes include p-OCH 3 -benzylpyridinium, three peptides containing the sequence EEPP (which cleave very efficiently at the E-P site), and three deoxynucleosides (dA, dG, and dC), which lose the neutral sugar to give the protonated base. We found that the matrix hardness/softness was consistent when comparing the analytes, with a consensus ranking from hardest to softest: CHCA Ͼ Ͼ DHB Ͼ SA Ϸ THAP Ͼ ATT Ͼ HPA. However, the exact ranking can be fluence-dependent, for example between ATT and HPA. Our goal here was to provide the scientific community with a detailed dataset that can be used to compare with theoretical predictions. We tried to correlate the consensus ranking with different matrix properties: sublimation or decomposition temperature (determined using thermogravimetry), analyte initial velocity, and matrix proton affinity. The best correlation was found with the matrix proton affinity. T he most exciting innovations in the last 15 years for the analysis of nonvolatile, high molecular weight compounds like peptides, proteins, oligonucleotides, carbohydrates, and synthetic polymers came from the development of two new ionization techniques for mass spectrometry: matrix-assisted laser desorption/ionization (MALDI) [1,2] and electrospray ionization (ESI) [3]. MALDI has established itself as a powerful analytical technique. However, despite its wide range of applications and many fundamental studies (for review articles, see [4 -7]), there is still a need for better understanding of the MALDI process, to improve ion yields, and to provide rational guidelines for matrix selection. The currently most widely used matrices were found empirically. An important step in the research for new (tailor-made) matrices is an improved knowledge of the role of the matrix in MALDI, and the establishment of criteria to predict matrix properties. The role of the matrix is 3-fold. (1) The matrix has to absorb the laser energy (via electronic excitation in case of UV-MALDI or vibrational excitation in case of IR-MALDI). (2) Disintegration of the condensed phase has to take place without excessive destructive heating of the embedded analyte molecules. (3) An efficient ionization of the analyte molecules has to be provided.It is well known experimentally that MALDI matrices can cause more or less analyte fragmentation, and matrices are therefore characterized as "hard" or "soft", respectively. While ionization with minimal internal energy deposition ("softness") is required for molecular mass determination, extensive fragmentation ("hardness") is advantageous for structural studies, and particularly for peptide sequencing. In addition to application issues, the study of the matrix influence on analyte fragmentation can also bring new elements to the picture of the MALDI mechanism. Several groups have therefore put some effort into the experimental characterization of ion internal...