Physiological proteomics: cells, organs, biological fluids, and biomarkers

Proteomic research is accelerating rapidly because of marked advances in protein labeling techniques, mass spectrometry (MS), and bioinformatics. Two-dimensional difference gel electrophoresis (2D-DIGE) is being used effectively in conjunction with liquid chromatography tandem MS (LC-MS/MS) and/or matrix-assisted laser desorption/ionization time-of-flight MS (MALDI-ToF MS) and database search software to quantify relative changes in the levels of proteins in two samples. It is now possible in a single study to identify and quantify large numbers of proteins and their posttranslational modifications in different biological samples. Comparisons can be made between groups of animals in different physiological states or in response to experimental treatment. Differences between normal individuals and those in disease states can form the foundation for elucidation of causative factors of disease and the identification of biomarkers for the diseased state. This symposium includes original research that compares the erythrocyte plasma membrane proteome in the normal and the sickle cell state, evaluates the anterior pituitary gland proteome in the ovariectomized rat in response to estrogen, and assesses proteomic methodology employed to identify potentially useful biomarkers in human cells and fluids for clinical medicine. It is directed not only to investigators working in these fields but also to a diverse group of scientists working in the biological and biomedical fields to stimulate cross-disciplinary awareness, interest, and collaboration.     

Parametric studies on droplet generation reproducibility for applications with biological relevant fluids

Although the great potential of droplet based microfluidic technologies for routine applications in industry and academia has been successfully demonstrated over the past years, its inherent potential is not fully exploited till now. Especially regarding to the droplet generation reproducibility and stability, two pivotally important parameters for successful applications, there is still a need for improvement. This is even more considerable when droplets are created to investigate tissue fragments or cell cultures (e.g. suspended cells or 3D cell cultures) over days or even weeks. In this study we present microfluidic chips composed of a plasma coated polymer, which allow surfactants‐free, highly reproducible and stable droplet generation from fluids like cell culture media. We demonstrate how different microfluidic designs and different flow rates (and flow rate ratios) affect the reproducibility of the droplet generation process and display the applicability for a wide variety of bio(techno)logically relevant media.     

Differential biological activity of disease-associated JAK2 mutants

The JAK2V617F mutation has been identified in most patients with myeloproliferative neoplasms (MPNs), including polycythemia vera, essential thrombocythemia and primary myelofibrosis. Although JAK2V617F is the predominant allele associated with MPNs, other activating Janus kinase 2 (JAK2) alleles (such as K539L, T875N) also have been identified in distinct MPNs. The basis for the differences in the in vivo effects of different JAK2 alleles remains unclear. We have characterized three different classes of disease-associated JAK2 mutants (JAK2V617F, JAK2K539L and JAK2T875N) and found significant differences in biochemical, signaling and transforming properties among these different classes of JAK2 mutants.     

The NMR study of human biological fluids for detection of pathologies NMR study of biological fluids

The paper deals with the NMR spectra obtained using preparations of five different human biological body fluids. Characteristic metabolite signals of blood, urine, tears, saliva, and sweat spectra have been determined and classified. The biological body fluid samples were used for search and identification of biomarkers of cardiovascular disease. Absolute functional biomarkers for diseases such as coronary heart disease (CHD) have not been recognized even in the case acute myocardial infarction. A hypothesis explaining reasons of lack of such markers has been formulated. The results of comparative analysis of blood and urine samples from humans and some laboratory animals are given. Identify and analyze signals of metabolites of pathogenic microflora and their dynamics in the urine from patients with urogenital diseases have been determined and analyzed and characteristic biomarkers have been recognized.     

Cell biological studies of the prion protein

Studying PrPC and PrPSc in cell culture systems is advantageous because such systems contain all the organelles, membranes, and molecular cofactors that are likely to play an important role in the biology of the proteins. Using cultured cells expressing PrPC, we have discovered that this isoform constitutively cycles between the cell surface and an endocytic compartment, a process that is mediated by clathrin-coated pits and a putative PrPC receptor. We have also constructed stably transfected lines of CHO cells that express PrP molecules carrying mutations that are associated with familial prion diseases. The mutant PrP molecules in these cells are spontaneously converted to the PrPSc state, a phenomenon which has allowed us to analyze several key features of prion formation.     

High-performance liquid chromatographic assay for laudanosine in biological fluids and tissue for neurotoxic studies in rats

A procedure for the determination of laudanosine, the central nervous system active metabolite of the neuromuscular blocking drug atracurium, in serum, cerebrospinal fluid and brain is described. The method uses a readily available internal standard, ethavrine, and a single-step protein precipitation with acetonitrile followed by high-performance liquid chromatographic separation with ultraviolet detection. Norlaudanosine, the major metabolite of laudanosine, can also be quantified. Linearity of detector response was obtained between 1 and 25 μg/ml or μg/g and the method is suitable for determining neurotoxic concentrations of laudanosine in experimental animals.     

Aggregation by lectins as an approach for exosome isolation from biological fluids: Validation for proteomic studies

Exosomes, a special type of microparticles produced by cells, are currently of considerable interest for researchers. The term “exosomes” denotes extracellular vesicles of less than 120 nm in size derived from intracellular multivesicular bodies. Multiple studies that address the distinctive features of exosome structure and biochemical composition in various pathological states imply the possibility of development of novel diagnostic techniques based on the detection of changes in the pool of proteins and nucleic acids transported by exosomes. However, methods for isolation and investigation of exosomes are rather difficult to develop because of a small size of these vesicles. A novel approach for preparative-scale isolation of exosomes based on the phenomenon of binding and aggregation of these particles in the presence of lectins has been put forward in the present study. The method developed is relatively cost-effective, allows for the isolation of exosomes from various biological fluids, and has been validated for the subsequent analysis of the protein composition of the exosomes in view of the possible clinical applications. The validation showed that the sedimentation of lectin-aggregated exosomes is a suitable approach for the isolation of these microvesicles from the complete conditioned culture medium in a research-laboratory setup.     

Altered protein dynamics of disease-associated lamin A mutants

Background  

Recent interest in the function of the nuclear lamina has been provoked by the discovery of lamin A/C mutations in the laminopathy diseases. However, it is not understood why mutations in lamin A give such a range of tissue-specific phenotypes. Part of the problem in rationalising genotype-phenotype correlations in the laminopathies is our lack of understanding of the function of normal and mutant lamin A. To investigate this we have used photobleaching in human cells to analyse the dynamics of wild-type and mutant lamin A protein at the nuclear periphery.     

Refinement of unbound protein docking studies using biological knowledge

In this work we present two methods for the reranking of protein-protein docking studies. One scoring method searches the InterDom database for domains that are available in the proteins to be docked and evaluates the interaction of these domains in other complexes of known structure. The second one analyzes the interface of each proposed conformation with regard to the conservation of Phe, Met, and Trp and their polar neighbor residues. The special relevance of these residues is based on a publication by Ma et al. (Proc Natl Acad Sci USA 2003;100:5772-5777), who compared the conservation of all residues in the interface region to the conservation on the rest of the protein's surface. The scoring functions were tested on 30 unbound docking test cases. The evaluation of the methods is based on the ability to rerank the output of a Fast Fourier Transformation (FFT) docking. Both were able to improve the ranking of the docking output. The best improvement was achieved for enzyme-inhibitor examples. Especially the domain-based scoring function was successful and able to place a near-native solution on one of the first six ranks for 13 of 17 (76%) enzyme-inhibitor complexes [in 53% (nine complexes) even on the first rank]. The method evaluating residue conservation allowed us to increase the number of good solutions within the first 100 ranks out of approximately 9000 in 82% of the 17 enzyme-inhibitor test cases, and for seven (41%) out of 17 enzyme-inhibitor complexes, a near native solution was placed within the first seven ranks.     

HDAPD: a web tool for searching the disease-associated protein structures

Background

PCR-restriction fragment length polymorphism (RFLP) assay is a cost-effective method for SNP genotyping and mutation detection, but the manual mining for restriction enzyme sites is challenging and cumbersome. Three years after we constructed SNP-RFLPing, a freely accessible database and analysis tool for restriction enzyme mining of SNPs, significant improvements over the 2006 version have been made and incorporated into the latest version, SNP-RFLPing 2.

Results

The primary aim of SNP-RFLPing 2 is to provide comprehensive PCR-RFLP information with multiple functionality about SNPs, such as SNP retrieval to multiple species, different polymorphism types (bi-allelic, tri-allelic, tetra-allelic or indels), gene-centric searching, HapMap tagSNPs, gene ontology-based searching, miRNAs, and SNP500Cancer. The RFLP restriction enzymes and the corresponding PCR primers for the natural and mutagenic types of each SNP are simultaneously analyzed. All the RFLP restriction enzyme prices are also provided to aid selection. Furthermore, the previously encountered updating problems for most SNP related databases are resolved by an on-line retrieval system.

Conclusions

The user interfaces for functional SNP analyses have been substantially improved and integrated. SNP-RFLPing 2 offers a new and user-friendly interface for RFLP genotyping that can be used in association studies and is freely available at http://bio.kuas.edu.tw/snp-rflping2.     

CA 125 in biological fluids

CA 125 is not a specific tumor marker, and is synthesized by normal and malignant cells of different origin (mainly in tissues derived from the müllerian epithelia) in a similar proportion. Abnormal CA 125 levels may be found in fluids of different origin (ascites, pleura, pericardium, amniotic fluid, cyst fluid, bronchoalveolar fluid, etc.) and in serum from patients with these fluids. Differences in serum CA 125 found in malignant or benign diseases may be related to the number of cells that synthesize the marker, and are highly dependent on the access to serum, where the marker is normally determined. Moreover, CA 125 is a very good tumor marker in ovarian and lung cancer. The sensitivity of CA 125 in ovarian cancer is related to stage (40-95%), histological type (lower levels in mucinous adenocarcinoma), and the marker is useful in the early detection of recurrence (sensitivity 80%) and in therapy monitoring. It's sensitivity in lung cancer is lower than in ovarian cancer, 39% in locoregional malignancies and 69% in metastatic disease, but clearly related to stage and histology (mainly in adenocarcinomas and large cell lung cancer) and it is useful in prognosis and disease monitoring.     

Biomarker discovery in biological fluids

Discovery of novel protein biomarkers is essential for successful drug discovery and development. These novel protein biomarkers may aid accelerated drug efficacy, response, or toxicity decision making based on their enhanced sensitivity and/or specificity. These biomarkers, if necessary, could eventually be converted into novel diagnostic marker assays. Proteomic platforms developed over the past few years have given us the ability to rapidly identify novel protein biomarkers in various biological matrices from cell cultures (lysates, supernatants) to human clinical samples (serum, plasma, and urine). In this article, we delineate an approach to biomarker discovery. This approach is divided into three steps, (i) identification of markers, (ii) prioritization of identified markers, and (iii) preliminary validation (qualification) of prioritized markers. Using drug-induced idiosyncratic hepatotoxicity as a case study, the article elaborates methods and techniques utilized during the three steps of biomarker discovery process. The first step involves identification of markers using multi-dimensional protein identification technology. The second step involves prioritization of a subset of marker candidates based on several criteria such as availability of reagent set for assay development and literature association to disease biology. The last step of biomarker discovery involves development of preliminary assays to confirm the bio-analytical measurements from the first step, as well as qualify the marker(s) in pre-clinical models, to initiate future marker validation and development.     

Measurement of peptidoleukotrienes in biological fluids

Samples of human bronchoalveolar lavage fluid (BALF) and urine were utilized to demonstrate methods for quantitation and validation of leukotrienes (LTs). These methods utilize an enzyme immunoassay (EIA) that uses commercially available reagents, the antibody recognizing LTC4, LTD4, LTE4, and N-acetyl LTE4. BALF containing epithelial lining fluid was collected from atopic asthmatics both before and 5 min after the subjects had been challenged with a local instillation of allergen into the airways. BALF samples collected without allergen challenge had low levels of immunoreactive LTs, whereas samples collected after allergen were markedly elevated. After high-performance liquid chromatography (HPLC) separation of LTs, EIA revealed the presence of LTC4. The identity was validated by incubating LTC4 with a bovine gamma-glutamyl transpeptidase with dipeptidase activity that converted added [3H]-LTC4 as well as LTC4 immunoreactivity to LTE4. Urine samples collected from six healthy volunteers, one patient with adult respiratory distress syndrome (ARDS), and three patients in status asthmaticus were also analyzed for LTs. After HPLC separation of LTs and quantitation by EIA, urine samples from healthy subjects were found to have low but measurable LTE4. In contrast, the urine samples from the patients in status asthmaticus and from the ARDS patient had large elevations of LTE4 levels compared with healthy subjects. When the HPLC fractions containing [3H]LTE4 and LT immunoreactivity in the ARDS sample were treated with acetic anhydride, HPLC analysis indicated that both radiolabel and immunoreactivity now eluted at the retention time of N-acetyl LTE4, the derivatized product of LTE4. The methods described are relatively easy and can be used to measure and validate the existence of peptidoleukotrienes in biological samples.     

Measurement of dimethylglycine in biological fluids

The method of quantitating N,N-dimethylglycine involves cation-exchange high-performance liquid chromatography and detection of dimethylglycine with dimethylglycine dehydrogenase. Dimethylglycine was added to plasma and urine and samples were assayed for dimethylglycine. Plasma and urine to which no dimethylglycine was added were also assayed. Recoveries of added dimethylglycine were 99 to 104% with no endogenous dimethylglycine found in rat plasma or normal human urine. The human plasma used contained a small amount of endogenous dimethylglycine. The cation-exchange chromatography separates dimethylglycine from other compounds which can serve as substrates for dimethylglycine dehydrogenase. Repeatability of the assay is +/- 10%. Using this method we have identified dimethylglycine in the urine of a 1-month-old female human patient.