Zbigniew Szewczuk

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.

Iron(III) and aluminum(III) complexes with hydroxypyrone ligands aimed to design kojic acid derivatives with new perspectives

With the aim to design new chelators for the clinical treatment of different diseases involving the trivalent metal ions Fe(III) and Al(III), we present the equilibria of kojic acid and its derivative 6-[5-hydroxy-2-hydroxymethyl-pyran-4-one]-5-hydroxy-2-hydroxymethyl-pyran-4-one with these two metal ions. Potentiometric and spectrophotometric techniques for iron, and potentiometry and (1)H NMR for aluminum were used, supported by X-ray, electrospray ionization-mass spectrometry (ESI-MS), calorimetry and quantum chemical calculations. In this work, evidence is given on the formation of MeL, MeL(2), and MeL(3) complexes of both metal ions with kojic acid, confirmed by the X-ray structure of the FeL(3) complex, and of variously protonated Me(2)L(2) and MeL(2) complexes of 6-[5-hydroxy-2-hydroxymethyl-pyran-4-one]-5-hydroxy-2-hydroxymethyl-pyran-4-one. The extremely good pFe value for this second ligand gives confidence to, and opens perspectives for, the search of new kojic acid derivatives.

Immunoregulatory properties of synthetic peptides, fragments of a proline-rich polypeptide (PRP) from ovine colostrum

It has been previously found that a proline-rich polypeptide (PRP) isolated from ovine colostrum has a regulatory effect on the immune response. A nonapeptide fragment Val-Glu-Ser-Tyr-Val-Pro-Leu-Phe-Pro was isolated from the chymotryptic digest of PRP. The nonapeptide showed biological activity similar to PRP. The determined amino acid sequence was now confirmed by synthesis. Synthetic nonapeptide as well as its C-terminal hexapeptide, Tyr-Val-Pro-Leu-Phe-Pro, showed biological activity similar to PRP and the nonapeptide obtained from PRP.

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.

Immunological properties of the thymopentin-like fragments of HLA-DQ

Class II human leukocyte antigens (HLA-II) are cell surface alpha beta heterodimers (M(r) approximately 60,000) that play a pivotal role in the immune response by presenting peptides derived from environmental antigens to the T-cell receptor. A 167-171 fragment of the beta 2-chain of the HLA-DQ molecule consists of the sequence RGDVY, 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. We synthesized and investigated the immunomodulatory activity of series of peptide fragments of HLA-DQ containing thymopentin-like sequences. The results indicate that all synthesized peptides suppress the cellular immune response. However, RGDV, RGDVY and QRGDVY show very weak stimulatory activity in humoral immunological response tests. In contrast to the shorter peptides, the nonapeptide fragment of HLA-DQ, TPQRGDVYT, shows significant immunosuppressive activity in all tests. A possible role of these fragments of the polypeptide chain of HLA-DQ in the regulation of HLA functions is discussed.

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.

To the problem of biologically active conformation of enkephalin

SummaryA semi-rigid structural analog of [Leu5] enkephalin, possessing the azo-bridge between Tyr1 and Phe4 residues, was synthesized, along with two other linear enkephalin analogs: [4′-amino Phe4] enkephalin and [4′hydroxyphenyl/-azo Phe4] enkephalin. The results of the determination of the analgesic activity of the synthesized compounds suggest that the biologically active conformation of the enkephalin molecule should be such that both aromatic rings, Tyr1 and Phe4, are situated in close proximity.

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.