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Microarrays of tagged combinatorial triazine libraries in the discovery of small-molecule ligands of human IgG.

A novel and highly diverse tagged triazine library incorporating a triethylene glycol-based linker was synthesized using an orthogonal combinatorial approach on the solid phase and covalently immobilized on a glass substrate as a small molecule microarray (SMM). The SMM was screened with a fluorophore-conjugated human IgG, and 4 novel binders from a library of 2688 compounds were identified from the fully spatially addressable array without the need for compound decoding. Using surface plasmon resonance (SPR) analysis, binding seen on the array was confirmed, and a binding constant as low as Kd = 2.02 x10(-6) M was measured.

Combinatorial Chemistry Techniques↗
PubMed2005

Screening microarrays of novel monoclonal antibodies for binding to T-, B- and myeloid leukaemia cells.

We have developed a microarray (DotScan) that enables rapid immunophenotyping and classification of leukaemias and lymphomas by measuring the capture of cells by immobilized dots of 82 CD antibodies [Belov, L., de la Vega, O., dos Remedios, C.G., Mulligan, S.P., 2001. Immunophenotyping of leukemia using a cluster of differentiation antibody microarray. Cancer Res. 61, 4483; Belov, L., Huang, P., Barber, N., Mulligan, S.P., Christopherson, R.I., 2003. Identification of repertoires of surface antigens on leukemias using an antibody microarray. Proteomics 3, 2147]. The DotScan technology has been used to investigate the properties of 498 new antibodies submitted to the HLDA8 Workshop. These antibodies have been applied as 10 nl dots to a film of nitrocellulose on a microscope slide to make an HLDA8 microarray. After blocking the remaining nitrocellulose surface, individual arrays were incubated with each of 7 cell types from a human leukaemia cell panel consisting of three cell lines, CCRF-CEM (a T-cell acute lymphocytic leukaemia), MEC-1 (derived from B-cell chronic lymphocytic leukaemia) and HL-60 (a promyelocytic leukaemia), and four leukaemias from patients: a T-cell prolymphocytic leukaemia, a B-cell chronic lymphocytic leukaemia, and two acute myeloid leukaemias. Leukaemia cells were captured by those immobilized antibodies for which they expressed the corresponding surface molecule. Unbound cells were gently washed off, bound cells were fixed to the arrays and dot patterns were recorded using a DotScan array reader and quantified using DotScan data analysis software. The data obtained show the unique expression profiles of the 7 cell types in the leukaemia cell panel obtained with the DotScan microarray, and the differential capture patterns for these 7 cell types screened against the 498 antibodies in the HLDA8 microarray constructed for this study.

Antibodies, Monoclonal↗
PubMed2006

Multiplexed and microparticle-based analyses: quantitative tools for the large-scale analysis of biological systems.

While the term flow cytometry refers to the measurement of cells, the approach of making sensitive multiparameter optical measurements in a flowing sample stream is a very general analytical approach. The past few years have seen an explosion in the application of flow cytometry technology for molecular analysis and measurements using microparticles as solid supports. While microsphere-based molecular analyses using flow cytometry date back three decades, the need for highly parallel quantitative molecular measurements that has arisen from various genomic and proteomic advances has driven the development in particle encoding technology to enable highly multiplexed assays. Multiplexed particle-based immunoassays are now common place, and new assays to study genes, protein function, and molecular assembly. Numerous efforts are underway to extend the multiplexing capabilities of microparticle-based assays through new approaches to particle encoding and analyte reporting. The impact of these developments will be seen in the basic research and clinical laboratories, as well as in drug development.

Coloring Agents↗
PubMed2005

Parallel analysis of multiple surface markers expressed on rat neural stem cells using antibody microarrays.

Neural stem cells are the attractive cell source for functional regeneration of damaged central nervous tissues by means of cell transplantation or in situ induction of differentiated neural cells. Such stem cell therapies require the prospective identification and isolation of neural stem cells. However they are difficult due to limited information on surface markers. This study aimed at developing an antibody microarray that permits parallel analysis of multiple surface antigens expressed on neural stem cells present in a neurosphere-forming cell population. A microarray was prepared by micro-spotting antibodies directed to surface antigens and ligands for membrane-associated receptors onto the patterned monolayer of alkanethiols self-assembled on a gold-evaporated glass plate. Neurosphere-forming cells were subjected to a cell-binding assay on the microarray followed by immunofluorescent staining of nestin, an intracellular marker of neural stem cells. It was demonstrated that such a cell based assay facilitated to examine the specificity of surface antigens for nestin-positive neural stem cells. Furthermore, the microarray could also be used to assess proliferation capability of cells bound to individual spots. These results suggest that the microarray-based strategy will provide a useful tool for the parallel analysis of surface markers expressed on a specific cell type in a heterogeneous population.

Animals↗
PubMed2006

Proteomic changes in hearts of kyphoscoliosis (ky) mutant mice in the absence of structural pathology: implication for the analysis of early human heart disease.

Complex molecular changes associated with early stage human heart disease are poorly understood and prevent the development of effective treatments of human cardiac disease. Relatively minor structural changes in early disease may accompany some conditions such as arrhythmias. Our objective was to determine if significant proteomic changes occur in heart tissues in the absence of structural pathology. We used a proteomic "pipeline" based on Ciphergen SELDI-TOF/MS, gel electrophoresis and MALDI-TOF/MS. The kyphoscoliosis (ky) mouse carries a mutation in a putative transglutaminase causing a primary skeletal muscle disease. The ky protein is expressed usually in skeletal and cardiac muscle but its absence from the ky heart causes no structural pathology making it a good model of "occult" heart disease. We discovered 20 statistically validated biomarkers discriminating ky from normal hearts, one cardiac troponin-I was reduced by 40% in ky hearts. A 17% deficit was confirmed subsequently by Western blot. Thus, the proteome of ky hearts was abnormal, giving support to our contention that this SELDI-based analytical approach is capable of making a significant contribution to the analysis of complex proteomic changes in early stage human heart disease.

Animals↗
PubMed2005

Microarray analysis on CYPs expression in pregnant rats after treatment with pregnenolone-16alpha-carbonitrile and phenobarbital.

We previously reported the protein expression profiles of nine cytochrome P450 isozymes (CYPs) in pregnant rat's liver, fetal liver, and placenta after treatment with pregnenolone-16alpha-carbonitrile (PCN), dexamethasone (DEX), or phenobarbital (PB). In this study, the gene expression of 40 CYPs and 2 orphan nuclear receptors for CYP inducers, that is, Nr1i2 (CYP3A subfamily inducible by PCN) and Nr1i3 (CYP2B subfamily inducible by PB), in pregnant rat's liver, fetal liver, and placenta was investigated at one time. Fischer 344 (F344) pregnant rats were daily treated intraperitoneally with 50 mg/kg of PCN or 80 mg/kg of PB from 13 to 16 days of gestation (DG). They were sacrificed on 17 DG, and microarray analysis using Affymetrix Rat Expression Array 230A was performed. Ten genes expression significantly increased in dam's liver in PCN group, and seven genes expression in PB group. On the other hand, four genes expression increased in fetal liver in PCN group, and three genes expression increased in PB group. Being common to dam's and fetal livers, the gene expression of Cyp3A1 (CYP3A subfamily) and cytochrome P-450e (CYP2B subfamily) increased in both PCN and PB groups. In placenta, the expression of Cyp3A1 gene was significantly induced in PB group, and it also showed a tendency to increase in PCN group. The expression of Nr1i2 gene was significantly elevated only in dam's liver of PCN group, while the expression of Nr1i3 gene showed no changes in all groups. The results of the present study of 40 CYPs gene expression mostly corresponded to our previous reports on 9 CYPs protein expression.

Animals↗
PubMed2005

Capture of a monoclonal antibody and prediction of separation conditions using a synthetic multimodal ligand attached on chips and beads.

A synthetic ligand called 2-mercapto-5-benzimidazolesulfonic acid has been successfully used for the specific chromatographic capture of antibodies from a cell culture supernatant. Adsorption occurred at physiological ionic strength and pH range between 5.0 and 6.0, with some binding capacity variations within this pH range: antibody uptake increased when the pH decreased. With very dilute feedstocks, as was the case with the cell culture supernatant under investigation, it was found that the pH had to be slightly lowered to get a good antibody sorption capacity. To optimize separation conditions, a preliminary study was made using ProteinChip Arrays that displayed the same chemical functionalities as the resin. Arrays were analyzed using SELDI-MS. By this mean, it was possible to cross-over simultaneously different pH conditions at the adsorption and the desorption steps. Best conditions were implemented for preparative separation using regular lab-scale columns. At pH 5.2, antibody adsorption was not complete, while at pH 5.0 the antibody was entirely captured. pH 9 was selected at elution, rather than pH 8.0 or 10.0, and resulted in a complete desorption of antibodies from the column. Benefits of the prediction of separation conditions of antibodies on MBI beads using SELDI-MS were a significant reduction in analysis time and in sample volume. This was possible because the separation of IgG on the chip surface did mimic very well the separation on beads.

Adsorption↗
PubMed2005

Analysis of differential protein expression in Acidithiobacillus ferrooxidans grown under different energy resources respectively using SELDI-ProteinChip technologies.

Surface-enhanced laser desorption/ionization (SELDI)-time of flight is an affinity-based mass spectrometric method in which proteins of interest are selectively absorbed to a chemically modified surface on a chip, which allows proteomic analysis with limited material requirements. This characteristic makes it a valuable technique for microbiologists handling problematic samples, such as low cell number cultures. In this study, we explored differential-expressed proteome of Acidithiobacillus ferrooxidans cultivated with Fe(2+) and elemental sulfur separately by adopting the protein biochip SELDI approach. The cell lysates of A. ferrooxidans were applied onto Ciphergen ProteinChip WCX2, SAX2 and IMAC-Cu arrays. Proteins bound to the chips were analyzed on a ProteinChip Reader Model PBS II. A summary of the molecular masses of the differentially regulated proteins found on WCX2, IMAC-Cu and SAX2 was obtained and 28 differentially expressed proteins were found on the molecular weight range of 5.0 to 25 kDa.

Acidithiobacillus↗
PubMed2006

Serum proteomic patterns for gastric lesions as revealed by SELDI mass spectrometry.

SELDI mass spectrometry was used to investigate protein expression in sera of patients with gastric cancer and gastritis compared to normal volunteers. Differences in peak morphology and intensity were observed in regions of 5910 Da, 5084 Da, 6640 Da and 8691 Da. Patients with gastric cancer exhibited an up-regulation of the 5910 Da peak and a down-regulation of the 8691 Da peak compared to the healthy volunteers; there was also some bi-partitioning and tri-partitioning at the 5084 Da peak. When comparing the sera of these cancer patients with those of gastritis, the former had an up-regulation of the 5910 Da peak and a down-regulation of the 6640 Da peak. This is the first report showing that SELDI sera analysis may be useful in the screening of gastric lesions.

Adenocarcinoma↗
PubMed2006

New analytical tools for studying autoimmune diseases.

Protein microarrays with immobilised proteins on their surface are new analytical tools to overcome the current limits with respect to sample volume and throughput. They have a great potential as well with respect to multiplexing of complex samples, as a research tool and in diagnostics. Based on recent advances in this technology, new applications for protein microarrays in studying autoimmune diseases were described. Required tools for bioinformatical analysis of protein microarrays concerning normalisation, clustering and classification methods are discussed. The huge potential of this technology as well as future requirements such as protein microarray based diagnostics are presented.

Animals↗
PubMed2006

Component-resolved diagnostics in food allergy.

PURPOSE OF REVIEW: The purpose of this paper is to review and discuss recent studies on component-resolved diagnostics in food allergy, involving panels of pure allergen molecules or arrays of peptides derived from allergen sequences, and to summarize the reporting of new food allergens during the past 2 years. RECENT FINDINGS: Several component-resolved diagnostic studies in food allergy suggest that the use of panels of allergen molecules may allow refined clinical information to be obtained on the likelihood or severity of an allergic food reaction and regarding diagnostic specificity. Further, in some studies the use of pure allergen molecules has led to a clearly higher sensitivity of the immunoglobulin E immunoassay compared with conventional allergen extracts. SUMMARY: While common diagnostic methods in allergy assess the presence or absence of allergen-specific sensitization, to date, no in-vitro or in-vivo test exists which exhibits full correlation with clinical food allergy. A multitude of recently reported findings and observations indicate that molecular analysis of allergen sensitization pattern may serve to enhance the clinical utility of immunoglobulin E antibody-based allergy diagnostics. Pure natural and recombinant allergen molecules as well as panels of synthetic peptides have been used for this purpose.

Allergens↗
PubMed2004

The development and application of methods for activity-based protein profiling.

Recent advances in genomic and proteomic technologies have begun to address the challenge of assigning molecular and cellular functions to the numerous protein products encoded by prokaryotic and eukaryotic genomes. In particular, chemical strategies for proteome analysis have emerged that enable profiling of protein activity on a global scale. Herein, we highlight these chemical proteomic methods and their application to the discovery and characterization of disease-related enzyme activities.

Animals↗
PubMed2002

Global proteome analysis of a human gastric carcinoma.

An approach that combines analysis of global protein digests (GPDs) of various subcellular fractions with a novel chromatographic-based method to map protein expression profiles is described. The KATO III gastric carcinoma cell line was fractionated into membrane and cytosol fractions. Each subcellular fraction was digested with trypsin to yield complex mixtures of global protein tags (GPTs). These mixtures were fractionated by two dimensions of chromatography, and GPTs were sequenced by microcapillary liquid chromatography-tandem mass spectrometry (LC-MS/MS), using two further complementary dimensions of chromatography. Additionally, a novel method of protein expression profiling was used to map the KATO III human gastric carcinoma cell line. This method uses the cells' natural proteolytic processes to derive in vivo peptide tags that represent proteins of every functional class and from all subcellular compartments. In one example, expressed protein tags (EPTs) are naturally displayed on the surface of cells by multiligand receptors. Isolation and sequence identification of EPTs is an efficient approach for protein profiling that is complementary to GPT analysis. The EPT approach also provides a further unique subcellular fraction of the biological starting material. Isolation of the multiligand receptors was by immunoaffinity chromatography (IAC). In the current study, five individual peptide maps (two EPTs and three GPTs) of the KATO III cell line were fractionated by multimodal chromatography, and sequenced by on-line multimodal microcapillary LC-MS/MS. This analysis led to the identification of 4291 individual peptide sequences, which defined 1966 unique proteins expressed by this human carcinoma cell line.

Chromatography, Affinity↗
PubMed2005

Proteohistography--direct analysis of tissue with high sensitivity and high spatial resolution using ProteinChip technology.

On the proteomic level, all tissues, tissue constituents, or even single cells are heterogeneous, but the biological relevance of this cannot be adequately investigated with any currently available technique. The analysis of proteins of small tissue areas by any proteomic approach is limited by the number of required cells. Increasing the number of cells only serves to lower the spatial resolution of expressed proteins. To enhance sensitivity and spatial resolution we developed Proteohistography. Laser microdissection was used to mark special areas of interest on tissue sections attached to glass slides. These areas were positioned under microscopic control directly on an affinity chromatographic ProteinChip Array so that cells were lysed and their released proteins bound on a spatially defined point. The ProteinChip System, surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS), allows the laser to be steered to up to 215 distinct positions across the surface of the spot, enabling a high spatial resolution of measured protein profiles for the analyzed tissue area. Protein profiles of the single positions were visually plotted over the used tissue section to visualize distribution proteohistologically. Results show that the spatial distribution of detectable proteins could be used as a Proteohistogram for a given tissue area. Consequently, this procedure can provide additional information to both a matrix-assisted laser desorption/ionization (MALDI)-based approach and immunohistochemistry, as it is more sensitive, highly quantitative, and no specific antibody is needed. Hence, proteomic heterogeneity can be visualized even if proteins are not known or identified.

Carcinoma, Hepatocellular↗
PubMed2006

Proteomic-based prediction of clinical behavior in adult acute lymphoblastic leukemia.

BACKGROUND: Response in adult acute lymphoblastic leukemia (ALL) can be achieved in a majority of patients. However, unlike pediatric ALL, recurrence is common in adult ALL, and the ability to predict at an early stage which patients are most likely to experience recurrence may help in devising new therapeutic approaches to prevent recurrence. METHODS: Peripheral blood plasma from 57 patients with confirmed ALL was obtained before induction therapy for proteomic analysis. Follow-up continued for a median period of 71 weeks. For each plasma sample, 4 fractions eluted from a strong anion column were applied to 3 different ProteinChip array surfaces, and 12 surface-enhanced laser desorption/ionization (SELDI) spectra were generated. Peaks that correlated with recurrence were identified and decision trees were constructed and evaluated, using only 2 peaks per predictive tree. RESULTS: The best decision trees provided strong positive prediction of recurrence, with correct predictions 84% to 92% of the time, whereas negative prediction of patients who did not experience recurrence was less robust, with 62% to 74% accuracy. Prediction of recurrence was independent of cytogenetics, bone marrow blast count, lactate dehydrogenase, beta-2-microglobulin, or surface markers. Positive prediction of L3 morphological classification was achieved in 80% of test cases. CONCLUSIONS: Peripheral blood plasma is adequate to predict clinical behavior in ALL patients irrespective of the percentage of bone marrow blasts. Proteomic analysis of plasma offers a useful approach for profiling patients with ALL.

Adolescent↗
PubMed2006

Host response to the attenuated poxvirus vector NYVAC: upregulation of apoptotic genes and NF-kappaB-responsive genes in infected HeLa cells.

NYVAC has been engineered as a safe, attenuated vaccinia virus (VV) vector for use in vaccination against a broad spectrum of pathogens and tumors. Due to the interest in NYVAC-based vectors as vaccines and current phase I/II clinical trials with this vector, there is a need to analyze the human host response to NYVAC infection. Using high-density cDNA microarrays, we found 368 differentially regulated genes after NYVAC infection of HeLa cells. Clustering of the regulated genes identified six discrete gene clusters with altered expression patterns. Clusters 1 to 3 represented 47.5% of the regulated genes, with three patterns of gene activation kinetics, whereas clusters 4 to 6 showed distinct repression kinetics. Quantitative real-time reverse transcription-PCR analysis of selected genes validated the array data. Upregulated transcripts correlated with genes implicated in immune responses, including those encoding interleukin-1 receptor 2 (IL-1R2), IL-6, ISG-15, CD-80, and TNFSF7. NYVAC upregulated several intermediates of apoptotic cascades, including caspase-9, correlating with its ability to induce apoptosis. NYVAC infection also stimulated the expression of NF-kappaB1 and NF-kappaB2 as well as that of NF-kappaB target genes. Expression of the VV host range K1L gene during NYVAC infection prevented NF-kappaB activation, but not the induction of apoptosis. This study is the first overall analysis of the transcriptional response of human cells to NYVAC infection and provides a framework for future functional studies to evaluate this vector and its derivatives as human vaccines.

Activating Transcription Factors↗
PubMed2005

Differential protein expression between esophageal squamous cell carcinoma and dysplasia, and prognostic significance of protein markers.

The aim of the current study was to evaluate the protein expression involved in the progression from dysplasia to invasive esophageal squamous cell carcinomas and to analyze the prognostic value of markers. Immunohistochemistry was performed for cell cycle regulators [p53, p21, p27, p16, cyclin D1, Rb], apoptosis-related proteins [Fas, Fas-L, FADD, TRAIL, DR4, DR5, caspase-8, caspase-3, bcl-2, Bax], tumor suppressor proteins [beta-catenin, E-cadherin, FHIT, Smad 4, VHL, PTEN, KAI-1], and oncoproteins [c-myc, COX-2, EGFR]. Caspase-3, TRAIL, Fas-L, Fas, Smad 4, VHL, E-cadherin, and EGFR revealed significant differences between dysplasia and their corresponding invasive cancer portion in 25 cases. In a total of 118 cases of invasive cancer, proteins with frequent (> or = 60% of the cases) alterations were p53 (overexpression in 64% of SCCs), p27 (loss in 91%), p16 (loss in 81%), and FHIT (loss in 75%). Early clinical stage and bcl-2 immunopositivity were related to the survival rate of patients. In conclusion, caspase-3, TRAIL, Fas-L, Fas, Smad 4, VHL, E-cadherin, and EGFR may be involved in the progression from dysplasia to invasive esophageal SCCs. Clinical stage and bcl-2 are independent prognostic factors throughout the multivariate analysis.

Adult↗