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Prestructured MALDI-MS sample supports have been developed that simplify high-throughput analysis of biomolecules and improve the detection sensitivity. The mass spectrometric sample support is coated with a thin layer of hydrophobic Teflon that carries an array of 200-microm gold spots, which provide hydrophilic sample anchors. Each transferred sample droplet contacts one anchor, on top of which, after solvent evaporation, the sample is exclusively deposited due to the strongly water repellent nature of the Teflon surface. The initial matrix concentration is kept low, enabling sample up-concentration by more than 2 orders of magnitudes before crystallization commences. As a result, the detection sensitivity is improved as documented by mass spectra recorded from 100 amol of various peptides, 1 fmol of a DNA 20 mer, and 5 fmol of a 130 bp PCR product. Size and spacing of the hydrophilic anchors are optimized for MALDI-MS performance (sample spot size approximately = laser irradiation spot size), for short analysis times (predetermined sample coordinates), and for high throughput sample preparation (sample anchor array according to the 1536 microtiter plate format).

Each cell of a living organism contains the whole genetic information in form of DNA molecules. The size of the DNA from a single cell, or genome, of human beings is 3 × 109 nucleotide base pairs. Although the DNA information is usually identical in each cell there are several hundred different cell types. This is due to the fact that genetic information is read out from genes and transcribed from DNA into a cell-specific population of mRNA molecules, which itself can be further translated into different types of proteins. Every step of these cellular processes includes complex interactions of DNA, RNA and protein (Alberts, Bray et al., 1994). To understand these interaction mechanisms scientists started to decode the genetic information (Dulbecco, 1986). This task became finally a major goal of the Human Genome Project (Cantor, 1990).

We developed a mass spectrometric method to precisely characterize the structures of the diethyl pyrocarbonate (DEP)-modified amino acid derivatives in intact peptides and proteins. Using acetate-buffered solutions for modification reactions improved the yields of DEP modification. UV quantification of carbethoxylation of angiotensin II was consistent with the degree of mass spectrometrically determined modification. Unequivocal identification of the modification sites in carbethoxylated angiotensin II derivatives was achieved by HPLC separation and mass spectrometric sequencing. With increasing concentrations of DEP, a gradual increase of carbethoxy groups, comprising biscarbethoxylation products, was detected in angiotensin II and in insulin. When using a high molar excess of DEP, histidine carbethoxylation was found together with modifications at alpha-amino groups and tyrosine residues. The sites of carbethoxylation in insulin were identified by MALDI-MS-peptide mapping analyses of the tryptic digestion mixtures from the nonreduced insulin derivatives and after reduction of disulfide bonds, demonstrating that histidine carbethoxylation was sufficiently stable during disulfide bond reduction and tryptic digestion at pH 7.5. The mass spectrometric identification of mono- and biscarbethoxylated histidine residues in insulin is in agreement with surface accessibilities of imidazolyl nitrogen atoms and seems to reflect the microenvironment of the protein tertiary structure. Thus, mass spectrometric peptide mapping analyses of carbethoxylated protein derivatives allowed both the simultaneous identification of histidine carbethoxylation in the presence of other modified groups and the detection of different chemical behavior of histidine residues by the unambiguous identification of mono- and bismodifications.

Immunosuppressed patients are highly susceptible to invasive fungal infections (IFI) such as invasive pulmonary aspergillosis, which is predominantly caused by the fungus Aspergillus fumigatus. An important component of the fungal cell wall is chitin, a polymer of N-acetyl-D-glucosamine (GlcNAc). Chitin is not produced by humans, however, the chitin degrading enzymes (chitinases) chitotriosidase (Chit-1) and acidic mammalian chitinase (AMCase) are. Chitinase is predominantly produced by activated macrophages, and may possibly aid in the defense against chitin-containing pathogens. We show that serum and bronchoalveolar lavage (BAL) chitinase levels are increased in patients with IFI, and in mice after pulmonary exposure to A. fumigatus conidia. Several different stimuli, including stimulation with chitin can lead to chitinase responses. In vitro stimulation of U937 human monocytes and RAW mouse macrophages with either chitin-particles (7-12 µm) or GlcNAc increased secreted and intracellular chitinase activity, the accumulation of intracellular Chit-1, and significantly increased the mRNA levels of Chit-1 and AMCase. Accordingly, we hypothesize that Chit-1 expression is regulated through a positive feedback mechanism involving the degradation of chitin to GlcNAc by host chitinases and GlcNAc recognition that in turn upregulates chitinase expression. This potential feedback mechanism of chitinase regulation may have utility in the diagnosis of IFIs.

We observed fragmentation of an essential proliferation-related human nuclear protein prothymosin alpha in the course of apoptosis induced by various stimuli. Prothymosin alpha cleavage occurred at the DDVD(99) motif. In vitro, prothymosin alpha could be cleaved at D(99) by caspase-3 and -7. Caspase hydrolysis disrupted the nuclear localization signal of prothymosin alpha and abrogated the ability of the truncated protein to accumulate inside the nucleus. Prothymosin alpha fragmentation may therefore be proposed to disable intranuclear proliferation-related function of prothymosin alpha in two ways: by cleaving off a short peptide containing important determinants, and by preventing active nuclear uptake of the truncated protein.

Immunocompromised mice were infected intranasally with Aspergillus fumigatus as part of a vaccine efficacy study. Although body temperature was measured throughout the study, a formal evaluation of its usefulness as an endpoint criterion was not performed. We retrospectively evaluated survival data and temperature records to determine whether body temperature can be used as an objective predictor of death and included in the humane endpoint criteria for this mouse model. CF1 mice were immunosuppressed with either cortisone acetate or by treatment with antiGR1 (a neutrophil-depleting antibody) and then intranasally challenged with A. fumigatus. Body temperature was measured by using an infrared noncontact thermometer a maximum of 3 times daily until death or euthanasia. A surface body temperature below 29.0 °C was correlated with a poor chance of survival, and using this cutoff point with signs of morbidity (hunched, ruffled fur, respiratory distress) reliably indicates mice for euthanasia without negatively affecting data collection. Using 2 subsequent readings of less than 31.0 °C as an endpoint would have led to premature euthanasia of only one mouse (2.2%). As a single reading, a body temperature of 28.8 °C had a sensitivity of 92.2% and specificity of 90.9%. Hypothermia proved to be a useful addition to the humane endpoint criteria for this mouse model, and veterinary and research groups should discuss their study needs in relation to animal welfare to best determine the most appropriate means of including this parameter.