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A nonmetallic sample support for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry enhances the positive ion yield by 2 orders of magnitude and generally affects the charge balance in the desorption plume. We interpret the effects of the target material and of the sample preparation on MALDI mass spectra as a result of photoelectrons emitted upon laser irradiation of a metal target covered by a thin sample layer. These electrons are shown to play an important role in MALDI and laser desorption/ionization because they decrease the yield of positive ions, reduce ions with higher oxidation states, and affect the ion velocity distribution as well as the mass resolution. Understanding the role of these photoelectrons helps to clarify previously obscure aspects of the ion formation mechanism in MALDI.

The mechanisms of the reduction of Cu(II) in matrix-assisted laser desorption/ionization mass spectrometry (MALDI) are studied. In MALDI mass spectra, ions cationized by copper mostly contain Cu(I) even if Cu(II) salts are added to the sample. It was found that Cu(II) was reduced to Cu(I) by gas-phase charge exchange with matrix molecules, which is a thermodynamically favorable process. Under some conditions, large amounts of free electrons are present in the plume. Cu(II) can be even more efficiently reduced to Cu(I) by free electron capture in the gas phase. The matrices studied in this work are nicotinic acid, dithranol, and 2,5-dihydroxybenzoic acid. ϩ ) were observed in the analysis of proteins [11,17,19,20] and polar synthetic polymers, e.g., polyethylene glycols (PEG) [12], whereas in the analysis of apolar synthetic polymers, e.g., polystyrene (PS), the reduction of divalent metal ions was dominant [13][14][15][16]18].In this work, we present a detailed study of the reduction of divalent copper used as a cationization agent in MALDI. Different explanations of this reduction in the gas phase have been given, namely electron capture [21] and charge exchange with matrix [22]. The origin and the amount of free electrons in MALDI has not been comprehensively investigated before and many questions were still open [22]. Recently, studies of the origin of the free electrons in MALDI from our laboratory [23,24] have shown that free electrons are largely formed by photoelectric emission from a metal/ dielectric-substance interface, not by photoionization of matrix [21]. Our studies also showed that the yield of electrons strongly depends on the thickness of the matrix layer. In the case of nonmetallic surfaces, few electrons are produced.According to these results, we are able to investigate the roles of free electrons and matrices as possible reducing agents in MALDI separately. Blocking the electron source by using a nonmetallic sample carrier or a metallic target covered with an insulating layer provides the possibility to examine the effect of the gasphase charge transfer between divalent metal ions and the matrices. For investigation of the effect of the free electron capture, experiments have to be done in the absence of matrix by using direct laser desorption/ ionization (LDI) of the sample.Under continuous extraction conditions, for example on a time-of-flight (TOF) mass spectrometer, free electrons as the most mobile species can either be extracted very quickly (in the negative ion mode) or pushed back towards the target (in the positive ion mode) and thus have less overlap with the plume, i.e., electrons will be less available for capture by positively charged species. On the other hand, delayed extraction allows time for secondary ion-molecule reactions to occur, e.g., the charge transfer reaction between neutral matrix molecules and copper ions. In order to investigate the influence of the high extraction field on the free electron capture and the charge exchange reaction, we have compared the results ...

Noncovalently bound complexes between basic sites of peptides/proteins and sulfonates are studied using Matrix Assisted Laser Desorption/Ionization (MALDI) Mass Spectrometry. Reactive sulfonate dyes such as Cibacron Blue F3G-A are known to bind to protonated amino groups on the exterior of a protein. In this work, we examine a wide range of other sulfonates with distinctly simpler structure and more predictable reactivity. Naphthalene-sulfonic acid derivatives were found to bind to arginine only, as opposed to expected binding to all basic sites (Arg, Lys and His). Detailed control experiments were designed to unambigously confirm this selectivity and to rule out nonspecific adduct formation in the gas phase. The data show that the number of complex adducts found equals the number of accessible arginine sites on the surface of folded peptides and proteins, plus the N-terminus. Lys and His are not complexed nor are buried residues with hindered access. MALDI-MS can therefore provide fast information related to the exposed surface of these biomolecules. Additional titration experiments with 1-anilino-naphthalene-8-sulfonic acid (ANS) revealed that this fluorescent dye, which was often hypothesized to bind to so-called molten globule states of proteins, behaved exactly like all other naphthalene-sulfonic acids. ANS binding thus occurs largely through the sulfonate group.

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