Piotr Stefanowicz

Capric Acid Secreted by S. boulardii Inhibits C. albicans Filamentous Growth, Adhesion and Biofilm Formation

Candidiasis are life-threatening systemic fungal diseases, especially of gastro intestinal track, skin and mucous membranes lining various body cavities like the nostrils, the mouth, the lips, the eyelids, the ears or the genital area. Due to increasing resistance of candidiasis to existing drugs, it is very important to look for new strategies helping the treatment of such fungal diseases. One promising strategy is the use of the probiotic microorganisms, which when administered in adequate amounts confer a health benefit. Such a probiotic microorganism is yeast Saccharomyces boulardii, a close relative of baker yeast. Saccharomyces boulardii cells and their extract affect the virulence factors of the important human fungal pathogen C. albicans, its hyphae formation, adhesion and biofilm development. Extract prepared from S. boulardii culture filtrate was fractionated and GC-MS analysis showed that the active fraction contained, apart from 2-phenylethanol, caproic, caprylic and capric acid whose presence was confirmed by ESI-MS analysis. Biological activity was tested on C. albicans using extract and pure identified compounds. Our study demonstrated that this probiotic yeast secretes into the medium active compounds reducing candidal virulence factors. The chief compound inhibiting filamentous C. albicans growth comparably to S. boulardii extract was capric acid, which is thus responsible for inhibition of hyphae formation. It also reduced candidal adhesion and biofilm formation, though three times less than the extract, which thus contains other factors suppressing C. albicans adherence. The expression profile of selected genes associated with C. albicans virulence by real-time PCR showed a reduced expression of HWP1, INO1 and CSH1 genes in C. albicans cells treated with capric acid and S. boulardii extract. Hence capric acid secreted by S. boulardii is responsible for inhibition of C. albicans filamentation and partially also adhesion and biofilm formation.

Electrospray tandem mass spectrometry of poly(3-hydroxybutanoic acid) end groups analysis and fragmentation mechanism

The electrospray ionization tandem mass spectrometry technique applied for the first time to aliphatic polyester analysis, allowed fast and reliable identification of the various macromolecules of poly(3-hydroxybutanoic acid) (PHB), synthesized via ring-opening polymerization of β-butyrolactone. It was demonstrated that the biomimetic polymers studied contain various end groups, depending on the anionic initiator employed, and show close similarity to the natural PHB produced by enzymes in living cells of prokaryotic and eukaryotic organisms.

Electrospray mass spectrometry and tandem mass spectrometry of the natural mixture of cyclic peptides from linseed.

The cyclic peptides from linseed are composed exclusively of the hydrophobic amino acids: Phe, Leu, Ile, Val, Met, Pro and Trp. Because these compounds do not contain functional groups which undergo protonation or deprotonation easily, their ionization in solvents usually used for peptide analysis is not efficient. A rapid and sensitive procedure for the detection and structure elucidation of the cyclic peptides based on ionization with Na+ and NH4+ ions is described. A cationization of methionine-containing peptides with methyl iodide has been described also. The extract of Linum utitatissimum seeds was analyzed directly using electrospray ionization mass spectrometry and neutral loss electrospray ionization tandem mass spectrometry techniques. The analysis confirms the presence of cyclolinopeptides reported previously: CLA [c(Pro–Pro–Phe–Phe–Leu–Ile–Ile–Leu–Val], and CLB [c(Pro–Pro–Phe–Phe–Val–Ile–Met–Ile–Leu)]. Cyclolinopeptides CLC, CLD, CLE and CLG, which contain methionine oxide, were detected in relatively small quantities. These peptides probably result from the oxidation of their precursors which were not reported: CLD′ [c(Pro–Phe–Phe–Trp–Ile–Met–Leu–Leu)], CLE′ [c(Pro–Leu–Phe–Ile–Met–Leu–Val–Phe)], CLF [c(Pro– Phe–Phe–Trp–Val–Met–Leu–Met)] and CLG [c(Pro–Phe–Phe–Trp–Ile–Met–Leu–Met)], present at higher concentrations in the extract protected from atmospheric oxygen. The sequences of the unreported cyclic peptides were proposed on the basis of collision-induced dissociation experiments and homology with peptides described by Morita1,2 and supported by the fragmentation of synthetic analogs of CLA of a known structure.

Detection of glycation sites in proteins by high-resolution mass spectrometry combined with isotopic labeling

The products of nonenzymatic glycation of proteins are formed in a chemical reaction between reducing sugars and the free amino group located either at the N terminus of the polypeptide chain or in the lysine side chain. Glycated proteins and their fragments could be used as markers of the aging process as well as diabetes mellitus and Alzheimers disease, making them an object of interest in clinical chemistry. In this article, we propose a new method for the identification of peptide-derived Amadori products in the mixtures obtained by enzymatic hydrolysis of glycated proteins. Two proteins, ubiquitin and human serum albumin (HSA), were modified with an equimolar mixture of glucose and [(13)C(6)]glucose and were subjected to enzymatic hydrolysis. The obtained enzymatic digests were analyzed by high-resolution mass spectrometry (HRMS), and the peptide-derived Amadori products were identified on the basis of specific isotopic patterns resulting from (13)C substitution. The number of glycated peptides in the digest of HSA detected by our procedure was in agreement with the data recently reported in the literature.

Derivatization of peptides as quaternary ammonium salts for sensitive detection by ESI-MS

A series of model peptides in the form of quaternary ammonium salts at the N‐terminus was efficiently prepared by the solid‐phase synthesis. Tandem mass spectrometric analysis of the peptide quaternary ammonium derivatives was shown to provide sequence confirmation and enhanced detection. We designed the 2‐(1,4‐diazabicyclo[2.2.2] octylammonium)acetyl quaternary ammonium group which does not suffer from neutral losses during MS/MS experiments. The presented quaternization of 1,4‐diazabicyclo[2.2.2]octane (DABCO) by iodoacetylated peptides is relatively easy and compatible with standard solid‐phase peptide synthesis. This methodology offers a novel sensitive approach to analyze peptides and other compounds. Copyright

The immunosuppressive activity and solution structures of ubiquitin fragments

Recently, ubiquitin was suggested as a promising anti-inflammatory protein therapeutic. We found that a peptide fragment corresponding to the ubiquitin(50-59) sequence (LEDGRTLSDY) possessed the immunosuppressive activity comparable with that of ubiquitin. CD and NMR spectroscopies were used to determine the conformational preferences of LEDGRTLSDY in solution. The peptide mixture, obtained by pepsin digestion of ubiquitin, was even more potent than the intact protein. Although the peptide exhibited a well-defined conformation in methanol, its structure was distinct from the corresponding 50-59 fragment in the native ubiquitin molecule. (c) 2009 Wiley Periodicals, Inc. Biopolymers 91: 423-431, 2009.

The Competition of Charge Remote and Charge Directed Fragmentation Mechanisms in Quaternary Ammonium Salt Derivatized Peptides—An Isotopic Exchange Study

Derivatization of peptides as quaternary ammonium salts (QAS) is a promising method for sensitive detection by electrospray ionization tandem mass spectrometry (Cydzik et al. J. Pept. Sci.2011, 17, 445–453). The peptides derivatized by QAS at their N-termini undergo fragmentation according to the two competing mechanisms – charge remote (ChR) and charge directed (ChD). The absence of mobile proton in the quaternary salt ion results in ChR dissociation of a peptide bond. However, Hofmann elimination of quaternary salt creates an ion with one mobile proton leading to the ChD fragmentation. The experiments on the quaternary ammonium salts with deuterated N-alkyl groups or amide NH bonds revealed that QAS derivatized peptides dissociate according to the mixed ChR-ChD mechanism. The isotopic labeling allows differentiation of fragments formed according to ChR and ChD mechanisms.

The immunosuppressory and adhesive miniregion of the human major histocompatibility protein, human leukocyte antigen DQ

Our previous studies showed that the nonapeptide fragment of human leukocyte antigen DQ with the TPQRGDVYT sequence strongly suppresses the immune response1 [Z. Szewczuk, I. Z. Siemion, and Z. Wieczorek (1996) Molecular Immunology, 33, 903–908]. The fragment contains the RGDVY sequence, which is very similar to thymopentin (pentapeptide RKDVY, an active fragment (32–36) of thymopoietin, an immune system activator produced in thymi), and at the same time contains the RGD sequence, known as an inhibitor of adhesion processes. In the present study we tested an influence of the nonapeptide and its shorter fragments on binding of activated platelets and K562 cells to fibrinogen and fibronectin, respectively. We also designed and synthesized a cyclic thymopentin‐like peptide, C*RGDVYC* (where C* indicates Cys participating in disulfide bridge) to restrict its conformation. The cyclization product strongly suppresses the humoral and cellular immune response and selectively inhibits the adhesion of K562 cells to fibronectin. The results are discussed in the light of CD conformational studies.

The hydrogen-deuterium exchange at α-carbon atom in N,N,N-trialkylglycine residue: ESI-MS studies.

Derivatization of peptides as quaternary ammonium salts (QAS) is a known method for sensitive detection by electrospray ionization tandem mass spectrometry. Hydrogens at α-carbon atom in N,N,N-trialkylglycine residue can be easily exchanged by deuterons. The exchange reaction is base-catalyzed and is dramatically slow at lower pH. Introduced deuterons are stable in acidic aqueous solution and are not back-exchanged during LC-MS analysis. Increased ionization efficiency, provided by the fixed positive charge on QAS group, as well as the deuterium labeling, enables the analysis of trace amounts of peptides.

Electron capture dissociation mass spectrometric analysis of lysine-phosphorylated peptides

Phosphorylation of proteins is an essential signalling mechanism in eukaryotic and prokaryotic cells. Although N-phosphorylation of basic amino acid is known for its importance in biological systems, it is still poorly explored in terms of products and mechanisms. In the present study, two MS fragmentation methods, ECD (electron-capture dissociation) and CID (collision-induced dissociation), were tested as tools for analysis of N-phosphorylation of three model peptides, RKRSRAE, RKRARKE and PLSRTLSVAAKK. The peptides were phosphorylated by reaction with monopotassium phosphoramidate. The results were confirmed by 1H NMR and 31P NMR studies. The ECD method was found useful for the localization of phosphorylation sites in unstable lysine-phosphorylated peptides. Its main advantage is a significant reduction of the neutral losses related to the phosphoramidate moiety. Moreover, the results indicate that the ECD–MS may be useful for analysis of regioselectivity of the N-phosphorylation reaction. Stabilities of the obtained lysine-phosphorylated peptides under various conditions were also tested.