Protein desolvation in UV matrix-assisted laser desorption/ionization (MALDI)

Abstract

We describe experiments in MALDI-TOF and MALDI-TOF-TOF showing that the ejection of protein-matrix cluster ions and their partial decay in the source occur in MALDI. The use of radial beam deflection and small size detector in linear mode allows detection of ions with higher time-of-flight and kinetic energy deficit. MALDI-TOF-TOF experiments were carried out by selecting chemical noise ions at m/z higher than that of a free peptide ion. Whatever the selected m/z (up to m/z 300) the molecular peptide ion appeared as the main fragment. The production of protein-matrix clusters and their partial decay in the source was found to increase with the size of the protein (MW from 1000 to 150,000 u), although it decreases with increasing charge state. These effects were observed for different matrices (HCCA and SA) and in a large laser fluence range. Experimental results and calculation highlight that a continuous decay of protein-matrix cluster ions occurs in the source. This decay-desolvation process can account for the high-mass tailing and peak shifting as well as the strong noise/background in the mass spectra of proteins. . This new ionization method allows the production of large intact molecular ions (protein, peptide, DNA, synthetic polymer, etc.) in the gas phase. Intact ions are then analyzed by a mass analyzer, essentially time-of-flights (TOFs).Although MALDI-TOF is now a well-established and powerful method, many questions are still open concerning (1) the conditions of a successful experiment and (2) the main mechanisms involved in the formation of free intact ions [2].Target preparation is a critical step in MALDI. Analyte is often supposed to be co-crystallized with a large excess of matrix. This incorporation of analyte into the small matrix crystals (co-crystallization) remains an open question [3,4]. However, a protein in the target is likely surrounded by matrix molecules.The ejection of material in the very first stage leads to a very dense plume (with a density similar to that of the target) becoming increasingly more diluted (gas) during the plume expansion.The main question is how a free ion is formed from such a dense plume and why analyte-matrix clusters are not the main products. Ejection of matrix-analyte clusters was already found as essential for the analyte ionization in MALDI [5][6][7][8]. Desorption of large clusters was also predicted from molecular dynamics simulations [9,10]. Other authors believe that ionization can be explained by an ion-molecule reaction in the plume [11]. Recent very interesting simulations, including desorption/ablation molecular dynamics and ionization processes modeling, showed that free ions and those in clusters are produced with t t close abundance in the plume [12].The ion analysis through axial-TOF instruments is probably the key point of the main observation of free ions in MALDI. A free ion formed in the source before the delay time is fully accelerated and is detected at a given time forming a resolved peak. The peak width is a function of the initial a...

Molecular dynamics simulations of MALDI: laser fluence and pulse width dependence of plume characteristics and consequences for matrix and analyte ionization

“…This is consistent with experiments that show significant portion of the ablated material may be in the form of aggregates rather than single molecules 15, 16. While any clusters or aggregates that can be mass analyzed or otherwise collected do not contribute directly to the desired signal of free, single ions, there have been suggestions that they play an important role in ion formation or release 3, 17–22…”

“…Experimental evidence for entrainment, late ion release and analyte embedding in clusters has been available for some time,56, 57, 74 and cluster‐based models followed soon after 3, 17. Massive clusters of analyte ions with matrix have been suggested, up to 50 000 Da and more,18 and various pathways for their decay into observable analyte ions suggested 9, 19–22…”

Electrochemical Aspects of Electrospray and Laser Desorption/Ionization for Mass Spectrometry

“…ISD was first observed and applied to the structure analysis of peptides and proteins in 1995 (75). Typically, it occurs less than 100 ns after the desorption/ionization step in the ion source (82,83) and results in the cleavage of the N-C α bond of the peptide backbone, producing c-and z-fragment types ( Figure 5) (83). The matrix employed is a key factor influencing the decay efficiency.…”

Ghost peaks observed after atmospheric pressure matrix-assisted laser desorption/ionization experiments may disclose new ionization mechanism of matrix-assisted hypersonic velocity impact ionization

“…An alternative and more reasonable model considers the escape of protonated analyte ions from hot molecular clusters [9],[10],[11] or nanoparticles which are produced in abundance during laser ablation. [12] In this cluster paradigm, [13],[14],[15],[16] the MALDI process starts with laser-assisted ablation and is completed with the release of protonated analytes from matrix clusters; the last stage is believed to be governed by processes similar to those responsible for ion desolvation in ESI. [17] A major problem in MALDI models is to explain the charge separation that accompanies the ablation.…”