RESUMO
The damage to blood coagulation factor XIII (FXIII) at different stages of its enzymatic activation under the action of various physiological amounts of hypochlorite ion was studied. The results obtained by HPLC-MS/MS, SDS-PAGE, and colorimetry showed that, during the conversion of FXIII to FXIIIa, the vulnerability of FXIII to hypochlorite-induced oxidation increased. FXIII oxidized with 150 µM hypochlorite completely retained its enzymatic activity inherent to the intact protein, whereas FXIIIa treated with 50 µM hypochlorite showed sharply reduced enzymatic activity. It was shown that a number of methionine and cysteine residues on the catalytic subunit can perform antioxidant function; additionally, the regulatory subunits of FXIII-B contribute to the antioxidant protection of the catalytic center of the FXIII-A subunit, which, together with the tight packing of the tetrameric structure of the FXIII proenzyme, are the three factors that provide high protein resistance to the oxidizing agent.
Assuntos
Fator XIII/química , Ácido Hipocloroso/farmacologia , Oxidantes/farmacologia , Domínio Catalítico , Ativação Enzimática , Humanos , Oxirredução , Espectrometria de Massas em Tandem/métodosRESUMO
Monitoring of peptides offers a promising approach for the discovery of novel biomarkers, which might be valuable for detection, treatment and prevention of large variety of diseases. Development of highly effective methods for plasma peptide extraction remains an important task. In the current study, we applied different types of plasma peptide extraction approaches to reveal efficient methods which would provide the highest peptide yield. We used different combinations of plasma treatment with acetonitrile and/or urea/guanidine, protein precipitation by acetone, gel-filtration, ultrafiltration, and two types of solid phase extraction. The extracted peptides were analyzed by LC-MS/MS. The obtained results suggest that several methods, including differential solubilization, organic precipitation, as well as some variants of ultrafiltration and solid phase extraction, provide effective plasma peptide enrichment convenient for further LC-MS/MS analysis. Alas, most of the identified peptides were extracted by only one of the applied methods. Hence, it seems reasonable to consider several methods to increase the possibility of novel biomarker discovery.
Assuntos
Peptídeos/sangue , Peptídeos/isolamento & purificação , Espectrometria de Massas em Tandem/métodos , Precipitação Química , Cromatografia Líquida/métodos , Proteoma/análise , Proteoma/isolamento & purificação , Extração em Fase Sólida , UltrafiltraçãoRESUMO
In order to find a peptide panel to differentiate close hypertensive conditions a case-control study was designed for 64 women from 4 groups: preeclampsia (PE), chronic hypertension superimposed with PE, chronic hypertension, and healthy individuals. Chromatography coupled with mass-spectrometry and subsequent bioinformatic analysis showed several patterns in the changes of the urine peptidome. There were 36 peptides common for four groups. Twenty two of them 22 belonged to alpha-1-chain of collagen I, nine peptides were from alpha-1-chain of collagen III, two from alpha-2-chain of collagen I, one from alpha-1/2-chain of collagen I, one from alpha-1-chain of collagen I/XVIII and one from uromodulin. Patients with hypertensive disorders had 34 common peptides: 12 from alpha-1-chain of collagen I, 10 from fibrinogen alpha-chain, eight from alpha-1-chain of collagen III, and 4 per other types of collagen. Comparative analysis revealed 12 peptides, which could be used as a diagnostic panel for confident discrimination of pregnant women with various hypertensive disorders.
Assuntos
Hipertensão Induzida pela Gravidez/urina , Peptídeos/urina , Pré-Eclâmpsia/urina , Estudos de Casos e Controles , Feminino , Humanos , Espectrometria de Massas , Gravidez , UrináliseRESUMO
Complexes of peptide fragment 1-16 of beta-amyloid with transition metals play an important role in the development of a broad class of neurodegenerative diseases, which determines the interest in investigating the structures of these complexes. In this work, we have applied the method of the deuterium/hydrogen exchange in combination with ultra-high-resolution mass spectrometry to study conformational changes in (1-16) beta-amyloid peptide induced by binding of zinc(II) atoms. The efficiency of the deuterium/hydrogen exchange depended on the number of zinc atoms bound to the peptide and on the temperature of the ionization source region. Deuterium/hydrogen exchange reactions have been performed directly in the ionization source. The number of exchanges decreased considerably with an increasing numbers of zinc atoms. The relationship has been described with a damped exponential curve, which indicated that the binding of zinc atoms altered the conformation of the peptide ion by making it less open, which limits the access to inner areas of the molecule.
Assuntos
Peptídeos beta-Amiloides/química , Fragmentos de Peptídeos/química , Acetato de Zinco/química , Medição da Troca de Deutério , Modelos Moleculares , Ligação Proteica , Estrutura Secundária de Proteína , Espectrometria de Massas por Ionização por Electrospray , TemperaturaRESUMO
By using the mass-spectrometry method, the oxidative modifications of the fibrinogen Aα, Bß, and γ polypeptide chains induced by its oxidation have been studied. The αC-region has been proven to be the most vulnerable target for the oxidizer (ozone) as compared with the other structural elements of the Aα chain. The Bß chain mapping shows that the oxidative sites are localized within all the structural elements of the chain in which the ß-nodule exhibits high susceptibility to oxidation. The γ chains are the least vulnerable to the oxidizer action. The results obtained demonstrate convincingly that the self-assembly centers dealing with interactions of knob "A": hole "a" are not involved in oxidative modification. It is concluded that the numerous oxidative sites revealed are mainly responsible for inhibiting lateral aggregation of protofibrils. The part of amino acid residues subjected to oxidation is supposed to carry out the antioxidant function.
Assuntos
Fibrinogênio/química , Fragmentos de Peptídeos/metabolismo , Fibrinogênio/metabolismo , Hidrólise , OxirreduçãoRESUMO
Preeclampsia (PE) is a pregnancy complication characterized by high blood pressure and proteinuria. The disorder usually occurs after the 20th week of pregnancy and gets worse over time. PE increases the risk of poor outcomes for both the mother and the baby. In the study we applied LC-MS/MS method for the analysis of the urine peptidome of women with PE. Samples were prepared using size-exclusion chromatography method which gives more than twice peptides identities if compared with solid phase extraction. Thirty urine samples from women with mild and severe preeclampsia and the control group were analyzed. In total 1786 peptides were identified using complementary search engines (Mascot, MaxQuant and PEAKS). A high level of agreement in peptide identification was observed with previously published data. Label-free data comparison resulted in 35 peptides which reliably distinguished a particular PE group (severe or mild) from controls. Our results revealed unique identifications (correlate to alpha-1-antitrypsin, collagen alpha-1(I) chain, collagen alpha-1 (III) chain, and uromodulin, for instance) that can potentially serve as early indicators of PE.