Spomenka Kovač

Polymer-dimer distribution in the electrochemical synthesis of polyaniline

The distribution of major products associated with the process of polyaniline (PANI) synthesis is discussed. On the basis of the results obtained in the cyclovoltammetric (cv) and constant potential (cp) synthesis the dependence of that distribution upon monomer concentration, switching potential (Eλ) and potential of synthesis, Esynth, are discussed, the corresponding reaction scheme is given and terms for the reaction rates derived. The conditions governing the polymer-dimer distribution are formulated. The origin of the middle peak, appearing during the PANI synthesis, is discussed in the light of the specific experimental conditions, and species corresponding to it are identified. A reaction scheme for the hydrolysis pathway of dimer is confirmed.

Application of proteomics in biotechnology--microbial proteomics.

The genomes of most economically important microbial cells are already sequenced and proteomic technologies can be applied during various process development steps, starting with the selection and optimization of the functions of the industrial strains, application of the knowledge of cell function in response to the changes of production parameters, validation of the downstream processing, and thorough characterization of the final product. Unfortunately, there are only a few direct examples in the literature that present the optimization of the production process based on proteomics. In this review, we discuss the potential of this technology for the design of future bioprocesses and for optimization of existing ones.

Proteomic analysis for process development and control of therapeutic protein separation from human plasma

The use of proteomics technology during the development of a new process for plasma protein separation was demonstrated. In a two‐step process, the two most abundant proteins, HSA and IgG, were removed in a first step of anion‐exchange chromatography using a gel with very high capacity. Subsequently, two fractions containing medium and low abundance proteins were re‐chromatographed on a smaller column with the same type of gel. Collected fractions were separated by SDS‐PAGE and 2‐D electrophoresis, and excised proteins were digested with trypsin and identified by LC‐ESI‐MS/MS. This proteomic analysis proved to be a useful method for detection of low abundance therapeutic proteins and potential harmful contaminants during process development. Based on this method, low abundance therapeutic proteins, such as vitamin‐K‐dependent clotting factors and inhibitors, could be identified as present in target fractions after chromatographic separation. In addition, the tracking of potentially dangerous impurities and designing proper steps for their removal are important outcomes when developing, refining or controlling a new fractionation schema. For the purpose of in‐process control, in‐solution digestion of complete fractions followed by protein identification with LC‐ESI‐MS/MS was demonstrated as a rapid and simple alternative to the entire analysis including 1‐D or 2‐D electrophoretic steps.

Proteomics as a tool for optimization of human plasma protein separation

The application of proteomics technology in purification of proteins from human plasma and for characterization of plasma-derived therapeutics has been recently discussed. However, until now, the impact of this technology on the plasma protein fractionation and analysis of the final product has not been realized. In the present work, we demonstrate the use of proteomic techniques the monitoring of the first step of the plasma fractionation by use of anion-exchange chromatography. This chromatographic method is frequently used in the purification scheme for isolation of vitamin K dependent clotting factors II, VII, IX and X, and clotting inhibitors protein C and protein S, as well as inter-alpha inhibitor proteins (IaIp). After the removal of immunoglobulin G and non-binding proteins in the flow-through fraction, albumin and weakly bound proteins were eluted with low concentration of sodium chloride. The proteins that strongly bind to the anion-exchange column were eluted by higher salt concentrations. The fractions of interest were analyzed, and proteins were identified by LC-ESI-MS/MS. By use of this method, not only candidates for therapeutic concentrates, but also some potentially harmful components were identified. This strategy was very helpful for further process optimization, fast identification of target proteins with relatively low abundance, and for the design of subsequent steps in their removal or purification.

Proteomic characterization of plasma-derived clotting factor VIII-von Willebrand factor concentrates.

Proteomic methods were used to identify the levels of impurities in three commercial plasma‐derived clotting factor VIII‐von Willebrand factor (FVIII/VWF) concentrates. In all three concentrates, significant amounts of other plasma proteins were found. In Octanate and Haemoctin, two concentrates developed in the 1990s, the major impurities identified were inter‐α inhibitor proteins, fibrinogen and fibronectin. These two concentrates were also found to contain additional components such as clotting factor II (prothrombin) that are known activators of FVIII. In Wilate, a recently developed FVIII/VWF concentrate, the amount of these impurities was significantly reduced. Batch‐to‐batch variations and differences between three investigated products were detected using iTRAQ, an isotope labeling technique for comparative MS, demonstrating the potential value of this technique for quality control analysis. The importance of thorough proteomic investigations of therapeutic FVIII/VWF preparations from human plasma is also discussed.

Changes in photosynthetic performance and antioxidative strategies during maturation of Norway maple (Acer platanoides L.) leaves

Different structural and functional changes take place during leaf development. Since some of them are highly connected to oxidative metabolism, regulation of reactive oxygen species (ROS) abundance is required. Most of the reactive oxygen species ROS in plant cells are produced in chloroplasts as a result of highly energetic reactions of photosynthesis. The aim of our study was to examine the changes in concentration of oxidative stress parameters (TBARS - thiobarbituric acid-reacting substances and protein carbonyls) as well as antioxidative strategies during development of maple (Acer platanoides L.) leaves in the light of their enhanced photosynthetic performance. We reveal that biogenesis of the photosynthetic apparatus during maple leaf maturation corresponded with oxidative damage of lipids, but not proteins. In addition, antioxidative responses in young leaves differed from that in older leaves. Young leaves had high values of non-photochemical quenching (NPQ) and catalase (CAT, EC 1.11.1.6) activity which declined during the maturation process. Developing leaves were characterized by an increase in TBARS level, the content of non-enzymatic antioxidants as well as ascorbate peroxidase activity (APX, EC 1.11.1.11), while the content of protein carbonyls decreased with leaf maturation. Fully developed leaves had the highest lipid peroxidation level accompanied by a maximum in ascorbic acid content and superoxide dismutase activity (SOD, EC1.15.1.1). These observations imply completely different antioxidative strategies during leaf maturation enabling them to perform their basic function.

SELDI-TOF as a method for biomarker discovery in the urine of aristolochic-acid- treated mice

Aristolochic acids (AAs) present in Aristolochia plants are substances responsible for Chinese herbs nephropathy. Recently, strong indications have also been presented, which dietary poisoning with AA is responsible for endemic (Balkan) nephropathy (EN), an enigmatic renal disease that affects rural population living in some countries in Southeastern Europe. A mouse model was applied to follow the effects of two forms of AA, AAI and AAII. SDS‐PAGE and SELDI‐TOF mass spectrometry with normal phase chips were used to evaluate changes in the urine of treated animals. These two methods are demonstrated to be comparable. The use of SELDI‐TOF MS for rapid analysis of a large number of samples and the combination of this method with nano‐LC‐ESI MS/MS for protein identification were demonstrated. Biomarker discovery after analysis of large cohort of EN patients will be the final aim of these investigations.

Therapeutic plasma proteins – application of proteomics in process optimization, validation, and analysis of the final product

An overview is given on the application of proteomic technology in the monitoring of different steps during the production of therapeutic proteins from human plasma. Recent advances in this technology enable the use of proteomics as an advantageous tool for the validation of already existing processes, the development and fine tuning of new production steps, the characterization and quality control of final products, the detection of both harmful impurities and modifications of the therapeutic protein and the auditing of batch‐to‐batch variations. Further, use of proteomics for preclinical testing of new products, which can be either recombinant or plasma‐derived, is also discussed.

Reversed‐Phase High Performance Liquid Chromatography of Proteins

Reversed‐phase HPLC (RP‐HPLC) is one of most important techniques for protein separations and the method of choice for peptide separation. RP‐HPLC has been applied on the nano, micro, and analytical scale, and has also been scaled up for preparative purifications, to large industrial scale. Because of its compatibility with mass spectrometry, RP‐HPLC is an indispensable tool in proteomic research. With modern instrumentation and columns, complex mixtures of peptides and proteins can be separated at attomolar levels for further analysis. In addition, preparative RP‐HPLC is often used for large‐scale purification of proteins. This unit provides protocols for packing and testing a column, protein separation by use of gradient or step elution, desalting of protein solutions, and separation of enzymatic digests before mass spectrometric analyses. A protocol is also provided for cleaning, regenerating, and storing reversed‐phase chromatography columns. Curr. Protoc. Protein Sci. 61:8.7.1‐8.7.22.

Volatile Organic Compounds from Centaurium erythraea Rafn (Croatia) and the Antimicrobial Potential of Its Essential Oil

GC and MS were used for the analysis of Croatian Centaurium erythraea Rafn essential oil (obtained by hydrodistillation) and headspace (applying headspace solid-phase microextraction). The headspace contained numerous monoterpene hydrocarbons (the major ones were terpinene-4-ol, methone, p-cymene, γ-terpinene and limonene). Oxygenated monoterpenes were present in the headspace and oil, while 1,8-cineole, bornyl acetate and verbenone were present only in the headspace. High headspace percentages of toluene and naphthalene were found, followed by hemimellitene. Lot of similarities were observed with Serbian C.erythraea oil [neophytadiene (1.4%), thymol (2.6%), carvacrol (6.1%) and hexadecanoic acid (5.7%)], but different features were also noted such as the presence of menthol, menthone and phytone. The oil fractionation enabled identification of other minor compounds not found in total oil such as norisoprenoides, alk-1-enes or chromolaenin. The essential oil showed antimicrobial potential on Escherichia coli, Salmonella enteritidis, Staphylococcus aureus and Bacillus cereus. On the other hand, no antibacterial activity of the oil was observed on Pseudomonas fluorescens and Lysteria monocytogenes.