Anna Smolira

Role of the support material on laser desorption/ ionization mass spectra

We report the results of experimental studies on the effects of sample supports in laser desorption/ionization mass spectrometry (LDI-MS). LDI time-of-flight (TOF) mass spectra obtained for C(60) and insulin samples deposited onto standard stainless steel substrate and/or onto some non-metallic materials (glass, scotch tape, floppy disc foil, Teflon foil, photocopy film), all recorded under identical, typical experimental conditions, have been compared with regard to their intensity and quality. The LDI investigations show that compared with stainless steel, glass and floppy disc foil sample supports boost (2-3.5 times) ion yields for C(60)(+) and C(60)(-) ions, respectively. The stainless steel and scotch tape sample supports are the best for the mass resolution of positive ions and the formation of (C(60))(n)(-) (n <or= 4) cluster ions, respectively. In the case of detection of insulin by matrix-assisted laser desorption/ionization (MALDI) we did not observe significant differences in sensitivity for the support materials tested. A mechanism of ion formation in the desorption plume is suggested.

Response of antimicrobial peptides from porcine neutrophils to pentoxifylline and antigens from Gram negative and Gram positive bacteria

Neutrophils, the main component of the defense against invading organisms have also been implicated in tissue damage in numerous inflammatory conditions. Neutrophil products can degrade the extracellular matrix and when excessively released are thought to cause some disorders. As it is known, pentoxifylline (PTX) can suppress a range of neutrophil responses. Cathelicidins are components of the early host defenses against infection, however, in most cases cleavage with elastase is necessary to obtain active forms. Thus, the aim of our study was to assess the usage of PTX as a factor which could inhibit some neutrophil functions, and to assess if PTX can lead to the impairment of the release from these cells active cathelicidins. For these purposes we determined neutrophil activity as well as expression of cathelicidins from porcine neutrophils in cultures under the influence of PTX. PTX exerted an inhibitory effect on elastase and MPO release from neutrophils. At lower concentrations of PTX, ALP release was inhibited both in cultures stimulated with PTX+fMLP and with PTX+LPS. Inhibition of superoxide generation was insignificant, whereas a decrease of NO production was noted. The MALDI TOF analysis revealed that in all cultures stimulated with PTX+fLMP and PTX+LPS there was no inhibition of the release of cathelicidins in comparison with cultures stimulated only with fMLP and only with LPS. Our study proved that although PTX in porcine neutrophils is able to suppress many neutrophil functions, the expression of cathelicidins is maintained.

Direct matrix-assisted laser desorption ionization time-of-flight mass spectrometric analysis of lysozyme contained in hen egg white

As a natural antibacterial peptide, lysozyme (LZ) is widely used in medicine and the food industry. Despite many years of research on this compound, its new antibacterial properties are still to be determined. The primary aim of this work is to demonstrate the application of the matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometric analysis of LZ directly in hen egg white samples without extraction thereof. The egg white samples were kept over 10 weeks at room temperature and measured every week. The resulting positive and negative ion mass spectra were then compared to determine the intensity of the LZ mass peak. Storage of the egg white for over 10 weeks did not influence the LZ mass peak intensity (both positive and negative). It can be concluded that the LZ concentration in the egg white samples did not vary with time. The effect of the matrix/sample ratio on LZ detection was also examined, and it was found to be different in the case of positive and negative ionization. The mass peaks of LZ oligomeric forms were observed in all mass spectra, so the MALDI method could be used in subsequent studies.

Quantification of the PR‐39 cathelicidin compound in porcine blood by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

RATIONALE The PR-39 porcine cathelicidin occurs naturally in animal neutrophils. Its main function is antimicrobial activity, which potentially can be used in antibiotic treatments in veterinary medicine. Investigations concerning such a use require the detection and quantification of PR-39 in a given sample. The aim of this work is to determine the concentration of PR-39 contained in porcine blood. METHODS Prior to matrix-assisted laser desorption/ionization (MALDI) analysis, the porcine blood sample was subjected to crude extraction in order to release the active form of PR-39 from the neutrophil granules. Next, gel filtration chromatography was performed to separate PR-39 from other cathelicidins present in porcine blood. Positive ion MALDI time-of-flight (TOF) mass spectra of the resulting portion of lyophilisate with unknown PR-39 content were acquired in linear mode. To quantify PR-39 in the lyophilisate sample, the standard addition method was applied. The PR-39 concentration obtained in the lyophilisate sample was then converted into the peptide concentration in porcine blood. RESULTS The linear fit function of the constructed calibration curve indicates an excellent correlation between the PR-39 peak intensity and the added quantity of synthetic PR-39 (R(2) = 0.994) and a low relative standard deviation of the slope = 1.98%. From the x-intercept of the straight line, we estimated the PR-39 concentration in porcine blood to be 20.5 ± 4.6 ng/mL. CONCLUSIONS The MALDI method was successfully applied for the quantitative analysis of PR-39 found in porcine blood. Compared with other available methods, it is relatively easy, inexpensive and not time-consuming. Despite the method having lower accuracy than the enzyme-linked immunosorbent assay (ELISA), the results obtained here, by a much simpler method, are in good agreement with the literature data.