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. Ï© ) 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 ...