Abstract. The analytical sensitivity in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is largely affected by the specific analyte-matrix interaction, in particular by the possible incorporation of the analytes into crystalline MALDI matrices. Here we used time-of-flight secondary ion mass spectrometry (ToF-SIMS) to visualize the incorporation of three peptides with different hydrophobicities, bradykinin, Substance P, and vasopressin, into two classic MALDI matrices, 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (HCCA). For depth profiling, an Ar cluster ion beam was used to gradually sputter through the matrix crystals without causing significant degradation of matrix or biomolecules. A pulsed Bi 3 ion cluster beam was used to image the lateral analyte distribution in the center of the sputter crater. Using this dual beam technique, the 3D distribution of the analytes and spatial segregation effects within the matrix crystals were imaged with sub-μm resolution. The technique could in the future enable matrix-enhanced (ME)-ToF-SIMS imaging of peptides in tissue slices at ultra-high resolution.
Time-of-flight secondary ion mass spectrometry (ToF-SIMS) was used to characterize the freeze-fracturing process of human epithelial PANC-1 and UROtsa cells. For this purpose, phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, and phosphatidylserine standard samples were investigated to find specific signals with both high specificity and signal intensity. The results were used to investigate single cells of subconfluent cell layers prepared with a special silicon wafer sandwich preparation technique. This freeze-fracturing technique strips cell membranes off the cells, isolating them on opposing silicon wafer substrates. Criteria were found for defining regions with stripped off cell membranes and, on the opposing wafer, complementary regions with the remaining cells. Measured ethanolamine/choline and serine/choline ratios in these regions clearly showed that in the freeze-fracturing process, the lipid bilayer of the plasma membrane is split along its central zone. Accordingly, only the outer lipid monolayer is stripped off the cell, while the inner lipid monolayer remains attached to the cell on the opposing wafer, thus allowing detailed analysis of a single lipid monolayer. Furthermore, it could be shown that using different washing procedures did not influence the transmembrane lipid distribution. Under optimized preparation conditions, it became feasible to detect lipids with a lateral resolution of approximately 100 nm. The data indicate that ToF-SIMS would be a very useful technique to study with very high lateral resolution changes in lipid composition caused, for example, by lipid storage diseases or pharmaceuticals that interfere with the lipid metabolism.
Time of flight secondary ion mass spectrometry (ToF-SIMS) is a powerful technique for the nanoanalysis of biological samples, but improvements in sensitivity are needed in order to detect large biomolecules, such as peptides, on the individual cell level at physiological concentrations. Two promising options to improve the sensitivity of SIMS to large peptides are the use of cluster primary ions to increase desorption of intact molecules or the use of matrix-assisted laser desorption/ionization (MALDI) matrices to increase the ionization probability. In this paper, the authors have combined these two approaches in order to improve understanding of the interaction between ionization and fragmentation processes. The peptides bradykinin and melittin were prepared as neat monolayers on silicon, in a Dextran-40 matrix and in two common MALDI matrices, 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxy cinnamic acid (HCCA). ToF-SIMS spectra of these samples were collected using a range of small Bi cluster primary ions and large Ar cluster primary ions. The trends observed in the molecular ion yield and the [M+H](+)/C4H8N(+) ratio with primary ion cluster size were sample system dependent. The molecular ion yield of the bradykinin was maximized by using 30 keV Bi3 (+) primary ions in a DHB matrix but in the HCCA matrix, the maximum molecular ion yield was obtained by using 30 keV Bi7 (+) primary ions. In contrast, the molecular ion yield for melittin in both matrices was greatest using 20 keV Ar2000 (+) primary ions. Improvements in the molecular ion yield were only loosely correlated with a decrease in small fragment ions. The data indicate a complex interplay between desorption processes and ion formation processes which mean that the optimal analytical conditions depend on both the target analyte and the matrix.
Silver nanoparticles (SNPs) are among the most commercialized nanoparticles because of their antibacterial effects. Besides being employed, e.g. as a coating material for sterile surfaces in household articles and appliances, the particles are also used in a broad range of medical applications. Their antibacterial properties make SNPs especially useful for wound disinfection or as a coating material for prostheses and surgical instruments. Because of their optical characteristics, the particles are of increasing interest in biodetection as well. Despite the widespread use of SNPs, there is little knowledge of their toxicity. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) and laser post-ionization secondary neutral mass spectrometry (Laser-SNMS) were used to investigate the effects of SNPs on human macrophages derived from THP-1 cells in vitro. For this purpose, macrophages were exposed to SNPs. The SNP concentration ranges were chosen with regard to functional impairments of the macrophages. To optimize the analysis of the macrophages, a special silicon wafer sandwich preparation technique was employed; ToF-SIMS was employed to characterize fragments originating from macrophage cell membranes. With the use of this optimized sample preparation method, the SNP-exposed macrophages were analyzed with ToF-SIMS and with Laser-SNMS. With Laser-SNMS, the three-dimensional distribution of SNPs in cells could be readily detected with very high efficiency, sensitivity, and submicron lateral resolution. We found an accumulation of SNPs directly beneath the cell membrane in a nanoparticular state as well as agglomerations of SNPs inside the cells.
Heterogeneous polymer coatings, such as those used in organic electronics and medical devices, are of increasing industrial importance. In order to advance the development of these types of systems, analytical techniques are required which are able to determine the elemental and molecular spatial distributions, on a nanometer scale, with very high detection efficiency and sensitivity. The goal of this study was to investigate the suitability of laser postionization secondary neutral mass spectrometry (Laser-SNMS) with a 157 nm postionization laser beam to image structured polymer mixtures and compare the results with time-of-flight secondary ion mass spectrometry (ToF-SIMS) measurements using Bi primary ions. The results showed that Laser-SNMS is better suited than ToF-SIMS for unambiguous detection and submicrometer imaging of the wide range of polymers investigated. The data also showed that Laser-SNMS has the advantage of being much more sensitive (in general higher by more than an order of magnitude and peaking at up to 3 orders of magnitude) than ToF-SIMS while also showing superior performance on topographically complex structured insulating surfaces, due to significantly reduced field effects and a higher dynamic range as compared to ToF-SIMS. It is concluded that Laser-SNMS is a powerful complementary technique to ToF-SIMS for the analysis of heterogeneous polymers and other complex structured organic mixtures, providing submicrometer resolution and high sensitivity.
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