No abstract

Age-related macular degeneration (AMD) is the primary cause of central blindness among the elderly. AMD is associated with progressive accumulation of lipofuscin granules in retinal pigment epithelium (RPE) cells. Lipofuscin contains bisretinoid fluorophores, which are photosensitizers and are phototoxic to RPE and neuroretinal cells. In the presence of oxygen, bisretinoids are also oxidized, forming various products, consisting primarily of aldehydes and ketones, which are also potentially cytotoxic. In a prior study, we identified that in AMD, bisretinoid oxidation products are increased in RPE lipofuscin granules. The purpose of the present study was to determine if these products were toxic to cellular structures. The physicochemical characteristics of bisretinoid oxidation products in lipofuscin, which were obtained from healthy donor eyes, were studied. Raman spectroscopy and time-of-flight secondary ion mass spectrometry (ToF–SIMS) analysis identified the presence of free-state aldehydes and ketones within the lipofuscin granules. Together, fluorescence spectroscopy, high-performance liquid chromatography, and mass spectrometry revealed that bisretinoid oxidation products have both hydrophilic and amphiphilic properties, allowing their diffusion through lipofuscin granule membrane into the RPE cell cytoplasm. These products contain cytotoxic carbonyls, which can modify cellular proteins and lipids. Therefore, bisretinoid oxidation products are a likely aggravating factor in the pathogenesis of AMD.

Ultraviolet matrix-assisted laser desorption/ionization (UV-MALDI) detection of ions with charge states as high as those usually seen with electrospray ionization (ESI), was recently demonstrated by Gologan, Cooks and co-workers in soft-landing experiments. 1 In theses experiments, multiply charged ions of proteins generated by ESI were landed in high vacuum (~10 -8 torr) onto self-assembled monolayer (SAM) surfaces. To analyze the deposited protein material, laser desorption mass spectrometry (LD-MS) was used. By comparing the mass spectra from various samples, it was found that from some SAMs, LD-MS shows multiply charged ions, with the charge states being the same as in the original electrospray used for deposition. The interpretation was that multiply charged ions generated by ESI survive during soft-landing and laser desorption if an optimized SAM surface is used. It was noted that such a sample preparation can be used for the enhancement of multiply charged ions yielded in MALDI and can be useful, for example, for applying mass spectrometers with limited m/z range.From the experiments cited above, it appears that the type and structure of the surface used for soft landing greatly infl uences the yield of multiply charged ions. Based on this, we decided to investigate whether organic compounds used as MALDI matrices could also be used for ESI deposition of multiply charged ions. This is often referred to as the electrospray sample preparation method for MALDI. [2][3][4] Simultaneously, we wanted to investigate whether electrospray deposition at atmospheric pressure would also give a significant yield of multiply charged ions in MALDI.The effi ciency of desorption/ionization in MALDI and, in particular, the formation of multiply charged ions strongly depends on the type of matrix and laser irradiance. It has been shown that using matrices such as α-cyano-4-hydroxycinamic acid (HCCA) in UV-MALDI and caffeic acid in infrared (IR)-MALDI and laser irradiance near the threshold the yield of multiply charged ions can be increased. 5-7 The sample preparation method is also known to shift the charge state distribution in MALDI, 8 although it is generally significantly lower compared with ESI. Electrospray deposition of bovine-insulin onto an α-cyano-4-hydroxycinamic acid (HCCA) matrix layer was investigated in detail. It was found that, using electrospray preparation, certain HCCA matrix layers and low laser irradiance showing a signifi cant shift of the insulin ion distribution to higher charge states (lower mass-to-charge values) can be observed.The experiments were all performed on a commercial MALDI time-of-flight mass spectrometer (Axima CRF, Kratos/Shimadzu, Manchester, UK) equipped with a nitrogen laser (337 nm emission wavelength, 10 ns pulse duration, 100 µm × 100 µm laser spot size). The laser irradiance was varied in the range of 3-5 MW cm -2 (near the MALDI threshold) using a computer-controlled attenuator. The actual values were determined from a calibration curve relating to the attenuator setting and i...