Laboratoire "Genetique Medicale et Genomique Fonctionnelle" Inserm UMR_S 910 Faculte de Medecine de la Timone 27, Bd Jean Moulin 13385 Marseille Cedex 05
Abstract: Proteomic analysis is a powerful tool to follow physiological modifications and phenotypes of mesenchymal stem cells (MSC). This approach generates informative data on expression and post-translational modifications of proteins which are of interest to assess the true potential of MSC in regenerative medicine. No matter the technologies used, proteomic analysis is always a challenge as the proteome is extremely diverse (in terms of constituents and concentrations), is changing with time, and is highly sensitive to pre-analytical conditions. In the framework of a European project (GENOSTEM http://www.genostem.org/), we have set up a multisite two dimensional gel electrophoresis (2DE) proteomic comparison of MSC. The goal is to compare cells from different origins, to follow their differentiation and to ultimately define a specific MSC proteomic signature. One important initial task is the optimization of 2DE protocols such that they are robust enough to be used in a multisite project. In this chapter, we detail these protocols which can be used not only for MSC but also for other cells in culture.
Abstract: To improve the etiological diagnosis of neurodegenerative dementias like Alzheimer's disease (AD) or frontotemporal dementia (FTD), we evaluated the value of individual and combined measurements of the following relevant cerebrospinal fluid (CSF) biomarkers: Tau, 181p-Tau, Aβ38, Aβ40, Aβ42, sAβPPα, and sAβPPβ. This study conducted in two centers included patients with FTD (n = 34), AD (n = 52), as well as a control group of persons without dementia (CTRL, n = 42). Identical clinical criteria and pre-analytical conditions were used while CSF biomarkers were measured using commercial single and multiplex quantitative immunoassays. Thorough statistical analyses, including ROC curves, logistic regressions, and decision trees, were performed. We validated in AD the specific increase of p-Tau levels and the decrease of Aβ42 levels, two biological hallmarks of this disease. Tau concentrations were highest in AD and intermediate in FTD when compared to CTRL. The most interesting results were obtained by focusing on amyloid biomarkers as we found out in FTD a significant decrease of sAβPPβ, Aβ38, and Aβ40 levels. Aβ38 in particular was the most useful biomarker to differentiate FTD subjects from the CTRL population. Combining p-Tau and Aβ38 led us to correctly classifying FTD patients with sensitivity at 85% and specificity at 82%. Significant changes in amyloid biomarkers, particularly for Aβ38, are therefore seen in FTD. This could be quite useful for diagnosis purposes and it might provide additional evidence on the interrelationship between Tau and AβPP biology which understanding is essential to progress towards optimal therapeutic and diagnostic approaches of dementia.
Abstract: Neurodegenerative diseases are often associated with misfolding and deposition of specific proteins in the nervous system. The prion protein, which is associated with transmissible spongiform encephalopathies (TSEs), is one of them. The normal function of the cellular form of the prion protein (PrP(C)) is mediated through specific signal transduction pathways and is linked to resistance to oxidative stress, neuronal outgrowth and cell survival. In TSEs, PrP(C) is converted into an abnormally folded isoform, called PrP(Sc), that may impair the normal function of the protein and/or generate toxic aggregates. To investigate these molecular events we performed a two-dimensional gel electrophoresis comparison of neuroblastoma N2a cells expressing different amounts of PrP(C) and eventually infected with the 22L prion strain. Mass spectrometry and peptide mass fingerprint analysis identified a series of proteins with modified expression. They included the chaperones Grp78/BiP, protein disulfide-isomerase A6, Grp75 and Hsp60 which had an opposite expression upon PrPC expression and PrP(Sc) production. The detection of these proteins was coherent with the idea that protein misfolding plays an important role in TSEs. Other proteins, such as calreticulin, tubulin, vimentin or the laminin receptor had their expression modified in infected cells, which was reminiscent of previous results. Altogether our data provide molecular information linking PrP expression and misfolding, which could be the basis of further therapeutic and pathophysiological research in this field.
Abstract: Cerebrospinal fluid (CSF) biomarkers are now widely used for diagnosis of Alzheimer Disease (AD) in atypical clinical forms, for differential and early diagnosis, or for stratification of patients in clinical trials. Among these biomarkers, different forms of amyloid peptides (Abeta) produced by the cleavage of a transmembrane precursor protein called APP (amyloid precursor protein) have a major role. Abeta peptides exist in different length the most common ones having 40 (Abeta40), 42 (Abeta42) or 38 (Abeta38) amino acids in length. APP processing by gamma-secretase releases also an amino-terminal secreted fragment called sAbetaPP-beta while an alternative non-amyloidogenic cleavage of APP, through an alpha-secretase, liberates an other fragment called sAbetaPP-alpha. To decipher the molecular and pathological mechanisms leading to the production and the detection of these entities is essential for the comprehension and the prevention of AD. In this report, we present the results of the multiplex measurement of CSF Abeta38, Abeta40, Abeta42, sAbetaPP-alpha and sAbetaPP-beta in 60 patients mostly with dementia eventually segregated between neurochemical dementia diagnostic (NDD) positive and negative groups. The NDD classification was based on our routine Tau, P-tau(181) and Abeta(42) cut off values. We confirmed previous findings regarding the correlation between sAbetaPP-alpha and sAbetaPP-beta, as well as the potential interest of these new biomarkers. We also studied the correlation between sAbetaPPs and Abeta peptides, as well as between Abeta peptides themselves. We observed a strong correlation between Abeta38 and sAbetaPP-beta which suggested that the production of this peptide was in direct relation with beta secretase activities. We also reported a strong correlation between Abeta38 and Abeta40, while Abeta42 was correlated to these fragments only in non pathological situations. These results enlighten the complex relationships between these molecular markers in both physiological and pathological situations. Our results are important for the further use of these analytes for AD diagnosis, as well as, to validate cell biological hypotheses of APP processing and Abeta fragment production.
Abstract: Given the current developments of therapeutic strategies in the field of neurodegenerative disorders, exact diagnosis, especially at an early stage, is becoming increasingly crucial for optimal patient care. Importantly, the new diagnosis criteria of dementia include functional imagery as well as CSF biomarkers. Proteomics investigations of these CSF biomarkers have produced quite promising results. The interest of CSF analysis resides in the anatomical and pathophysiological characteristics of this fluid. In this article, we will review current proteomics investigations of CSF which have led to the discovery and validation of biomarkers, mainly in the field of neurodegenerative disorders in Alzheimer's disease.
Abstract: Depletion of major blood proteins is one of the most promising approaches to access low abundant biomarkers using proteomics. Immunocapture columns often used for this purpose exist in different formats depending on the number of major proteins removed. In this article, we compared the relative interest of depleting either one (albumin), six (albumin, IgG, IgA, transferrin, alpha1-antitrypsin, and haptoglobin), twelve (the previous six and apo A-I and -II, orosomucoid, alpha2-macroglobulin, fibrinogen, IgM) or twenty blood proteins (the previous twelve and IgD, ceruloplasmin, apo B, complement C1q, C3, C4, plasminogen, and prealbumin). Such study raises interesting issues related to the reproducibility, practicability, specificity of the immunocapture, and to the impact of removing not only the selected molecules, but also associated peptides and proteins. Depleted sera were here analysed using different proteomic approaches, including two dimensional electrophoresis and SELDI-TOF. Altogether, our results clearly confirmed the interest of depleting major blood proteins for the proteomic detection of low abundant components. However, we observed that increasing the number of depleted proteins from twelve to twenty had a limited beneficial impact and might increase drawbacks in removing associated peptides and proteins. This conclusion is however related to the technologies that we have used, and we believe that it is necessary to adapt the immunocapture to the analytical method employed, and to the ratio between wanted and unwanted proteins removed.
Abstract: Mesenchymal stem cells (MSC) are adult multipotential progenitors which have a high potential in regenerative medicine. They can be isolated from different tissues throughout the body and their homogeneity in terms of phenotype and differentiation capacities is a real concern. To address this issue, we conducted a 2-DE gel analysis of mesenchymal stem cells isolated from bone marrow (BM), adipose tissue, synovial membrane and umbilical vein wall. We confirmed that BM and adipose tissue derived cells were very similar, which argue for their interchangeable use for cell therapy. We also compared human mesenchymal to embryonic stem cells and showed that umbilical vein wall stem cells, a neo-natal cell type, were closer to BM cells than to embryonic stem cells. Based on these proteomic data, we could propose a panel of proteins which were the basis for the definition of a mesenchymal stem cell proteomic signature.
Abstract: Understanding the virus-host interactions that lead to approximately 20% of patients with acute Hepatitis C Virus (HCV) infection to viral clearance is probably a key towards the development of more effective treatment and prevention strategies. Acute hepatitis C infection is usually asymptomatic and therefore rarely diagnosed. Nevertheless, HCV Nucleic Acid Testing carried out on all blood donations detects donors who have resolved their HCV infection after seroconversion. Here we have employed SELDI-TOF-MS technology to compare, at a proteomic level, plasma samples respectively from donors with HCV clearance, from donors with chronic HCV infection and from unexposed healthy donors (n= 15 / group). A candidate marker of about 9.4 kDa was detected as differentially expressed in the three groups. After purification we identified by nanoLC-Q-TOF-MS/MS this candidate marker as Apolipoprotein C-III (ApoC-III). The identification was confirmed by Western Blot analysis. Levels of ApoC-III were then determined in the 45 plasma samples by immunoturbidimetric assay. ApoC-III was found to be higher in donors who had resolved their HCV infection than in donors with chronic infection; results which were consistent with SELDI-TOF-MS data. ApoC-III is the first reported candidate biomarker in plasma associated with the spontaneous resolution of HCV infection.
Abstract: Stromal cells from bone marrow and adipose tissue are attractive sources of adult progenitors for cell-based therapy. However, whether those cell populations represent intrinsically different cell types is still largely under debate. The aim of this study was to systematically and quantitatively compare adipose-derived stromal cells (ADSC) and bone marrow-derived multipotent mesenchymal-stromal cells (BM-MSC). The quantitative comparison was realized using Taqman Low Density Array, 2D electrophoresis and differentiation functional assays in vitro. Furthermore, cells engineered to express TGFbeta1 were injected into the intra-articular space of mouse knee joints in order to determine whether they were able to form new differentiated tissues in vivo. Our data revealed cell specific differences at transcriptional and proteomic levels between both cell types according to their tissue origin as well as functional differences in their differentiation processes towards adipogenic, osteogenic and chondrogenic programs. Nevertheless, in vitro as well as in vivo ADSC displayed the same ability than MSC to differentiate towards chondrocytes/osteoblasts, comforting the status of both cell sources as promising regenerative cells. In summary, our observations indicate that ADSC and MSC are fundamentally different cell types and differently committed cells.
Abstract: The cerebrospinal fluid (CSF) circulates within the CNS where it plays an essential physiological role in homeostasis of neuronal cells. This biological fluid has an important protein diversity that results from both filtration of serum through the blood-brain barrier and production/secretion of neuronal peptides and proteins. Changes in CSF composition depend on blood proteome, CSF circulation alterations, as well as physiological or pathological brain status. Hence, CSF proteomic analysis gives a unique opportunity to detect and describe biomarkers in neurological affections. Although lumbar puncture is considered as invasive, post lumbar puncture events remain rare and minor. Nevertheless, CSF biological analysis is currently limited to a small number of parameters and clinical situations suggesting mainly meningitis, malignancies or dementia. Few CSF proteomic studies have been performed in comparison to those on blood. In this review, we will provide a proteomics description of the CSF, summarize the current clinical use of this fluid and describe its clinical proteomics examination.
Abstract: OBJECTIVE: Bone marrow (BM) mesenchymal stem cells (MSCs) are being considered as potential therapeutic agents in various inflammatory autoimmune diseases for their tissue-repair and anti-inflammatory tissue-protective properties. This study investigates the reserves and function, the molecular and proteomic profile and the differentiation potential of BM MSCs in patients with active rheumatoid arthritis (RA). METHODS: We evaluated the frequency of MSCs in the BM mononuclear cell fraction using a limiting dilution assay, the proliferative/clonogenic potential and the capacity of cells to differentiate towards the osteogenic/chondrogenic/adipogenic lineages using appropriate culture conditions. We also assessed the molecular and proteomic characteristics in terms of inflammatory cytokine gene and protein expression, the relative telomere length and the survival characteristics of BM MSCs. RESULTS: MSCs from patients with RA (n = 26) and age- and sex-matched healthy individuals (n = 21) were similar in frequency, differentiation potential, survival, immunophenotypic characteristics, and protein profile. Patient MSCs, however, had impaired clonogenic and proliferative potential in association with premature telomere length loss. Transcriptome analysis revealed differential expression of genes related to cell adhesion processes and cell cycle progression beyond the G1 phase. Previous treatment with methotrexate, corticosteroids, anti-cytokine and biological agents or other disease-modifying anti-inflammatory drugs did not correlate with the clonogenic and proliferative impairment of BM MSCs. CONCLUSION: In spite of some restrictions related to the impaired clonogenic and proliferative potential, our findings support the use of autologous BM MSCs in RA and may have important implications for the ongoing efforts to repair tissue injury commonly seen in the course of the disease.
Abstract: Detection of autoantibodies, which are involved in tissue injury and/or the reporters from the immune system of various pathologic events, has an important potential for diagnosis, prognosis, disease staging and treatment selection. This explains the interest for new proteomics technologies, such as the high-density protein microarray used here, that allow a high-throughput, multiplexed and sensitive detection of specific autoantibodies. So far, most of the research has been performed on blood. In this note, we focus on the cerebrospinal fluid in an attempt to address autoimmune events associated with neurological disorders. Importantly, the cerebrospinal fluid is quite different from the blood in terms of protein composition and concentration. We had therefore to adapt the available blood protocols. We present here the result of our optimization that will be useful to carry out full scale immunological studies of the cerebrospinal fluid using high-density protein microarrays.
Abstract: Radioactive compounds incorporated in tissues can have biological effects resulting from energy deposition in subcellular compartments. We addressed the genetic consequences of [(3)H] or [(14)C]thymidine incorporation into mammalian DNA. Low doses of [(3)H]thymidine in CHO cells led to enhanced sensitivity compared with [(14)C]thymidine. Compared with wild-type cells, homologous recombination (HR)-deficient cells were more sensitive to lower doses of [(3)H]thymidine but not to any dose of [(14)C]thymidine. XRCC4-defective cells, however, were sensitive to both low and high doses of [(3)H] and [(14)C]thymidine, suggesting introduction of DNA double-strand breaks, which were confirmed by gamma-H2AX focus formation. While gamma rays induced measurable HR only at toxic doses, sublethal levels of [(3)H] or [(14)C]thymidine strongly induced HR. The level of stimulation was in an inverse relationship to the emitted energies. The RAD51 gene conversion pathway was involved, because [(3)H]thymidine induced RAD51 foci, and [(3)H]thymidine-induced HR was abrogated by expression of dominant negative RAD51. In conclusion, both HR and non-homologous end-joining pathways were involved after labeled nucleotide incorporation (low doses); genetic effects were negatively correlated with the energy emitted but were positively correlated with the energy deposited in the nucleus, suggesting that low-energy beta-particle emitters, at non-toxic doses, may induce genomic instability.
Abstract: Micro-environment seems to exert an important influence on human mesenchymal stem cell (MSC) differentiation and proliferative capacity in bone marrow as well as in culture ex vivo. Oct-4, Rex-1, and TERT genes are well-known for the maintenance of pluripotentiality differentiation and the proliferative capacity of embryonic stem cells. Some previous data report expression of these embryonic factors in selected clones from bone marrow adult stem cells. Our goal was to study expression of Oct-4, Rex-1, and TERT in primary cultured human MSC according to the serum concentration. In addition, we have studied the expression of Gata-4 since this factor plays a key role in organogenesis. We hypothesized that low serum concentration with appropriate growth factors may induce an undifferentiated status with a re-expression of embryonic factors and extend differentiation capacity. Thus, using a defined culture medium, we report on the increased expression of Oct-4, Rex-1, and Gata-4 in human MSC. We have correlated this expression to an increase in differentiation efficiency towards osteogenic and adipogenic phenotypes. Our data suggest that the culture medium used permits the emergence of adult stem cells with a high differentiation capacity and expression of embryonic factors. These cells may have important implications for cell therapy.
Abstract: This review focuses on "clinical proteomics" which represents an emerging discipline in biomedical research. "Clinical proteomics" relies on the analysis of the proteome, i.e. the entire set of peptides and proteins present in a biological sample, to provide relevant data for diagnosis, prognosis or therapeutic strategies of human pathologies. This new type of approach has tremendous potential for the diagnosis of complex pathologies or for the early detection of cancers. This article reports the conclusions of a workgroup of the French Society for Clinical Biology (SFBC) 2004-2006 which evaluated the status, the impact and the future development of proteomics in the clinical field. It provides therefore a broad view going from the methods already present in the clinical laboratories (multiplex technologies...), to the tools for clinical and basis research including bioinformatics.
Abstract: BACKGROUND: Surface-Enhanced Laser Desorption/Ionization - Time Of Flight (SELDI-TOF) has been proposed as new approach for blood biomarker discovery. However, results obtained so far have been often disappointing as this technique still has difficulties to detect low-abundant plasma and serum proteins. RESULTS: We used a serum depletion scheme using chicken antibodies against various abundant proteins to realized a pre-fractionation of serum prior to SELDI-TOF profiling. Depletion of major serum proteins by immunocapture was confirmed by 1D and 2D gel electrophoresis. SELDI-TOF analysis of bound and unbound (depleted) serum fractions revealed that this approach allows the detection of new low abundant protein peaks with satisfactory reproducibility. CONCLUSION: The combination of immunocapture and SELDI-TOF analysis opens new avenues into proteomic profiling for the discovery of blood biomarkers.
Abstract: Tissue and functional regeneration takes place in the body at various stages throughout life. However, bone, cartilage, tendons, blood vessels and cardiac muscle have a limited capacity for self repair and, after injury or disease, the regenerative ability of these adult tissues is often insufficient and leads to nonfunctional scar tissue. In this context, mesenchymal stem cells, which are adult multipotential progenitors of mesoderm cells (osteoblasts, chondrocytes, adipocytes and stroma cells), represent a major hope for tissue-engineered replacement and regenerative medicine. Furthermore, the autologous use of these cells prevents immunological responses against new tissues and the risks of disease transmission from donors, which are both common problems of organ transplantation. While the existence of mesenchymal stem cells is undisputed, many questions remain regarding their self-renewal and capacity to differentiate, their homogenous nature as a cell population throughout the body and their true potential in regenerative medicine. In this article, the proteomics studies carried out to characterize mesenchymal stem cells and to help understand their physiology are reviewed.
Abstract: This report completes a previous study on the growth and metabolism of fetal bovine epiphyseal chondrocytes cultured, within native or cross-linked collagen sponges carried out without the addition of fresh ascorbate. At low initial cell density (2.3 x 10(6)cells/cm(3)) cell proliferation and a low matrix deposition were observed, whereas at high initial cell density (2.3 x 10(7)cells/cm(3)) there was an absence of cell proliferation, but the deposition of a cartilage-like matrix was measured. In both cases, only traces of type I collagen (marker of chondrocyte dedifferentiation) were detected. In this report, we observed, after 1 month in culture with ascorbate, in both type of scaffolds and initial cell densities, an increase in cell proliferation (2-fold) and in expression of genes encoding for collagen types I, II, X and MMP-2 and -13, but no change in the level of matrix deposition (collagen and GAG). With regard to the proteins present, the main differences with or without ascorbate concerned the increase of neosynthesised type I collagen (up to 35% of the total collagen deposited in the sponge) and of the MMP-2 active form. In conclusion, these results show that ascorbate is an important factor to consider when preparing cartilage constructs for its action on chondrocyte phenotype modulation and proliferation.
Notes: Ronziere, Marie Claire xD;Roche, Stephane xD;Gouttenoire, Jerome xD;Demarteau, Olivier xD;Herbage, Daniel xD;Freyria, Anne Marie xD;Research Support, Non-U.S. Gov't xD;England xD;Biomaterials xD;S0142961202004180 xD;Biomaterials. 2003 Feb;24(5):851-61.
Abstract: Collagen-based biomaterials in the form of sponges (bovine type I collagen, both native and cross-linked by treatment with diphenylphosphorylazide, noted control and DPPA sponges respectively) were tested as three-dimensional scaffolds to support chondrocyte proliferation with maintenance of the phenotype in order to form neocartilage. Control and DPPA sponges were initially seeded with 10(6) or 10(7) foetal bovine epiphyseal chondrocytes and maintained for 4 weeks in culture under static conditions in RPMI/NCTC medium with 10% FCS and without addition of fresh ascorbic acid. Both supports were always present during the study and a partial decrease in size and weight was detected only with control sponges, both seeded and unseeded. Cell proliferation was only noted in the 10(6) cells-seeded sponges (4-fold increase after 4 weeks of culture). Specific cartilage collagens (types II and XI) were deposited in the matrix throughout the culture and traces of type I collagen were noticed only in the culture medium after 2-3 weeks and 4 weeks in the case of 10(6) and 10(7) cells-seeded sponges, respectively. Glycosaminoglycans accumulated in the matrix, up to 1.8 and 9.8% of total dry weight after one month with both seeding conditions, which was much lower than in the natural tissue. In the 10(7) cells-seeded sponges, mineral deposition, observed with unseeded sponges, was significantly decreased (2- to 3-fold). These in vitro results indicate that both collagen matrices can support the development of tissue engineered cartilage.
Abstract: Phenotypic expression of chondrocytes can be modulated in vitro by changing the culture technique and by agents such vitamins and growth factors. We studied the effects of ascorbic acid, retinoic acid (0.5 and 10 microM), and dihydrocytochalasin B (3, 10, 20 microM DHCB), separately or in combination (ascorbic acid + retinoic acid or ascorbic acid + DHCB), on the induction of maturation of fetal bovine epiphyseal chondrocytes grown for up to 4 weeks at high density in medium containing 10% fetal calf serum and the various agents. In the absence of any agent or with retinoic acid or DHCB alone, the metabolic activity of the cells remained very low after day 6, with no induction of type I or X collagen synthesis nor increase in alkaline phosphatase activity. Chondrocytes treated with fresh ascorbic acid showed active protein synthesis associated with expression of types I and X after 6 and 13 days, respectively. This maturation was not accompanied by obvious hypertrophy of the cells or high alkaline phosphatase activity. Addition of retinoic acid to the ascorbic acid-treated cultures decreased the level of type II collagen synthesis and delayed the induction of types I and X collagen, which were present only after 30 days. A striking increase in alkaline phosphatase activity (15-20-fold) was observed in the presence of both ascorbic acid and the highest dose of retinoic acid (10 microM). DHCB was also a potent inhibitor of the maturation induced by treatment with ascorbic acid, as the chondrocytes maintained their rounded shape and synthesized type II collagen without induction of type I or X collagen. The pattern of protein secretion was compared under all culture conditions by two-dimensional gel electrophoresis. The different regulations of chondrocyte differentiation by ascorbic acid, retinoic acid, and DHCB were confirmed by the important qualitative and quantitative changes in the pattern of secreted proteins observed by two-dimensional gel electrophoresis along the study.
Abstract: Proteomic analysis of mesenchymal stem cells (MSC) is an useful way to identify physiological modification of cell behaviour and phenotypes through analysis of protein expressions and post-translational modifications. The data generated can be use to categorize MSC and to assess their true potential in regenerative medicine. No matter the technologies used, proteomic analysis is always a challenge as the proteome is extremely diverse (in terms of constituents and concentrations), changing with time, and sensitive to pre-analytical conditions. In the framework of an European project (GENOSTEM http://www.genostem.org/), we have set up a multisite 2DE proteomic comparison of MSC. The goal was to compare MSC from different origins, follow their differenciation and finally define a proteomic signature specific for these cells. One initial task was to define optimized 2DE protocols, robust enough to be used in a multicentric project. In this chapter we detail these protocols which can be used not only for MSC but also for other cells in culture.
Abstract: Introduction: Human serum and plasma have an important clinical value for identification and detection of biomarkers. However, the analysis of these biological fluids is analytically challenging due to the high dynamic concentration range (over 10 orders of magnitude) of constituent protein/peptide species. Profiling technologies such as Surface-Enhanced Laser Desorption/Ionization (SELDI-TOF, Ciphergen) and ClinProt chromatographic microbeads (Bruker) permit to focus on a particular sub-proteome relying on mass spectrometry to detect proteins and peptides initially selected by binding to various chromatographic matrices (anionic, cationic, IMAC, hydrophobic). In this work, we compared with proprietary algorithm serum profiles obtained using SELDI-TOF and Clinprot technologies. xD; xD;Methods: The same samples were analyzed on comparable weak cation exchange chromatographic surface (CM10, SELDI-TOF) or microbeads (WCX, Clinprot). 10 µl of sera from 12 different mice were analyzed on both systems following classical published protocols and using a CHCA matrix. Data acquisition was performed on the PBSIIc mass spectrometer for SELDI-TOF and on an Ultraflex I mass spectrometer for Clinprot. Raw spectrum data were exported to the statistical software R and analyzed in parallel. Peak detection was based on sign changes of the derivated spectra and was followed by peak alignment using hierarchical clustering of all the detected peaks. xD; xD;Results: An equivalent number of peaks (80 in average) were detected from the SELDI-TOF and the Clinprot profiles. 25 m/z peaks were common to both technologies with an intensity correlation of 0.84 ± 0.1 (0.66 to 0.96). Peak distribution in regards to the m/z value was not homogeneous. In fact 83% of the peaks for the SELDI-TOF and 96% for Clinprot were detected in 1,500 to 5,000 m/z range. Above 5,000 m/z, SELDI-TOF spectra show 3.6 ± 1.3 time more peaks than those of Clinprot. On the other hand, the background signal variability was more homogeneous in Clinprot than in SELDI-TOF spectra (noise standard deviation respectively of 0.001 and 0.006). This is likely related to differences between the mass spectrometer tested (i.e. PBSIIc vs Ultraflex I) which explain also why we observed a lower peak resolution with SELDI-TOF than with Clinprot in this series of experiments. xD; xD;Conclusion: SELDI-TOF and Clinprot technologies could achieve a comparable proteomic profiling from unfractionated serum which could then be used for detection of potential blood biomarkers. Interestingly, although some peaks appeared to be present in both profiles using the two technologies, many differences still exist. It remains to be determined the exact origin of these differences and their implication for the use of one approach or the other.
Abstract: Human serum and plasma have an important clinical value for identification and detection of biomarkers. However, the analysis of these biological fluids is analytically challenging due to the high dynamic concentration range (over 10 orders of magnitude) of constituent protein/peptide species. In addition, the few most abundant blood proteins constitute 95% of the bulk mass of proteins but they represent less than 0.1% of the total number of proteins. These high abundant proteins mask or interfere with the detection of the other low amount protein components. Surface-Enhanced Laser Desorption/Ionization (SELDI-TOF) focuses on a particular sub-proteome that relies on mass spectrometry to detect proteins and peptides initially selected by binding to various chromatographic surfaces (anionic, cationic, IMAC, hydrophobic). However, additional pre-fractionation methods of blood (for review see Issaq et al, 2002) could still present some interest with this technique. We used the IgY12 microbeads (ProteomLab IgY12, Beckman) on serum samples as recommended by the manufacturer. xD;We controlled and quantified the outcome of the capture by electrophoresis on agarose (Hydragel, Sebia) and 2D Electrophoresis. We demonstrated that the unbound protein profile was dramatically modified when compared those of the initial and the bound fraction, which were alike. xD;Initial, bound and unbound serum fractions were then submitted to SELDI-TOF CM10 ProteinChips analysis (Ciphergen, Fremont, CA). The unbound fraction revealed many new peaks. We repeated several time the analysis and obtained satisfactory reproducibility with Pearson factor above 0.9. Hierarchical Cluster analysis of the SELDI-TOF data revealed the presence of two main clusters, one being composed by serum and bound protein profiles and the second by unbound profiles. This clearly illustrates the fact that some new protein peaks were retained by the column, while many were detected only in the unbound fraction. To illustrate the potency of this approach, we spiked serum with 0.1 ng/µl of recombined IL-8 and repeated the SELDI-TOF analysis. This IL-8 was hardly detectable when the whole serum was analyzed, while it was readily identify in the unbound fraction. xD;In conclusion, we showed that an approach combining immunocapture of major serum proteins followed by SELDI-TOF is reproducible, versatile, can be applied to a large number of samples and we believe presents a major interest for blood proteome analysis, profiling and biomarker discovery.
Abstract: Introduction: Human serum and plasma have an important clinical value for identification and detection of biomarkers. However, the analysis of these biological fluids is analytically challenging due to the high dynamic concentration range (over 10 orders of magnitude) of constituent protein/peptide species. Profiling technologies such as Surface-Enhanced Laser Desorption/Ionization (SELDI-TOF, Ciphergen) and ClinProt chromatographic microbeads (Bruker) permit to focus on a particular sub-proteome relying on mass spectrometry to detect proteins and peptides initially selected by binding to various chromatographic matrices (anionic, cationic, IMAC, hydrophobic). In this work, we compared with proprietary algorithm serum profiles obtained using SELDI-TOF and Clinprot technologies. xD; xD;Methods: The same samples were analyzed on comparable weak cation exchange chromatographic surface (CM10, SELDI-TOF) or microbeads (WCX, Clinprot). 10 µl of sera from 12 different mice were analyzed on both systems following classical published protocols and using a CHCA matrix. Data acquisition was performed on the PBSIIc mass spectrometer for SELDI-TOF and on an Ultraflex I mass spectrometer for Clinprot. Raw spectrum data were exported to the statistical software R and analyzed in parallel. Peak detection was based on sign changes of the derivated spectra and was followed by peak alignment using hierarchical clustering of all the detected peaks. xD; xD;Results: An equivalent number of peaks (80 in average) were detected from the SELDI-TOF and the Clinprot profiles. 25 m/z peaks were common to both technologies with an intensity correlation of 0.84 ± 0.1 (0.66 to 0.96). Peak distribution in regards to the m/z value was not homogeneous. In fact 83% of the peaks for the SELDI-TOF and 96% for Clinprot were detected in 1,500 to 5,000 m/z range. Above 5,000 m/z, SELDI-TOF spectra show 3.6 ± 1.3 time more peaks than those of Clinprot. On the other hand, the background signal variability was more homogeneous in Clinprot than in SELDI-TOF spectra (noise standard deviation respectively of 0.001 and 0.006). This is likely related to differences between the mass spectrometer tested (i.e. PBSIIc vs Ultraflex I) which explain also why we observed a lower peak resolution with SELDI-TOF than with Clinprot in this series of experiments. xD; xD;Conclusion: SELDI-TOF and Clinprot technologies could achieve a comparable proteomic profiling from unfractionated serum which could then be used for detection of potential blood biomarkers. Interestingly, although some peaks appeared to be present in both profiles using the two technologies, many differences still exist. It remains to be determined the exact origin of these differences and their implication for the use of one approach or the other.
Abstract: Detection of autoantibodies, which are involved in tissue injury and/or the reporters from the immune system of various pathologic events, has an important potential for diagnosis, prognosis, disease staging and treatment selection. This explains the interest for new proteomics technologies, such as the high-density protein microarray used here, that allow a high-throughput, multiplexed and sensitive detection of specific autoantibodies. So far, most of the research has been performed on blood. In this note, we focus on the cerebrospinal fluid in an attempt to address autoimmune events associated with neurological disorders. Importantly, the cerebrospinal fluid is quite different from the blood in terms of protein composition and concentration. We had therefore to adapt the available blood protocols. We present here the result of our optimization that will be useful to carry out full scale immunological studies of the cerebrospinal fluid using high-density protein microarrays.
