Nina Kočevar

Comparison of capillary electrophoresis and high performance liquid chromatography for determination of flavonoids in Achillea millefolium

Flavonoids represent an important bioactive component in Achillea millefolium. The comparison of the most commonly used analytical methods for the identification and quantification of flavonoids, capillary electrophoresis (CE) and high performance liquid chromatography (HPLC), is presented. The methods were optimized and validated. Using a 20 mM borate buffer with 30% (v/v) of methanol (pH 9.3) in the CE analysis and a gradient elution with water-acetonitrile mobile phase in the HPLC analysis, sufficient separation of the analytes was achieved. A relatively high injection volume in the CE analysis (30 mbar x 30s) enabled low limit of detection (LOD) (0.3-0.7 mg/L). Repeatability of both methods was acceptable (relative standard deviation of peak area were <6%). Additionally, the amount of flavonoids in a real sample of the dried herbal drug was determined.

Comparative study of robustness between micellar electrokinetic capillary chromatography and high-performance liquid chromatography using one-variable-at-a-time and a new multi-variable-at-a-time approach

Micellar electrokinetic capillary chromatography and reverse-phase liquid chromatography methods were developed in order to perform robustness testing to determine the caffeine content in beverages. Both methods were fully validated and two different robustness approaches were applied. One-variable-at-a-time (OVAT) approach at eleven levels (0; +/-1; +/-2; +/-3; +/-4; +/-5 units) was carried out and compared with multi-variable-at-a-time (MVAT) approach at three levels (+/-1 unit per investigated parameter). Four analysts in two laboratories on two capillary electrophoresis and two RPLC equipments have tested the samples. Robustness was statistically analyzed using peak area, migration or retention time, symmetry, and resolution of caffeine and sulfacetamide as internal standard, and presented as R.S.D. values. The RPLC method was found to be more sensitive than the MEKC method. Both methods showed acceptable robustness level for OVAT approach, whilst MEKC method was more robust when the determination of real samples coming from different beverages was based on the MVAT approach.

Proteomic approaches in biomarker discovery: new perspectives in cancer diagnostics.

Despite remarkable progress in proteomic methods, including improved detection limits and sensitivity, these methods have not yet been established in routine clinical practice. The main limitations, which prevent their integration into clinics, are high cost of equipment, the need for highly trained personnel, and last, but not least, the establishment of reliable and accurate protein biomarkers or panels of protein biomarkers for detection of neoplasms. Furthermore, the complexity and heterogeneity of most solid tumours present obstacles in the discovery of specific protein signatures, which could be used for early detection of cancers, for prediction of disease outcome, and for determining the response to specific therapies. However, cancer proteome, as the end-point of pathological processes that underlie cancer development and progression, could represent an important source for the discovery of new biomarkers and molecular targets for tailored therapies.

The progress of proteomic approaches in searching for cancer biomarkers

Biomarkers are indicators of a specific biological state. Their detection in pathological conditions, such as cancer, is important for clinical disease management. One of their greatest values could be in early diagnosis and detection of neoplasms when the cancer is more manageable. Protein biomarkers are expected to be reliable predictors of pathological conditions, as they represent the endpoint of biological processes. The proteomic methodology has rapidly evolved in the past ten years, thus enabling discovery of a vast amount of potential biomarker candidates. However, the majority of novel candidates have not yet reached the integration into clinical environment. To do that, well-constructed large population validation studies are necessary as well as development of new algorithms for deciphering complex biological interactions and their involvement in pathological processes. This review focuses on advances in classical proteomic approaches and emerging high-throughput proteomic technologies for identifying cancer biomarkers.

Proteomic analysis of gastric cancer and immunoblot validation of potential biomarkers

AIM To search for and validate differentially expressed proteins in patients with gastric adenocarcinoma. METHODS We used two-dimensional gel electrophoresis and mass spectrometry to search for differentially expressed proteins in patients with gastric adenocarcinoma. A set of proteins was validated with immunoblotting. RESULTS We identified 30 different proteins involved in various biological processes: metabolism, development, death, response to stress, cell cycle, cell communication, transport, and cell motility. Eight proteins were chosen for further validation by immunoblotting. Our results show that gastrokine-1, 39S ribosomal protein L12 (mitochondrial precursor), plasma cell-induced resident endoplasmic reticulum protein, and glutathione S-transferase mu 3 were significantly underexpressed in gastric adenocarcinoma relative to adjacent non-tumor tissue samples. On the other hand, septin-2, ubiquitin-conjugating enzyme E2 N, and transaldolase were significantly overexpressed. Translationally controlled tumor protein was shown to be differentially expressed only in patients with cancer of the gastric cardia/esophageal border. CONCLUSION This work presents a set of possible diagnostic biomarkers, validated for the first time. It might contribute to the efforts of understanding gastric cancer carcinogenesis.

Improved acylation method enables efficient delivery of functional palmitoylated cystatin into epithelial cells.

The effective delivery of therapeutic proteins to the site of action is of great importance in achieving an effective therapy. Due to hydrophilicity, proteins are generally poorly transported across biological membranes. Chemical acylation represents one of the basic methods for improving their membrane permeability. A novel method for acylation is presented, based on the formation of palmitoylchloride dispersion in aqueous acetonitrile solution, using chicken cystatin as a model protein. We examined the effects of palmitoylchloride/cystatin molar ratio, reaction pH and introduction of successive palmitoylation cycles on the protein modification degree. The reaction products were analysed by capillary electrophoresis and SDS‐PAGE, and the in vitro inhibitory activity was determined by N‐benzoyl‐d,l‐arginine‐β‐naphthylamide assay. Using cell culture‐based assays, we examined the transport properties of unmodified and palmitoylated cystatin, its efficiency to inhibit intracellular enzymes, and its cytotoxicity. We demonstrated that palmitoylated cystatin rapidly internalized into the cell and caused a complete loss of cathepsin B activity. In contrast, the unmodified control cystatin was unable to inhibit the intracellular enzymes. These results strongly suggest protein palmitoylation to be a very effective strategy for improving cell internalization.

TRIM28 and β-Actin Identified via Nanobody-Based Reverse Proteomics Approach as Possible Human Glioblastoma Biomarkers

Malignant gliomas are among the rarest brain tumours, and they have the worst prognosis. Grade IV astrocytoma, known as glioblastoma multiforme (GBM), is a highly lethal disease where the standard therapies of surgery, followed by radiation and chemotherapy, cannot significantly prolong the life expectancy of the patients. Tumour recurrence shows more aggressive form compared to the primary tumour, and results in patient survival from 12 to 15 months only. Although still controversial, the cancer stem cell hypothesis postulates that cancer stem cells are responsible for early relapse of the disease after surgical intervention due to their high resistance to therapy. Alternative strategies for GBM therapy are thus urgently needed. Nanobodies are single-domain antigen-binding fragments of heavy-chain antibodies, and together with classical antibodies, they are part of the camelid immune system. Nanobodies are small and stable, and they share a high degree of sequence identity to the human heavy chain variable domain, and these characteristics offer them advantages over classical antibodies or antibody fragments. We first immunised an alpaca with a human GBM stem-like cell line prepared from primary GBM cultures. Next, a nanobody library was constructed in a phage-display vector. Using nanobody phage-display technology, we selected specific GBM stem-like cell binders through a number of affinity selections, using whole cell protein extracts and membrane protein-enriched extracts from eight different GBM patients, and membrane protein-enriched extracts from two established GBM stem-like cell lines (NCH644 and NCH421K cells). After the enrichment, periplasmic extract ELISA was used to screen for specific clones. These nanobody clones were recloned into the pHEN6 vector, expressed in Escherichia coli WK6, and purified using immobilised metal affinity chromatography and size-exclusion chromatography. Specific nanobody:antigen pairs were obtained and mass spectrometry analysis revealed two proteins, TRIM28 and β-actin, that were up-regulated in the GBM stem-like cells compared to the controls.

Two‐dimensional gel electrophoresis of gastric tissue in an alkaline pH range

2DE in combination with MS has facilitated the discovery of several proteins with altered abundance in gastric cancer. While acidic and wide pH ranges have been widely investigated, analysis in the alkaline pH range has not been specifically performed in gastric cancer to date. In the present study, we initially optimized the 2DE in alkaline pH range (pH 7–11) for gastric tissue samples. Using a modified lysis buffer, we analyzed pooled nontumor and tumor samples for proteins with altered abundance in gastric adenocarcinoma. We successfully identified 38 silver‐stained spots as 24 different proteins. Four of these were chosen for investigation with immunoblotting on individual paired samples to determine whether the changes seen in 2DE represent the overall abundance of the protein or possibly only a single form. While mitochondrial trifunctional protein (MTP) subunits were decreased in 2DE gels, immunoblotting identified their overall abundance as being differently dysregulated: in the gastric tumor samples, the MTP‐α subunit was decreased, and the MTP‐β subunit was increased. On the other hand, heterogenous nuclear ribonucleoprotein M and galectin‐4 were increased in the gastric tumor samples in both 2DE and immunoblotting.