Ceramide kinase profiling by mass spectrometry reveals a conserved phosphorylation pattern downstream of the catalytic site.

Ceramide kinase (CERK) is essential for production of ceramide-1-phosphate (C1P), a bioactive lipid whose formation critically modulates ceramide levels. To explore how CERK is regulated, we used insect cell-expressed, recombinant hCERK and searched for post-translational modifications, using mass-spectrometry techniques. This led to identification of two phosphorylated serine residues, at positions 340 and 408. Point mutations preventing phosphorylation at either of these sites did not lead to detectable changes in subcellular localization or activity. However, preventing phosphorylation at S340 resulted in CERK instability as revealed by the behavior of the S340A mutant protein under various assay conditions in vitro. Phosphorylation of a cognate serine residue in sphingosine kinases was previously shown to be important. Therefore, phosphorylation within a conserved "regulation loop" downstream of the catalytic domain emerges as a new paradigm for regulation of kinases of the diacylglycerol kinase family. This "regulation loop" is reminiscent of the "activation loop" that controls AGC protein kinases, being a similar distance from the critical ATP binding site determinants in the primary sequence.

Improved enrichment and proteomic analysis of brain proteins with signaling function by heparin chromatography.

Detection of low-abundance proteins with signaling function is essential for the identification of biomarkers and novel drug targets. We present a protocol for specific enrichment of secreted proteins with signaling function by combining subcellular fractionation with heparin chromatography. The subcellular fractionation includes the preparation of a fraction enriched in cytosolic proteins. A further enrichment was achieved by heparin chromatography. The proteins eluted from the heparin column were analyzed by MudPIT tandem mass spectrometry and identified with the use of an in silico algorithm. Forty-eight percent of the identified proteins (188 out of 391) bound to the heparin matrix. Fifty-four percent of them (101) are secreted proteins with signaling function and 23% (44) of the enriched signaling proteins had not been detected by 2D PAGE without application of the heparin enrichment step. The heparin chromatography method can be combined with other proteomics enrichment approaches, such as ion exchange or reversed phase chromatography.

Mass spectrometric evaluation of upstream and downstream process influences on host cell protein patterns in biopharmaceutical products.

For production of different monoclonal antibodies (mAbs), biopharmaceutical companies often use related upstream and downstream manufacturing processes. Such platforms are typically characterized regarding influence of upstream and downstream process (DSP) parameters on critical quality attributes (CQAs). CQAs must be monitored strictly by an adequate control strategy. One such process-related CQA is the content of host cell protein (HCP) which is typically analyzed by immunoassay methods (e.g., HCP-ELISA). The capacity of the immunoassay to detect a broad range of HCPs, relevant for the individual mAb-production process should be proven by orthogonal proteomic methods such as 2D gel electrophoresis or mass spectrometry (MS). In particular MS has become a valuable tool to identify and quantify HCP in complex mixtures. We evaluate up- and DSP parameters of four different biopharmaceutical products, two different process variants, and one mock fermentation on the HCP pattern by shotgun MS analysis and ELISA. We obtained a similar HCP pattern in different cell culture fluid harvests compared to the starting material from the downstream process. During the downstream purification process of the mAbs, the HCP level and the number of HCP species significantly decreased, accompanied by an increase in diversity of the residual HCP pattern. Based on this knowledge, we suggest a control strategy that combines multi product ELISA for in-process control and release analytics, and MS testing for orthogonal HCP characterization, to attain knowledge on the HCP level, clusters and species. This combination supports a control strategy for HCPs addressing safety and efficacy of biopharmaceutical products.

Identification of beta cell dysfunction at the pre-symptomatic stage of diabetes mellitus by novel analytical system: liquid biopsy measurements in femtograms.

BACKGROUND: Diabetes mellitus is produced and progresses as a consequence of complex and gradual processes, in which a variety of alterations of the endocrine pancreas, are involved and which mainly result in beta cell failure. Those molecular alterations can be found in the bloodstream, which suggests that we could quantify specific biomarkers in plasma or serum by very sensitive methods before the onset diabetes mellitus is diagnosed. However, classical methods of protein analysis such as electrophoresis, Western blot, ELISA, and liquid chromatography are generally time-consuming, lab-intensive, and not sensitive enough to detect such alteration in a pre-symptomatic state of the disease. METHOD: A very sensitive and novel analytical detection conjugate system by using the combination of polyfluorophor technology with protein microchip method was developed. RESULTS: This innovative system facilitates the use of a very sensitive microchip assays that measure selected biomarkers in a small sample volume (10 µL) with a much higher sensitivity (92%) compare to common immune assay systems. Further advances of the application of this technology combine the power of miniaturization and faster quantification (around 10 min). CONCLUSION: The power of this technology offers great promise for point-of-care clinical testing and monitoring of specific biomarkers for diabetes in femtogram level in serum or plasma. In conclusion, the results indicate that the technical performance of this new technology is valid and that the assay is able to quantified PPY-specific antigens in plasma at femtogram levels which can be used for identification of beta cell dysfunction at the pre-symptomatic stage of diabetes mellitus.

Proteomics in brain research: potentials and limitations.

The advent of proteomics techniques has been enthusiastically accepted in most areas of biology and medicine. In neuroscience, a host of applications was proposed ranging from neurotoxicology, neurometabolism, determination of the proteome of the individual brain areas in health and disease, to name a few. Only recently, the limitations of the method have been shown, hampering the rapid spreading of the technology, which in principle consists of two-dimensional gel electrophoresis with in-gel protein digestion of protein spots and identification by mass-spectrometrical approaches or microsequencing. The identification, including quantification using specific software, of brain protein classes, like enzymes, cytoskeleton proteins, heat shock proteins/chaperones, proteins of the transcription and translation machinery, synaptosomal proteins, antioxidant proteins, is a clear domain of proteomics. Furthermore, the concomitant detection of several hundred proteins on a gel allows the demonstration of an expressional pattern, rather generated by a reliable, protein-chemical method than by immunoreactivity, proposed by protein-arrays. An additional advantage is that hitherto unknown proteins, so far only proposed from their nucleic acid structure, designated as hypothetical proteins, can be identified as brain proteins. As to shortcomings and disadvantages of the method we would point to the major problem, the failure to separate hydrophobic proteins. There is so far no way to analyse the vast majority of these proteins in gels. Several other analytical problems need to be overcome, but once the latter problem can be solved, there is nothing to stop the method for a large scale analysis of membrane proteins in neuroscience.

Hypothetical proteins with putative enzyme activity in human amnion, lymphocyte, bronchial epithelial and kidney cell lines.

A myriad of predicted proteins have been described based upon nucleic acid sequences but the existence of these structures has not been confirmed at the protein level. The aim of the study was therefore to show expression of hypothetical proteins in several cell lines and to provide the analytical basis for their identification and characterisation. We used two-dimensional gel electrophoresis with in-gel digestion of high protein spots and subsequent MALDI-TOF analysis of cell lysates from human amnion, lymphocyte, bronchial epithelial and kidney cell lines. A pI range from 3 to 10 was selected and second dimension was run using 9-16% gradient gels. A series of structures that have not been described before at the protein level were identified in several cell lines and were assigned to major enzyme systems including proteolysis (proteases, peptidases, ubiquitin), intermediary metabolism and oxidoreductases. We conclude that the proteomic approach used serves as a suitable tool to verify the existence of predicted/hypothetical proteins. The herein identified enzymes may contribute to several pathways/cascades in the human organism. Furthermore, analytical data given are of major relevance as pIs, a prerequisite to find proteins in a map, cannot be predicted from nucleic acid sequences.

HTRF: a technology tailored for biomarker determination-novel analytical detection system suitable for detection of specific autoimmune antibodies as biomarkers in nanogram level in different body fluids.

BACKGROUND: Classical methods of gene product analysis such as binding assays (e.g., ELISA, protein chip technology) are generally time-consuming, lab-intensive, less sensitive, and lack high-throughput capacity. In addition, all existing methods used to measure proteins necessitate multiple divisions of the original sample and individual tests carried out for each substance, with an associated cost for each test. METHOD: Together with a small biotech company, we developed a new and innovative analytical detection system based on homogenous time-resolved fluorescence (HTRF) technology. Our system facilitates the development of immune assays that measure selective different analytes such as selected biomarkers in a small sample volume at less than 20 min with a much higher sensitivity compared to common binding assay systems such as enzyme-linked immunosorbent assay (ELISA). Recent advances of the application of this novel detection system combine the power of miniaturization, microfluidics, better linear range, and faster quantification. RESULTS: The power of the HTRF technology offers great promise for point-of-care clinical testing and monitoring of many important analytes such as disease-specific biomarkers in the nanogram level in different human body fluids such as CSF, blood, serum, plasma, and saliva. The linear dynamical range of our HTRF assay was determined between 2.5 and 100 ng/mL. Precision and accuracy calculated for inter- as well as intra-assays was less than ± 10 %. Intra-assay and inter-assay precision for high, medium, and low analyte concentrations show mean CV values less than ± 10 %. Intra- and inter-assay accuracy for all three concentrations show mean recovery values of 80-120 %. CONCLUSION: The aim of this work is to describe the development and establishment of this novel HTRF system that allows the very fast detection and quantification of biomarkers in different human body fluids. Furthermore, a specific antibody combination that assures a specific binding of the correct refolded autoimmune IgG is evaluated.

Proteomic characterization of the human cortical neuronal cell line HCN-2.

Neuronal marker proteins are widely used for characterization and identification of normal and tumor tissue of the central nervous system, but the most commonly used neuronal markers have inherent methodological problems. We used a proteomic approach with two-dimensional (2-D) gel electrophoresis and subsequent MALDI identification to identify possible new marker proteins in the human cortical neuronal cell line HCN-2. We found 14 proteins that previously were predicted only on the basis of open reading frames from mRNA sequences or DNA. We could unambiguously identify the following proteins: Bab55091, BK65A6.2 (Novel Sushi Domain (Scr repeat), glucosidase II alpha subunit, glucosidase II precursor (KIAA0088 protein), HSPC108, hypothetical 35.8 kDa protein, hypothetical 40.7 KDA protein, hypothetical 49.4 kDa protein, leucine-zipper protein FKSG13, lysophospholipase homolog, mitofilin (fragment), P1.11659_4, reticulocabin precursor and one unknown protein for MGC:10432. We could prove the existence of these 14 proteins in the human neuron, extending the list of proteins with neuronal expression and identify possible new candidate marker proteins.

Aberrant expression of peroxiredoxin subtypes in neurodegenerative disorders.

An increasing body of evidence indicates that oxidative stress and damage play a role in the pathogenesis of a number of diseases associated with neurodegeneration, including Down syndrome (DS), Alzheimer's disease (AD) and Pick's disease (PD). Although oxidative stress is a common element in these diseases, specific clinico-pathological phenotypes have been described for each disorder. Development of these phenotypes might be linked, among others, to differences in antioxidant response. The present study is designed to investigate expression of peroxiredoxins (Prxs), the newly characterized family of highly conserved antioxidant enzymes, and other antioxidant enzymes in frontal cortex and cerebellum of DS, AD and PD patients using the technique of proteomics. Levels of Prx I, Mn superoxide dismutase (SOD2) and glutathione-S-transferase omega1 in DS, AD and PD were not significantly different from that of controls in both brain regions investigated. In contrast, Prx II was significantly increased (P<0.05) in frontal cortex of DS, AD and PD, whereas Prx III was decreased in frontal cortex of DS (P<0.01) and PD (P<0.001). Interestingly, Prx VI displayed a significant increase (P<0.05) only in PD frontal cortex. The present data indicate that differential regulation of antioxidant enzymes exist in DS, AD and PD, suggestive of the diversity as well as distinct functional roles of these proteins. Moreover, while up-regulation of Prx II appears to provide evidence for the existence of compensatory response in increased cell loss, up-regulation of Prx VI may be used to discriminate PD from AD as well as DS.

Human brain cytosolic histamine-N-methyltransferase is decreased in Down syndrome and increased in Pick's disease.

Histamine-N-methyltransferase (HMT) inactivates the neurotransmitter histamine. Central histaminergic deficits may contribute to the cognitive impairment of neurodegenerative disorders including Alzheimer's disease (AD) and Down syndrome (DS). However, there is no evidence for histaminergic deficits in Pick's disease (PiD). HMT levels were measured in the frontal cortex and cerebellum of brains of patients with AD, DS, and PiD, and normal aged subjects using proteomics techniques. In frontal cortex, HMT was significantly decreased in DS, but significantly increased in PiD compared with controls. HMT levels were comparable in cerebellum of all groups. Elevated HMT in PiD could lead to increased histamine degradation that in turn would be in agreement with impaired cognitive functions of PiD. Decreased HMT in DS would be compatible with findings of decreased histamine synthesis, thus reflecting a compensation mechanism to antagonize reduced synthesis by decreased degradation.

Overexpressed protein disulfide isomerase in brains of patients with sporadic Creutzfeldt-Jakob disease.

Earlier studies have failed to detect covalent modifications in beta-sheet-rich scrapie isoform prion protein (PrP(Sc)) and have concluded that the conversion of alpha-helix-rich cellular form prion protein (PrP(C)) to PrP(Sc) represents purely conformational transition not involving chemical reactions. However, recent studies have shown that the intradisulfide bond of PrP(C) can play an important role for instability and conformational change to PrP(Sc). Interestingly, we found overexpressed protein disufide isomerase (PDI) in brains of sporadic Creutzfeldt-Jakob disease (sCJD, human prion disease) patients using two dimensional electrophoresis and Western blot analysis but not in other neurodegenerative disorders as Down Syndrome and Alzheimer's disease. However, proteinase K digestion and plasminogen binding assay of brain homogenates incubated with PDI suggest that PDI has no effect on either proteinase resistance or conformational change of PrP. Overexpression of PDI protein in sCJD brain may simply reflect a cellular defense response against the altered prion protein.

Expressional pattern of known and predicted signaling proteins in seven human cell lines.

Although a variety of signaling systems and signaling proteins have been described, cell specific expression of these structures has not yet been systematically studied. Human amnion, bronchial epithelial, fibroblast, glial, kidney, lymphocyte and mesothelial cells were subjected to two-dimensional-gel electrophoresis followed by analysis of protein spots by MALDI-TOF and subsequent identification by specific software. A series of well-documented signaling proteins showed cell specific expressional patterns. Five hypothetical proteins--hypothetical 37.5 kDa protein, similar to calsyntenin 1, hypothetical armadillo repeat/plakoglobulin ARM-repeat profile containing protein, 11 days embryo cDNA clone 2700084k13, hypothetical protein flj22171--so far predicted from their nucleic acid sequence only, were identified, complementing already reported signaling cascades. An analytical tool for the concomitant determination of a large series of signaling structures by an antibody independent protein-chemical method is provided.

Identification of tumour-related proteins as potential screening markers by proteome analysis-protein profiles of human saliva as a predictive and prognostic tool.

The analysis of biomarkers in saliva as a clinical application offers an attractive, simple and rapid diagnostic tool for the short- and long-term monitoring of pathological disorders and drug therapy. The collection of saliva, either in the pure or in its fractionated form, is a relatively easy and non-invasive procedure that is not harmful to the patients and has no complications at all. However, the fluid collection must be clearly defined due to variations in saliva composition, flow rate and day-to-day variability. In order to minimise possible variations, saliva from five patients without squamous cell carcinoma (SCC) pathology and five with suspicion of oral squamous carcinoma (OSCC) were collected and matched at different days and analysed by two-dimensional polyacrylamide gel electrophoresis (2DE-PAGE). Approximately 800 spots were identified, corresponding to 151 different gene products. The list of identified proteins includes a large number of structural proteins like keratins, keratin subunits, enzymes and enzyme inhibitors, cytokines, immunoglobulins as well as amylase and other salivary specific glycoproteins. The majority of proteins that are localised in oral epithelia cells were found as unsolved debris in saliva. One of the identified proteins was significantly overexpressed in OSCC and was selected for further validation by Western blot analysis.

Improved prognostic diagnosis of systemic lupus erythematosus in an early stage of disease by a combination of different predictive biomarkers identified by proteome analysis.

BACKGROUND: Since the original characterizations of the pathological features defining glomerulonephritis in systemic lupus erythematosus (SLE) were reported, numerous studies have linked the development of pathology to the abnormal expression of protein in urine. The determination of proteinuria is important and necessary; however, this alone is not predictive enough to confirm a suspected diagnosis, especially in an early state of disease when symptoms are not yet observed. Furthermore, several studies have already highlighted the pitfalls of proteinuria both as a clinical prognostic marker and as a factor predicting the progressive loss of renal function. Therefore, the identification of more accurate and predictive biomarkers is urgently needed. To address this, comparative urinary and kidney profiling was performed in the MRL-lpr/lpr mouse as a model of lupus tubulointerstitial nephritis and lupus glomerulonephritis corresponding to SLE in humans. RESULTS: Tamm-Horsfall glycoprotein (THG; uromodulin) and beta2-microglubulin (ß2M) were identified as immune process-related molecules in the urine and kidney of the MRL-lpr/lpr mouse model. Furthermore, we show that the combinatory expression profile of THG and ß2M as biomarkers, normalized by the proteinuria level, is more predictive than proteinuria determination alone. Data were confirmed by comparative urinary profiling of SLE in mice by Western blot and quantitative polymerase chain reaction (qPCR) analysis. CONCLUSION: Based on our results, we are able to diagnose SLE in the MRL-lpr/lpr mouse in a very early state of disease, when the proteinuria level alone is not able to confirm a suspected diagnosis. The pre-validation of our urinary biomarkers is associated with clinical outcomes of glomerulonephritis in humans and merits additional investigation. Further conformations of our predictive biomarkers in the urine of SLE patients in the course of a clinical study are still ongoing.

Pitfalls and limitations in translation from biomarker discovery to clinical utility in predictive and personalised medicine.

Since the emergence of the so-called omics technology, thousands of putative biomarkers have been identified and published, which have dramatically increased the opportunities for developing more effective therapeutics. These opportunities can have profound benefits for patients and for the economics of healthcare. However, the transfer of biomarkers from discovery to clinical practice is still a process filled with lots of pitfalls and limitations, mostly limited by structural and scientific factors. To become a clinically approved test, a potential biomarker should be confirmed and validated using hundreds of specimens and should be reproducible, specific and sensitive. Besides the lack of quality in biomarker validation, a number of other key issues can be identified and should be addressed. Therefore, the aim of this article is to discuss a series of interpretative and practical issues that need to be understood and resolved before potential biomarkers become a clinically approved test or are already on the diagnostic market. Some of these issues are shortly discussed here.

Advanced proteomics procedure as a detection tool for predictive screening in type 2 pre-Diabetes.

It has been suggested that a more precise selection of predictive biomarkers may prove useful in the early diagnosis of type 2 diabetes (T2D), even when glucose tolerance is normal. This is vital since many T2D cases may be preventable by avoiding those factors that trigger the disease process (primary prevention) or by use of therapy that modulates the disease process before the onset of clinical symptoms (secondary prevention) occurs. The selection of predictive markers must be carefully assessed and depends mainly on three important parameters: sensitivity, specificity and positive predictive value. Unfortunately, biomarkers with ideal specificity and sensitivity are difficult to find. One potential solution is to use the combinatorial power of different biomarkers, each of which alone may not offer satisfactory specificity and sensitivity. Recent technological advances in proteomics and bioinformatics offer a great opportunity for the discovery of different potential predictive markers. In this review, we described a cellular T2D model as an example with the intent of providing specific enrichment and new identification strategies, which might have the potential to improve predictive biomarker identification and to bring accuracy in disease diagnosis and classification, as well as therapeutic monitoring in the early phase of T2D.

The role of laboratory medicine in healthcare: quality requirements of immunoassays, standardisation and data management in prospective medicine.

In the last 10 years, the area of ELISA and protein-chip technology has developed and enthusiastically applied to an enormous variety of biological questions. However, the degree of stringency required in data analysis appears to have been underestimated. As a result, there are numerous published findings that are of questionable quality, requiring further confirmation and/or validation. In the course of feasibility and validation studies a number of key issues in research, development and clinical trial studies must be outlined, including those associated with laboratory design, analytical validation strategies, analytical completeness and data managements. The scope of the following review should provide assistance for defining key parameters in assay evaluation and validation in research and clinical trial projects in prospective medicine.

Economic concerns about global healthcare in lung, head and neck cancer: meeting the economic challenge of predictive, preventive and personalized medicine.

The introduction of biological science into the practice of medicine was a big transforming event for the profession, leading to different new medical models such as predictive, preventive and personalized medicine. Each of them is a rapidly emerging field that helps us to determine the risk for individuals to develop specific diseases, detect the disease's earliest onset and prevent or intervene early enough to provide maximum benefit for each patient. However, to realize this new potential, new healthcare models must be created, improved and validated. New healthcare models that are more proactive than reactive because prevention is less expensive than reactive medicine. Current knowledge about predictive, preventive and personalized medicine is already sufficient to implement this approach, but there are no effective practice models, delivery systems and appropriate reimbursement mechanisms. In the course of this review, we describe the economic components and benefits of a predictive, preventive and personalized health plan for lung as well as head and neck cancer and show how prospective care could relate to a community or group of covered individuals.

Potential involvement of MYC- and p53-related pathways in tumorigenesis in human oral squamous cell carcinoma revealed by proteomic analysis.

Oral squamous cellular carcinoma is a malignant tumor with poor prognosis. Discovery of early markers to discriminate between malignant and normal cells is of high importance in clinical diagnosis. Subcellular fractions from 10 oral squamous cell carcinoma and corresponding control samples, enriched in mitochondrial and cytosolic proteins, as well as blood from the tumor were analyzed by proteomics, two-dimensional gel electrophoresis, followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Three-hundred and fifty different gene products were identified. Twenty proteins showed deranged levels in oral squamous cell carcinoma in comparison with the control samples and are potentially involved in tumor growth and metastasis. Of these, 16 proteins were upregulated. By applying pathway analysis, we found 8 of the upregulated gene products to be linked to three main locus genes, p53, MYC, and MYCN, and could be candidate biomarkers for OSCC. The findings of this pilot study show that OSCC gene ontology combined with proteomic analysis is a powerful tool in systems biology for the elucidation of the complexity of expression profiles in cellular processes. Application of such pathway analysis has the potential to generate new insights into complex molecular mechanisms underlying disease related processes and could therefore significantly contribute to the efficient performance of the entire discovery process.