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Purpose: Proteomics technologies are well suited for harnessing the immune response to tumor antigens for diagnostic applications as in the case of breast cancer. We previously reported a substantial impact of hormone therapy (HT) on the proteome. Here, we investigated the effect of HT on the immune response toward breast tumor antigens., Experimental Design: Plasmas collected 0-10 months prior to diagnosis of ER+ breast cancer from 190 postmenopausal women and 190 controls that participated in the Women's Health Initiative Observational Study were analyzed for the effect of HT on IgG reactivity against arrayed proteins from MCF-7 or SKBR3 breast cancer cell line lysates following extensive fractionation., Results: HT user cases exhibited significantly reduced autoantibody reactivity against arrayed proteins compared to cases who were Not Current users. An associated reduced level of IL-6 and other immune-related cytokines was observed among HT users relative to nonusers., Conclusion and Clinical Relevance: Our findings suggest occurrence of a global altered immune response to breast cancer-derived proteins associated with HT. Thus a full understanding of factors that modulate the immune response is necessary to translate autoantibody panels into clinical applications., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Serum and plasma from which serum is derived represent a substantial challenge for proteomics due to their complexity. A landmark plasma proteome study was initiated a decade ago by the Human Proteome Organization (HUPO) that had as an objective to examine the capabilities of existing technologies. Given the advances in proteomics and the continued interest in the plasma proteome, it would timely reassess the depth and breadth of analysis of plasma that can be achieved with current methodology and instrumentation. A collaborative project to define the plasma proteome and its variation, with a plan to build a plasma proteome database would be timely., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We have developed a proteome database (DB), BiomarkerDigger (http://biomarkerdigger.org) that automates data analysis, searching, and metadata-gathering function. The metadata-gathering function searches proteome DBs for protein-protein interaction, Gene Ontology, protein domain, Online Mendelian Inheritance in Man, and tissue expression profile information and integrates it into protein data sets that are accessed through a search function in BiomarkerDigger. This DB also facilitates cross-proteome comparisons by classifying proteins based on their annotation. BiomarkerDigger highlights relationships between a given protein in a proteomic data set and any known biomarkers or biomarker candidates. The newly developed BiomarkerDigger system is useful for multi-level synthesis, comparison, and analyses of data sets obtained from currently available web sources. We demonstrate the application of this resource to the identification of a serological biomarker for hepatocellular carcinoma by comparison of plasma and tissue proteomic data sets from healthy volunteers and cancer patients.

The identification of human tumor antigens has potential utility in the diagnosis and treatment of cancers. We demonstrate here a complete strategy to profile immunoreactivity and identify tumor antigens from proteins derived from tumor cell lines. Microarrays of proteins produced from 2-D LC fractionation of prostate tumor cell-line lysates were used to profile immunoreactivity in the sera of prostate cancer patients and control subjects. Cancer-associated immunoreactivity to distinct groups of chromatography fractions was present in about 50% of the patients, with greater immunoreactivity present in patients with non-organ-confined cancer than in patients with organ-confined cancer. We grouped the immunoreactive fractions by similarities in elution order and patterns of immunoreactivity to guide and interpret the MS analysis of selected fractions, which was used to identify the proteins that may be responsible for the immunoreactivity. As a complementary method to further characterize and validate the immunoreactivity of the proteins identified by mass spectrometry, we demonstrate the use of focused microarrays of recombinant proteins. Disease-associated immunoreactivity was confirmed for one of the identified proteins, human Kallikrein 11. These results demonstrate a practical approach to screening, identifying, and validating immunoreactive proteins that could be applied to diverse studies on humoral immune responses., (Copyright © 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The broad characterization of the immune responses elicited by tumors has valuable applications in diagnostics and basic research. We present here the use of microarrays of tumor-derived proteins to profile the antibody repertoire in the sera of prostate cancer patients and controls. Two-dimensional liquid chromatography was used to separate proteins from the prostate cancer cell line LNCaP into 1760 fractions. These fractions were spotted in microarrays on coated microscope slides, and the microarrays were incubated individually with serum samples from 25 men with prostate cancer and 25 male controls. The amount of immunoglobulin bound to each fraction by each serum sample was quantified. Statistical analysis revealed that 38 of the fractions had significantly higher levels of immunoglobulin binding in the prostate cancer samples compared to the controls. Two fractions showed higher binding in the control samples. The significantly higher immunoglobulin reactivity from the prostate cancer samples may reflect a strong immune response to the tumors in the prostate cancer patients. We used multivariate analysis to classify the samples as either prostate cancer or control. In a cross-validation study, recursive partitioning classified the samples with 84% accuracy. A decision tree with two levels of partitioning classified the samples with 98% accuracy. Additional studies will allow further characterization of tumor antigens in prostate cancer and their significance for diagnosis. These results suggest that microarrays of fractionated proteins could be a powerful tool for tumor antigen discovery and cancer diagnosis.

The in depth-mining of the proteome necessitates the comprehensive analysis of proteins in individual subcellular compartments to uncover interesting patterns of protein expression that include assessment of protein location, trafficking and of post-translational modifications that are location specific. One of the compartments of substantial interest from a diagnostic and therapeutic point of view is the plasma membrane which contains intrinsic membrane proteins and other proteins expressed on the cell surface. Technologies are currently available for the comprehensive profiling of the cell surface proteome that rely on protein tagging of intact cells. Studies are emerging that point to unexpected patterns of expression of specific proteins on the cell surface, with a common occurrence of proteins previously considered to occur predominantly in other compartments, notably the endoplasmic reticulum. The profiling of the cell surface and plasma membrane proteomes will likely provide novel insights and uncover disease related alterations.

Megakaryocytes undergo an unusual cell cycle during differentiation that results in polyploidy through largely unknown mechanism(s). It has been shown that serine phosphorylation of oncoprotein 18 (Op18) is required for cell cycle progression specifically at the G2/M transition. Moreover, mutant forms of Op18 that are defective in one or more of the four serine residues induce G2/M arrest and subsequent polyploidization. Op18 phosphorylation is rapidly induced with phorbol myristate acetate (PMA) treatment in a wide range of human cells. In this study, we investigated the role of Op18 in PMA induced polyploidization during megakaryocyte differentiation of the human erythroleukemia cell line. Crucial to the molecular analysis of megakaryocyte differentiation, is the ability to fractionate cell populations with different ploidy levels. We have utilized cell elutriation as a fractionation strategy to analyze Op18 expression in synchronized cell subpopulations in different phases of the cell cycle or with progressive megakaryocyte polyploidization. In the absence of PMA, increased phosphorylation of Op18 was observed in HEL cells during cell cycle progression, as for other proliferating cells. However, in contrast to Jurkat leukemia cells chosen as control, HEL cells exhibited a lack of Op18 phosphorylation in response to PMA, which was accompanied by polyploidization and differentiation along the megakaryocytic lineage. To further determine the role of Op18 in polyploidization, HEL cells were transfected with different Op18 expression constructs. Differences in cell survival and polyploidization were observed between high and low Op18 expressors. An increased Op18 level reduced cell survival during the early stage of PMA induced megakaryocyte differentiation, but enhanced polyploidization efficiency. Our findings suggest that maintenance of a high level of unphosphorylated Op18 is required for efficient polyploidization during the differentiation program of megakaryocytes.

We have developed a comprehensive approach to identifying molecular changes in lung cancer that includes both genomic and proteomic analyses. The related effort has produced a large amount of data pertaining to gene expression at the RNA and protein levels. As a result, we have constructed a database that contains protein expression data on lung cancer as well as other relevant data including DNA microarray derived data. A large number of proteins that are expressed in different types of lung cancer have been identified and have been correlated with the expression measures for their corresponding genes at the RNA level. The database is intended to facilitate our effort at developing novel classification schemes for lung cancer and the identification of novel markers for early diagnosis.

A novel proteomic approach for probing cell and tissue proteome, which combines liquid phase protein separations with microarray technology has been developed. Proteins in cell and tissue lysates or in cellular subfractions are separated using any one of a number of separation modes which may consist of ion exchange liquid chromatography (LC), reverse phase LC, carrier ampholyte based separations, e.g. the use of Rotofor, affinity based separations, or gel based separations. Each first-dimension fraction obtained using one separation mode can be further resolved using one or more of the other separation modes to yield either purified protein in solution or liquid fractions with substantially reduced protein complexity. The advantage of a liquid based separation system is that proteins in hundreds of individual fractions can be arrayed directly and used as targets for a variety of probes. Constituent proteins in reactive fractions are identified by mass spectrometry and may be further resolved to determine the nature of the reactive protein(s). We present in this report initial data based on microarray analysis of individual Rotofor fractions obtained from lung adenocarcinoma cell line A549 lysates which have been probed with antibodies against specific proteins.

A common therapy for nonorgan-confined prostate cancer involves androgen deprivation. To develop a better understanding of the effect of androgen on prostatic cells, we have analyzed gene expression changes induced by dihydrotestosterone (DHT) in the androgen responsive prostate cancer line LNCaP, at both RNA and protein levels. Changes at the RNA level induced by DHT were determined by means of serial analysis of gene expression (SAGE), and protein profiling was done by means of quantitative two-dimensional polyacrylamide gel electrophoresis. Among 123,371 transcripts analyzed, a total of 28,844 distinct SAGE tags were identified representing 16,570 genes. Some 351 genes were significantly affected by DHT treatment at the RNA level (p < 0.05), of which 147 were induced and 204 repressed by androgen. In two independent experiments, the integrated intensity of 32 protein spots increased and 12 decreased at least two-fold in response to androgen, out of a total of 1031 protein spots analyzed. The change in intensity for most of the affected proteins identified could not be predicted based on the level of their corresponding RNA. Our study provides a global assessment of genes regulated by DHT and suggests a need for profiling at both RNA and protein levels for a comprehensive evaluation of patterns of gene expression.

There is currently much interest, as we enter the postgenome era, in studying gene expression at the protein level. Two-dimensional electrophoresis (2-DE) using immobilized pH gradients (IPG), coupled with mass spectrometry (MS), is currently the most widely utilized approach for the analysis of whole tissue proteins. The methodology for IPG-based 2-DE, since the introduction of the technique in the 1980s, is reviewed. In its present form the IPG methodology is mostly useful as a research tool. In general, high reproducibility and high resolution have been achieved. However, the lack of substantial automation and the limited sensitivity of the current overall methodology continue to represent drawbacks for biomedical applications. Further developments to increase throughput and to reduce sample requirement would substantially benefit the application of IPG-based 2-DE to biomedicine and would enhance the prospects for introducing the methodology into the clinical laboratory.

We have developed a simplified procedure for the separation of enzyme-digested genomic DNA, or of complex mixtures of cloned DNA, into two dimensions. The procedure relies on the use of precast gels for horizontal electrophoretic separations. Precast agarose-type gels are used for the first-dimensional separation of fragments based on size. Precast polyacrylamide gels are used for the second-dimensional separation of fragments. The separated fragments are subjected to enzymatic digestion in situ prior to their transfer to the second-dimensional gel. Applications of this procedure include the analysis of DNA libraries, analysis of yeast artificial chromosomes (YACs) as well as other similar preparations, and the screening of genomic DNA for the occurrence of multi-copy DNA fragments as in the case of genomic amplifications in cancer.

There is a considerable need to develop comprehensive, systematic mechanisms to analyze the vast number of proteins that orchestrate various cellular functions and to identify proteins associated with disease or that are affected by pharmacological agents. Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) continues to be relied upon to analyze protein constituents of cells and tissues. We have developed a Laboratory Information Processing System (LIPS) as a computer-based tool for capturing quantitative and qualitative changes in thousands of proteins detected in 2-D gels of various types. Protein databases have been developed to serve as a repository for data processing of the basic and derived data and of findings derived from different studies. There have been remarkable advances both in database technology as well as in the computer hardware that have benefited our effort at mining protein data from 2-D gels. We here review our current efforts aimed at improving the performance and features of our 2-D related protein databases, with particular emphasis on the tools we utilize for database mining via a systematic analysis of information known as post-planned analysis.

We have undertaken an effort in the past five years aimed at developing a database of lymphoid proteins detectable by two-dimensional (2-D) polyacrylamide gel electrophoresis. The database contains 2-D patterns and derived information pertaining to: (i) polypeptide constituents of unstimulated and stimulated mature T cells and immature thymocytes; (ii) cultured T cells and cell lines that have been manipulated by transfection with a variety of constructs or by treatment with specific agents; (iii) single cell-derived T and B cell clones; (iv) cells obtained from patients with lymphoproliferative disorders and leukemia; and (v) a variety of other relevant cell populations. The database has experienced a substantial expansion in 2-D patterns it contains, numbering currently 9167 individual 2-D patterns. This number represents a fraction of the 30,682 2-D patterns maintained in our databases. The capacity to design and undertake experiments, produce high-quality 2-D patterns, and to undertake simple or rudimentary analyses of 2-D patterns to meet the basic needs of the experiments for which the 2-D gels were produced has exceeded the capacity to fully and uniformly integrate information generated from any gel image or experiment, across all images and experiments. While only a fraction of the information in the 2-D patterns contained in the lymphoid database has been mined, novel findings derived from querying the database point to the merits of this protein based approach. Additional resources have recently been put into place to mine more effectively data pertaining to protein expression in lymphoid cells.

Two-dimensional (2-D) electrophoretic methods have been available that allow separation of the protein constituents of a cell population. It has also become feasible to electrophoretically separate in two dimensions and to display DNA fragments derived from genomic digests. Through the appropriate choice of restriction enzymes, the functional component of the genome that encompasses CpG islands can be preferentially visualized in 2-D gels. The same computerized approach for the analysis of 2-D patterns can be applied to investigations at either the protein or DNA levels. Our group has utilized 2-D electrophoresis to investigate both protein and DNA changes in cancer. The emphasis to date has been on the identification of proteins, the abundance of which is related to specific biological features of the tumors analyzed and of DNA fragments encompassed in genomic amplifications, as the latter commonly contain growth-related genes. Findings derived from our analysis of neuroblastoma tumors and cell lines using 2-D approaches are reviewed. Data for four proteins observed in 2-D gels are presented because of our demonstrated association of these proteins with differentiation and proliferation properties of neuroblastoma. At the genomic level, the detection of amplifications using 2-D gels has necessitated an understanding of the variability displayed by multi-copy genomic fragments, which we have accomplished to a large part and which we present. An important benefit of 2-D approaches is the efficiency of scale and the ease with which abundant proteins or multicopy genomic fragments can be detected, identified and quantitatively analyzed.

There is a continuing need for more efficient methods to examine human (and other) populations for altered germinal and somatic cell mutation rates. To this end, we have explored the potential usefulness of two-dimensional (2-D) electrophoresis of human DNA fragments obtained from restriction-enzyme-digested genomic DNA, using samples from father/mother/child trios. On a single 2-D DNA preparation, approximately 2000 DNA fragments varying in size from 1.0 to 5.0 kbp in the first dimension and 0.3 to 2.0 kbp in the second dimension are visualized. To enter into a genetic analysis of quantitative variation, these fragments must exhibit positional and quantitative stability. With respect to the latter, if spots that are the product of two homologous DNA fragments are to be distinguished with the requisite accuracy from spots that are the product of only one fragment, the coefficient of variation of spot intensity should be approximately < or = 0.12. At present, 482 of the spots in our preparations meet these standards. In an examination of preparations based on three Japanese mother/father/child trios, 43 of these 482 spots were found to exhibit variations that segregated within families according to Mendelian principles. Additionally, of the 2000 spots, 1114 (of which the aforementioned 482 are a subset) were deemed appropriate for the study of qualitative variation. A total of 142 variable spots were identified; the heterozygosity index for these DNA fragments was 4.4%. The genetic nature of the additional variants was again established by their segregation according to Mendelian principles. We have established the feasibility of cloning fragments from such gels and determining their nucleotide sequence. This technology should be highly efficient in monitoring for mutation resulting in loss/gain/rearrangement events in DNA fragments distributed throughout the genome.

A laboratory information processing system (LIPS) has been developed to provide support for various laboratory activities related to two-dimensional electrophoresis. In this paper we present the relational schema for each level of activity and the application development of LIPS, in terms of the relational database management system being used. We also discuss our experience with the current system.

An approach for the computer-assisted analysis of two-dimensional gels has been developed as a part of our laboratory information processing system (LIPS). This approach relies in part on an algorithm for the pairwise matching of protein spots. The matching process initially matches spots based on a cross-correlational measure of how well neighboring spots align. While this first pass correctly determines most spot correspondences and noncorrespondences, it can make errors. Higher accuracy is obtained by monitoring the consistency of spot match decisions in a second pass, which demands that neighboring spot pairs that align spatially must also have been found to match in the first pass. Pairwise comparisons of gels are combined into n-way comparisons by matching spot lists of gels to "master" gel spot lists, which in turn are matched to higher level masters, resulting in a hierarchy of matched spots. After each pairwise match the results are reviewed and corrected with the assistance of a graphical match-editor. Results are given for 19 single-cell-derived lymphoid clones in which the presence of a mutation had previously been established, each processed in duplicate. Only one of 46 spot changes failed to be detected, which demonstrates that the strategy is sensitive and efficient for detecting qualitative spot differences.

The effect of temperature, at which isoelectric focusing with immobilized pH gradients is performed, on spot positions and pattern quality in two-dimensional (2-D) electrophoresis was examined. Increased temperatures revealed improved 2-D patterns with respect to sample entry, resolution, and background staining. Focusing at 20 degrees C was superior to focusing at 10 and 15 degrees C. Even at 30 degrees C, a pattern of well-resolved polypeptide spots with a minimum amount of horizontal streaking at the basic end was observed. A computer-based analysis showed that a substantial proportion of polypeptides assumed altered positions in the 2-D pattern in relation to temperature. Mobility shifts of polypeptides were more variable on the neutral part than on the acidic or basic end. The mobility shifts were not restricted to one direction for all the spots whose migration was altered. However, for any given spot, the direction was the same with subsequent increments of temperature. The results clearly demonstrate that a defined temperature for first-dimensional isoelectric focusing is a requirement for the reproducibility of 2-D electrophoresis. After elimination of the cathodic drift, a major source of variability in 2-D patterns, associated with carrier ampholytes, temperature control becomes a critical parameter.

Horizontal two-dimensional (2-D) electrophoresis with immobilized pH gradients (IPG) in the first dimension for buffer soluble proteins and for complex proteins solubilized in the presence of Nonidet P-40 (Görg et al., Electrophoresis 1987, 8, 45-51), has been extended to analyze basic proteins of yeast cells focused under non-equilibrium and equilibrium conditions. Transient state isoelectric focusing (IEF) in IPG gels revealed sample smearing and background staining, displaying horizontal streaks in the resultant 2-D patterns. Inclusion of 0.5% carrier ampholytes (CA) to the IPG gel (IPG-CA), resulted in the formation of many sharp protein bands after transient state IEF with resultant distinct spots in the 2-D patterns; however, resolution was poor and the gel contained heavy background staining. With prolonged focusing time, background staining disappeared and there was less difference in the final steady state IEF patterns obtained with IPG and IPG-CA. Reduction of the Immobiline concentration to one third the manufacturer's recommended amount did not improve IEF resolution with respect to streaking and background staining under either transient state or equilibrium conditions. In general, spot intensities were less on 2-D gels using diluted IPG gels than with "standard" IPG gels. Optimization of 2-D electrophoresis with IPGs in the first dimension was strongly related to IEF conditions. The use of IPG gels focused to equilibrium should not only improve inter-gel reproducibility and resolution but also the quality of the final 2-D patterns with respect to background staining and horizontal streaking.

The effect of salt and buffer ions in the sample or in an immobilized pH gradient (IPG) on sample entry into the gel and on the final focused pattern are presented. During the initial phase of electrofocusing, ions present in the gel, either as counter ions to the immobilized charge groups of the IPG gel or added to the gel matrix during the rehydration process, are transported toward the electrodes. For ions present at a concentration exceeding approximately 1 mM the transport can be followed by the refractile line marking the trailing edge of an ion-containing zone. Gradual sample entry may be achieved by applying the sample at a site (near the anode or cathode) opposite to that from which the sharpest refractile line, marking the ion present in the highest concentration, approaches the sample. Additionally, lateral band spreading of the sample is avoided. Thus, sample applied at the cathode for IPG gels rehydrated with 1-2 mM Tris base, or at the anode for gels rehydrated with 1-2 mM acetic acid or sodium acetate, enters the gel matrix gradually without lateral band spreading. In contrast, sample applied at the anode, for Tris-containing gels, or at the cathode, for acetate-containing gels, enters rapidly in a sharp zone when the refractile line reaches the sample zone. This results in a high local protein concentration in the zone immediately behind the boundary with lateral band spreading.(ABSTRACT TRUNCATED AT 250 WORDS)

The feasibility of detecting quantitative genetic variants based on a decrease in the integrated intensity of polypeptide spots in two-dimensional polyacrylamide gels of human lymphoblastoid cell clones was investigated. A battery of 65 spots on 115 gels was studied to determine the distribution of quantitative measures for spots where no mutation had occurred. The corresponding distribution for spots which have decreased integrated intensity as a result of a mutation at one of two alleles coding for the spot was investigated by quantitating spots for which mutations were known to have occurred. These two distributions allowed the estimation of the rates of false positive and false negative errors for any particular strategy aimed at detecting null mutations, and thus provides a basis for the design of efficient strategies. Our silver stained gels have sufficient reproducibility of spot integrated intensities so that, for situations in which the mutation rate is relatively high, it is practical to monitor a subset of spots for null variants using the same digitized images as are used to detect structural variants.