The Matrix Suppression Effect and Ionization Mechanisms in Matrix-assisted Laser Desorption/Ionization

Abstract

At appropriate matrix:analyte mixing ratios, small to moderate sized analyte ions (lOOO-20OOOu) can fully suppress positively charged matrix ions in matrix-assisted laser desorptiodionization (MALDI) mass spectra. This is true for all matrix species, including radical cations and adducts with protons or alkali-metal ions. Full matrix suppression is also observed, regardless of the preferred d y t e ion form, be it protonated or an alkali adduct. These facts lead us to propose a mechanism for prompt, primary (not secondary gas-phase) MALDI ionization in which excited matrix molecules are the key species. At least two such excited molecules are believed to be necessary for free ion generation. This model is found to be consistent with the available data, as well as making several predictions which are confinned by new observations. The model also predicts that the matrix suppression effect will not be observable with heavy analytes because their large excluded volume precludes desorption at the neceSSary mixing ratios.Although it is not widely discussed in the literature, practitioners of matrix-assisted laser desorptionlionization (MALDI) have often noted a remarkable phenomenon: when analytes of moderate size (1000-20 OOO u) are mixed with matrix in relatively high molar ratio (1O:l to about 2OOO:l matrix-to-analyte ratio), the appearance of positive matrix ions in the mass spectrum can be completely suppressed.'.' Along with the fact that analyte fragmentation is typically weak in MALDI, this leads to nearly ideal mass spectra: strong analyte molecular ions but no other signals of any kind. Since the effect has been observed with a number of matrices'.' including nicotinic acid, a-cyanoChydroxycinnamic acid (CCA) and 2,5 dihydroxybenzoic acid (DHB), it seems to be a general phenomenon in MALDI. It would clearly be advantageous to understand the effect, so as to extend it to the widest possible range of analytes. At the same time we can also expect to learn something about the molecular-level mechanisms of the MALDI method.In their paper describing CCA as a MALDI matrix, Beavis et aL3 presented a spectrum of substance P (1 347 u) in which the matrix peaks were nearly absent. They did not specifically note this partial matrix suppression effect, nor did they study the matrix-to-analyte mixing ratio dependence of the matrix signals, but they did propose a disproportionation ionization mechanism involving nonionized matrix as an intermediate. They also noted the possibility of proton generation from the matrix as a first step, followed by efficient transfer to the analyte, but did not go on to verifj these hypotheses.Matrix-ion suppression was explicitly studied by Chan er aL,' using nicotinic acid as the matrix, and insulin (5807 u), lysozyme (-14 300 u) and myoglobin (-18 OOO u) as the analytes. They investigated matrix-to-analyte ratios of about 600 to 6OO0, and found that larger analytes required larger ratios for good suppression. They tentatively proposed a suppression model based on the competition between ma...

Sodium Cation Affinities of Commonly Used MALDI Matrices Determined by Guided Ion Beam Tandem Mass Spectrometry

“…Cationization by alkali metal cations such as Na + and K + is one of the secondary ionization processes that occur in MALDI [25][26][27][28][29][30][31][32][33]. Cationization is also believed to influence protonation, deprotonation, and electron transfer ionization processes that occur in MALDI.…”

Investigating the Quantitative Nature of MALDI-TOF MS

“…Another phenomenon hindering the use of MALDI for quantitative measurements is the suppression effect, which has also been discussed in a number of publications (19,29,30,32,33,44). Suppression can especially distort the data when complex biological samples are analyzed that contain thousands of components in a wide concentration range.…”

“…For example, preparations with ␣-cyano-4-hydroxycinnamic acid (CHCA) 1 deliver relatively homogenous samples (27) (which is why CHCA was used as the matrix in our experiments and in most other quantitative studies). On the other hand, dramatically nonlinear relationships of relative signal intensities and concentrations have been described despite the preparation of homogenous samples (32,33). The most common attempt to overcome the problem of poor reproducibility is the use of internal standards (5, 6, 8, 10, 11, 19 -22, 34).…”

Trapping of wide range mass-to-charge ions and dependence on matrix amount in internal source MALDI-FTMS

“…PEG ions resulting from these distributions should be expected to have the same ionization efficiency and would logically be detected in similar abundance. However, in practice it is difficult to obtain such a result due to the influence of various factors such as matrix concentration, [19 -21] sample morphology, [22] crystallite size, [23] buffer type, [24,25] laser energy, [26] delayed extraction, [27] cation choice, [28,29], and laser fluence [30 -32]. Figure 1 contains MALDI-FTMS spectra obtained for an equimolar mixture of PEG 2K, 4K, 6K, and 8K as a function of relative amount of DHB matrix added.…”