Protein Analysis by Mass Spectrometry

Opisana je analiza bioloških spojeva spektrometrijom masa (MS) uz ionizaciju elektroraspršenjem (ESI) i matricom pomognutu ionizaciju uz desorpciju laserskim zračenjem (MALDI). Objašnjena je karakterizacija proteinskih posttranslacijskih modifikacija i identifikacija proteina na temelju određivanja molekulske mase peptida (engl. peptide mass fingerprint, PMF) te identifikacija na temelju fragmentiranja peptida (kolizijom inducirana fragmentacija i poslijeionizacijska fragmentacija). Također je dan pregled metoda za kemijsku modifikaciju peptida, čime se omogućuje kvantifikacija i de novo sekvenciranje proteina.Soft ionization techniques, electrospray (ESI) and matrix-assisted laser desorption/ionization (MALDI) make the analysis of biomolecules by mass spectrometry (MS) possible. MS is used for determination of the molecular weight of peptides and protein, sequence analysis, characterization of protein-ligand interactions etc. The detection limit, resolution and mass accuracy depend on instrument used (Table 1). Impurities (buffers, salts, detergents) can reduce the ion intensities or even totally suppress them, so a separation method (chromatography, 2D-gel electrophoresis) must be used for purification of the sample. Molecular mass of intact protein can be determined by ESI or MALDI MS. Multiply charged ions are produced by ESI MS, while singly charged ions are predominant in MALDI spectra (Fig. 2). Sequence analysis of proteins by MS can be performed using peptide mass fingerprint. In this method, proteins are separated by 2-D gel electrophoresis and digested with specific protease (Table 2) or digested and then separated by two-dimensional chromatography (Fig. 1). The obtained peptide mixtures are analyzed by MS or MALDI-TOF technique. The masses determined by MS are compared with calculated masses from database entries. Different algorithms have been developed for protein identification. Example of posttranslational modifications (N- and O-glycosylation) and protein sequence complex analysis after dual digestion (endoproteinase digestion followed by endoglycosidase digestion) is shown in Fig. 3. It is known that detection of peptides by MS is influenced by intrinsic properties like amino acid composition, the basicity of the C-terminal amino acid, hydrophobicity, etc. Arginine-containing peptides dominate in MS spectra of tryptic digest, so the chemical derivatization of lysine terminal residue by O-methilisourea or 2-methoxy-4,5-1H-imidazole was suggested (Fig. 4). The peptide mass fingerprint method can be improved further by peptide fragmentation using tandem mass spectrometry (collision-induced dissociation, CID or post-source decay, PSD). Different types of fragments and main fragmentation paths are shown in Figs. 6 and 8. Fragmentation pathway of a doubly charged tryptic pentapeptide to b- and y-ions by collision-induced dissociation inside the mass spectrometer is described more in details in Fig. 7. All types of fragment ions are summarized in table 3. Since the any of the peptide bonds can be broken in several ways, the MS/MS spectra are complex, and quite difficult to interpret. Chemical derivatization is used to obtain only or predominantly one type of fragment ions. Sulfonation of N-terminal amino group enhance PSD sequencing, producing mainly y-type fragment ions. The mass difference of two consecutive y-ions corresponds to an amino acid mass, so the peptide sequence can be obtained with minimal or no assistance from genomic data, e. g. de novo protein sequencing is possible. Fig. 9 represents the strategy for the protein identification by mass spectrometry. Various chemical modifications on the peptide fragmentation patterns are shown in Fig. 10

Nonleptonic Ω−\Omega^{-} decays and the Skyrme model

Nonleptonic $\Omega^{-}$ decay branching ratios are estimated by means of the QCD enhanced effective weak Hamiltonian supplemented by the SU(3) Skyrme model used to estimate the nonperturbative matrix elements. The model has only one free parameter, namely the Skyrme charge $e$, which is fixed through the experimental values of the octet-decuplet mass splitting $\Delta$ and the axial coupling constant $g_{A}$. The whole scheme is equivalent to that which works well for the nonleptonic hyperon decays. The ratios of calculated amplitudes are in agreement with experiment. However, the absolute values are about twice too large if short-distance corrections and only ground intermediate states are included.Comment: 4 pages, 1 figure, 1 table, version to appear in Phys.Rev.

Bioethanol Production from Dilute-acid Pre-treated Wheat Straw Liquor Hydrolysate by Genetically Engineered Saccharomyces cerevisiae

Sustainable recycling of lignocellulosic biomass includes utilization of all carbohydrates present in its hydrolysates. Since wheat straw is a xylose-rich raw material, utilization of xylose from obtained liquid part (liquor) of hydrolysates improves overall bioprocess efficiency. In this work, dilute acid pre-treatment of wheat straw was performed in high-pressure reactor at different temperatures (160 °C – 200 °C), residence times (1 min – 10 min), and acids (H2SO4 and H3PO4) concentrations. During dilute acid pre-treatment, hemicellulose is degraded to pentose sugars that cannot be used by industrial ethanol-producing yeasts. Therefore, genetically engineered Saccharomyces cerevisiae strain that can utilize xylose was used. Fermentations were performed on different xylose-rich liquor wheat straw hydrolysates in shake-flasks and in horizontal rotating tubular bioreactor. The efficiency of fermentations carried out in shake flasks using xylose-rich liquor wheat straw hydrolysates were in the range of 19.61 – 74.51 %. However, the maximum bioprocess efficiency (88.24 %) was observed during fermentation in the HRTB on the liquor wheat straw hydrolysate obtained by pre-treatment with 2 % w/w phosphoric acid

Community-acquired pneumonia

Introduction: Community-acquired pneumonia (CAP) today, as well as a few decades ago, is a current medical problem considering the incidence and the mortality rate of the population, despite the availability of new and powerful antimicrobials and vaccines effectiveness. Objective: Analysis of outpatients diagnosed with pneumoniae, determination of the most common risk factors for their development, analysis of the success of outpatients' treatments and complications. Methods: Medical exams of 38 patients were analyzed. Each case is chosen by following previously prepared protocol, including patients with respiratory symptoms and infectious syndrome, positive auscultatory findings on the lungs which are radiologicaly confirmed and laboratory treated (SE, Le, FBC, the first and the tenth day of the therapy). Demographic data and associated illnesses, as well as a severity assessment of the illness, were made at the first medical examination, when pneumonia was suspected. Results: In the period from 01.11.2014 to 01.05.2015, there were 33 diagnosed pneumoniae. Associated illnesses, in population older than 65 years, were present in 92.85% of patients and some of them had two or three comorbidities. CRB65 proved itself as a good parameter in assessment of the disease severity for both groups. Applied antibiotic therapy proved to be effective in 80% of patients. There is no significant difference in pneumonia presentation with regards to gender and age. In data proccessing, descriptive statistics methods and no parameter x2 test were used for statistical significance assessment. Conclusion: All patients with clear indications should be hospitalized, but large percentage of patients can be treated in outpatints' facilities, with good patient cooperation. Also vaccination, as an available resource, seems to have not received a significant place in our environment

Utjecaj hijaluronske kiseline, kalcijeva hidroksida i dentinskih adheziva na odontoblaste i fibroblaste štakora

The aim of this study was to investigate the effects and efficiency of pulp capping preparations based on hyaluronic acid, calcium hydroxide, and dentin adhesive on the pulp tissue of Sprague-Dawley rats. The rats were killed and extracted teeth sectioned transversely through the pulp. The slices were placed in a RPMI 1640 cell culture medium supplemented with 10 % foetal calf serum. During 14 days of cultivation cultures were treated with preparations that contained hyaluronic acid (Gengigel Prof®), and calcium hydroxide (ApexCal®), or with dentin adhesive (Excite®). Cellularity and viability of fibroblasts and odontoblasts was analysed using a haemocytometer. Hyaluronic acid proved most efficient and the least toxic for direct pulp capping. Even though calcium hydroxide and dentin adhesive demonstrated a higher degree of cytotoxicity, their effects were still acceptable in terms of biocompatibility.Cilj ovog rada bio je istražiti djelovanje preparata na bazi hijaluronske kiseline i kalcijeva hidroksida te dentinskog adheziva na pulpno tkivo Sprague-Dawley štakora u svrhu procjene učinkovitosti navedenih materijala kod direktnog prekrivanja pulpe. Izvađeni zubi transverzalno su podijeljeni kroz pulpu. Naresci su uzgajani u RPMI 1640 staničnom mediju obogaćenom s 10 % fetalnoga telećeg seruma u plastičnim bočicama za staničnu kulturu. Kulture su tijekom 14 dana tretirane preparatima s hijaluronskom kiselinom (Gengigel Prof®), kalcijevim hidroksidom (ApexCal®) i dentinskim adhezivom (Excite®). Nakon 14 dana pristupilo se analizi staničnosti i vijabilnosti s pomoću hemocitometra. Iako su preparati na bazi kalcijeva hidroksida i dentinski adheziv pokazali nešto viši stupanj citotoksičnosti, dobiveni su rezultati u granicama biokompatibilnosti. Primjena preparata na bazi hijaluronske kiseline postigla je najbolje rezultate te se ovaj materijal pokazao najboljim za direktno prekrivanje pulpe između tri ispitivana preparata