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Proteomic maps of the cancer-associated infectious agents.

The number of infectious agents associated with cancer is increasing. There is a need to develop approaches for the early detection of the infected host which might lead to tumor development. Recent advances in proteomic approaches provide that opportunity, and it is now possible to generate proteomic maps of cancer-associated infectious agents. Protein arrays, interaction maps, data archives, and biological assays are being developed to enable efficient and reliable protein identification and functional analysis. Herein, we discuss the current technologies and challenges in the field, and application of protein signatures in cancer detection and prevention.

Animals↗
PubMed2004

A technical triade for proteomic identification and characterization of cancer biomarkers.

Biomarkers are needed to elucidate the biological background and to improve the detection of cancer. Therefore, we have analyzed laser-microdissected cryostat sections from head and neck tumors and adjacent mucosa on ProteinChip arrays. Two differentially expressed proteins (P = 3.34 x 10(-5) and 4.6 x 10(-5)) were isolated by two-dimensional gel electrophoresis and identified as S100A8 (calgranulin A) and S100A9 (calgranulin B) by in-gel proteolytic digestion, peptide mapping, tandem mass spectrometry analysis, and immunodepletion assay. The relevance of these single marker proteins was evaluated by immunohistochemistry. Positive tissue areas were reanalyzed on ProteinChip arrays to confirm the identity of these proteins. As a control, a peak with low P was identified as calgizzarin (S100A11) and characterized in the same way. This technical triade of tissue microdissection, ProteinChip technology, and immunohistochemistry opens up the possibility to find, identify, and characterize tumor relevant biomarkers, which will allow the movement toward the clonal heterogeneity of malignant tumors. Taking this approach, proteins were identified that might be responsible for invasion and metastasis.

Biomarkers, Tumor↗
PubMed2006

Exploring the regulation of human neural precursor cell differentiation using arrays of signaling microenvironments.

Cells of a developing embryo integrate a complex array of local and long-range signals that act in concert with cell-intrinsic determinants to influence developmental decisions. To systematically investigate the effects of molecular microenvironments on cell fate decisions, we developed an experimental method based on parallel exposure of cells to diverse combinations of extracellular signals followed by quantitative, multi-parameter analysis of cellular responses. Primary human neural precursor cells were captured and cultured on printed microenvironment arrays composed of mixtures of extracellular matrix components, morphogens, and other signaling proteins. Quantitative single cell analysis revealed striking effects of some of these signals on the extent and direction of differentiation. We found that Wnt and Notch co-stimulation could maintain the cells in an undifferentiated-like, proliferative state, whereas bone morphogenetic protein 4 induced an 'indeterminate' differentiation phenotype characterized by simultaneous expression of glial and neuronal markers. Multi-parameter analysis of responses to conflicting signals revealed interactions more complex than previously envisaged including dominance relations that may reflect a cell-intrinsic system for robust specification of responses in complex microenvironments.

Animals↗
PubMed2006

Proteomic analysis of fast and slow muscles from normal and kyphoscoliotic mice using protein arrays, 2-DE and MS.

A proteomic strategy based upon the integrated use of SELDI-TOF/MS, 2-DE and MALDI-TOF/MS has been used to identify a panel of fast muscle protein markers: MLC1F, MLC3F, fast troponin C (STNC) and slow muscle markers: MLC1SB and MLC2v. MLC3F, MLC1F and STNC were virtually absent in the physiologically pure slow soleus muscle of kyphoscoliotic mutant mice compared to control BDmice, whereas MLC2v increased threefold. A SELDI-TOF/MS peak at 18,012 Da in spectra from strong anionic exchange protein array fractions of fast vastus muscle was confirmed as STNC by its specific depletion from crude extracts of vastus muscle using an anti-TNC mAb. SELDI-TOF/MS also identified MLC2F phosphorylation in crude muscle extracts after treatment with alkaline phosphatase. High probability protein identifications were achieved by SELDI-TOF/MS PMF based upon the resolution of large peptides formed by partial cleavage and high peptide coverage. When the pI from 2-D gels and molecular weight estimations from SELDI-TOF/MS were entered into the TagIdent algorithm, high probability protein identity predictions were obtained that were confirmed later by PMF. We confirm that SELDI-TOF/MS can be integrated with other proteomics techniques for the efficient analysis of protein expression changes and PTMs associated with physiological changes in skeletal muscle.

Animals↗
PubMed2002

Single framework recombinant antibody fragments designed for protein chip applications.

High-throughput proteomics, based on the microarray platform, requires stable, highly functional components that will yield a highly sensitive read-out of low abundance proteins. Although antibodies are the best characterized binding molecules for this purpose, only a fraction of them appear to behave satisfactorily in the chip format. Therefore, high demands need to be placed on their molecular design. In the present study, we have focused on recombinant antibody design based on a single framework for protein chip applications, aiming at defining crucial molecular probe parameters. Our results show that engineered human recombinant scFv antibody fragments that displayed appropriate biophysical properties (molecular [functional] stability in particular) can be generated, making them prime candidates for high-density antibody arrays. In fact, a superior framework that displays both multifaceted adsorption properties and very high functional stability over several months on chips (stored in a dried-out state) was identified. Taken together, designed scFv fragments based on a single molecular scaffold, readily accessible in large phage display libraries, can undoubtedly meet the requirements of probe content in antibody microarrays, particularly for global proteome analysis.

Antibodies, Monoclonal↗
PubMed2006

Human spontaneous labor without histologic chorioamnionitis is characterized by an acute inflammation gene expression signature.

OBJECTIVE: The purpose of this study was to identify which biological processes may be involved in normal labor. STUDY DESIGN: Transcriptional profiles for chorioamniotic membranes (n = 24) and blood (n = 20) were generated from patients at term with no labor (TNL) and in labor (TIL). RESULTS: Expression of 197 transcripts (P < or = .02) differentiated TIL and TNL chorioamniotic membrane samples. Gene Ontology analysis indicated that TIL samples had increased expression of multiple chemokines and transcripts associated with neutrophil and monocyte recruitment. Microarray results were verified using quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) with independent samples. Transcriptional profiles from blood RNA revealed no Gene Ontology category enrichment of discriminant probe sets. CONCLUSION: Labor induces gene expression changes consistent with localized inflammation, despite the absence of histologically detectable inflammation.

Adult↗
PubMed2005

New advances in microarrays: finding the genes causally involved in disease.

Much progress has been made in the technologies available to assess global alterations in mRNA levels in clinical research samples. Although such transcript profiling can provide a powerful research tool, the broad range of options can be bewildering for the inexperienced investigator, and more often than not the limitations and pitfalls of this approach are not fully appreciated. It is important to recognize that the major goal of transcript profiling experiments can be to identify novel therapeutic targets or delineate complex patterns of gene expression that provide a potentially pathognomonic phenotype. Microarrays are therefore potentially powerful tools to address the need for high-throughput analysis techniques, but a careful postanalysis follow-up and validation of microarray experiments will be needed soon (1). A projected decrease in cost and complexity may facilitate increased availability of this technology in the physician's office, and future applications may then allow tailoring of clinical diagnosis and targeted therapeutic strategies.

Arteriosclerosis↗
PubMed2006

Data merging for integrated microarray and proteomic analysis.

The functioning of even a simple biological system is much more complicated than the sum of its genes, proteins and metabolites. A premise of systems biology is that molecular profiling will facilitate the discovery and characterization of important disease pathways. However, as multiple levels of effector pathway regulation appear to be the norm rather than the exception, a significant challenge presented by high-throughput genomics and proteomics technologies is the extraction of the biological implications of complex data. Thus, integration of heterogeneous types of data generated from diverse global technology platforms represents the first challenge in developing the necessary foundational databases needed for predictive modelling of cell and tissue responses. Given the apparent difficulty in defining the correspondence between gene expression and protein abundance measured in several systems to date, how do we make sense of these data and design the next experiment? In this review, we highlight current approaches and challenges associated with integration and analysis of heterogeneous data sets, focusing on global analysis obtained from high-throughput technologies.

Animals↗
PubMed2003

Fabrication of a reversible protein array directly from cell lysate using a stimuli-responsive polypeptide.

We report a new method to reversibly bind proteins to a surface in a functionally active orientation directly from cell lysate by exploiting a thermodynamically reversible hydrophilic-hydrophobic lower critical solution temperature (LCST) transition exhibited by a recombinant, stimuli-responsive elastin-like polypeptide (ELP). An ELP is covalently micropatterned on a glass surface against an inert BSA background. The ELP-patterned surface is incubated with the soluble fraction of E. coli lysate containing an expressed ELP fusion protein, which is appended with the same ELP as on the surface. The LCST transition of the grafted ELP and the ELP fusion protein is simultaneously triggered by an external stimulus. The LCST transition results in capture of the ELP fusion protein from solution onto the immobilized ELP by hydrophobic interactions between the grafted ELP and the ELP fusion protein. The captured ELP fusion protein is oriented such that the fusion partner is accessible to binding of its target from solution. We also demonstrate that TRAP is reversible; the bound protein-ligand complex is released from the surface by reversing the LCST transition. The triggered control of interfacial properties provided by an immobilized stimuli-responsive polypeptide at the solid-water interface is an enabling technology that allows reversible and functional presentation of ELP fusion proteins on a surface directly from cell lysate without the necessity of intermediate purification steps and subsequent recovery of the protein-ligand complex for downstream analysis by other analytical techniques. TRAP has application in lab-on-a-chip bioanalytical devices as well as in the fabrication of peptide and protein arrays.

Cell-Free System↗
PubMed1989

Application of high performance liquid chromatography combined with diode-array detection for analysis of proteins and peptides in human cerebrospinal fluid.

Different strategies for HPLC separation, including molecular sieving, ion-exchange, and hydrophobic interaction as well as reversed phase chromatography, were used to study molecular components in human cerebrospinal fluid (CSF). The separations were followed by photodiode-array UV detection, which is a recently developed technique allowing a direct and rapid discrimination between peptides and proteins differing in their content of aromatic amino acids. By the various HPLC techniques in conjunction with diode-array detection it was possible to identify and characterize several protein and peptide components present in CSF. The procedure also allowed quantitative analysis of CSF proteins using minute amounts of the fluid.

Cerebrospinal Fluid Proteins↗
PubMed2004

[Expression profile and prognostic significance of CD24, p53 and p21 in lymphomas. A tissue microarray study of over 600 non-Hodgkin lymphomas].

BACKGROUND AND OBJECTIVE: Tissue microarrays allow the simultanous, rapid and standardized in-situ analysis of up to 1000 different tissue samples on a single glass slide. We investigated the expression profil and the prognostic significance of CD24, p53 and p21 using a Non-Hodgkin-Lymphoma tissue microarray. METHODS: Over 600 well documented Non-Hodgkin-Lymphomas, consisting of 341 diffuse large B-cell lymphomas, 86 follicular lymphomas, 81 chronic lymphatic leukemias/small lymphocytic lymphomas, 51 primary mediastinal diffuse large B-cell lymphomas, 26 mantle cell lymphomas, 8 lymphoplasmocytic lymphomas, 8 T-cell-lymphomas NOS und 2 Burkitt lymphomas were brought into array format. The expression of CD24, p53 and p21 was analysed semiquantitatively by immunohistochemistry. The immunophenotype p53+/p21- (Deltap53) was used as a surrogat for p53 gene mutations. The expression profile was compared to clinical data and the overall survival in the subgroup of diffuse large B-cell lymphomas. RESULTS: 91% of the analyzed Non-Hodgkin-lymphomas (473 of 522 cases) showed CD24 positivity. CD24 Expression was not associated with survival. Deltap53 was found in thirteen percent of all lymphomas (70 of 539 cases), the subgroup of the diffuse large B-cell lymphomas demonstrated the highest Deltap53 (21%). In a multivariate cox regression analysis, a high international prognostic index and Deltap53 were independent markers of bad survival. CONCLUSION: The TMA-technology allows also in lymphoma research the simultanous, cost-effective and standardized analysis of large patient cohorts. Deltap53 revealed as an independent negative prognostic factor in diffuse large B-cell lymphomas. It can easily be determined in daily routine practice.

Adolescent↗
PubMed2003

Molecular analysis of cancer using DNA and protein microarrays.

In conclusion, array-based technologies have emerged that contribute to profiling tissues at the genomic, transcriptomic and proteomic levels. Analytical tools are needed to mine the vast amount of data generated. Ultimately the molecular analysis of cancer at a genome and proteome scale will allow better classification of disease and tailored individualized therapy for individual patients.

Biotechnology↗
PubMed2006

On-chip protein synthesis for making microarrays.

Protein microarrays are a miniaturized format for displaying in close spatial density hundreds or thousands of purified proteins that provide a powerful platform for the high-throughput assay of protein function. The traditional method of producing them requires the high-throughput production and printing of proteins, a laborious method that raises concerns about the stability of the proteins and the shelf life of the arrays. A novel method of producing protein microarrays, called nucleic acid programmable protein array (NAPPA), overcomes these limitations by synthesizing proteins in situ. NAPPA entails spotting plasmid DNA encoding the relevant proteins, which are then simultaneously transcribed and translated by a cell-free system. The expressed proteins are captured and oriented at the site of expression by a capture reagent that targets a fusion protein on either the N- or C-terminus of the protein. Using a mammalian extract, NAPPA expresses and captures 1000-fold more protein per feature than conventional protein-printing arrays. Moreover, this approach minimizes concerns about protein stability and integrity, because proteins are produced just in time for assaying. NAPPA has already proven to be a robust tool for protein functional assays.

Animals↗