Immobilized pH gradients: Analytical and preparative use

The use of immobilized pH gradients for two-dimensional electrophoresis overcomes several of the limitations of carrier ampholyte-based isoelectric focusing. Procedures followed in the authors' laboratory for the analytical or preparative use of immobilized pH gradients are presented.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29096/1/0000132.pd

Towards an integrated proteomic and glycomic approach to finding cancer biomarkers

Advances in mass spectrometry have had a great impact on the field of proteomics. A major challenge of proteomic analysis has been the elucidation of glycan modifications of proteins in complex proteomes. Glycosylation is the most structurally elaborate and diverse type of protein post-translational modification and, because of this, proteomics and glycomics have largely developed independently. However, given that such a large proportion of proteins contain glycan modifications, and that these may be important for their function or may produce biologically relevant protein variation, a convergence of the fields of glycomics and proteomics would be highly desirable. Here we review the current status of glycoproteomic efforts, focusing on the identification of glycoproteins as cancer biomarkers

A statistical analysis of spot variation using the two-dimensional polyacrylamide gel electrophoresis

For the development of valid algorithms for matching protein spots between two-dimensional gels, it is essential that one has an understanding of the relative roles of the many sources of variability affecting the location of spots. We first consider the contribution of observers to the measurement variability of spot location, arriving at a simple model for these effects. Next we present an analysis of the variability in spot locations for a sample of gels containing duplicate gels for each sample. Our data indicate that both differences between duplicates and between samples are considerable, and that the size of the effects depends on the region of the gel being considered. In the discussion we examine several matching strategies that match large groups of gels based on algorithms which match two gels at a time.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26270/1/0000355.pd

Overexpressed Genes/ESTs and Characterization of Distinct Amplicons on 17823 in Breast Cancer Cells

Abstract17823 is a frequent site of gene amplification in breast cancer. Several lines of evidence suggest the presence of multiple amplicons on 17823. To characterize distinct amplicons on 17823 and localize putative oncogenes, we screened genes and expressed sequence tags (ESTs) in existing physical and radiation hybrid maps for amplification and overexpression in breast cancer cell lines by semiquantitative duplex PCR, semiquantitative duplex RT-PCR, Southern blot, Northern blot analyses. We identified two distinct amplicons on 17823, one including TBX2 and another proximal region including RPS6KB1 (PS6K) and MUL. In addition to these previously reported overexpressed genes, we also identified amplification and overexpression of additional uncharacterized genes and ESTs, some of which suggest potential oncogenic activity. In conclusion, we have further defined two distinct regions of gene amplification and overexpression on 17823 with identification of new potential oncogene candidates. Based on the amplification and overexpression patterns of known and as of yet unrecognized genes on 17823, it is likely that some of these genes mapping to the discrete amplicons function as oncogenes and contribute to tumor progression in breast cancer cells

Systemic Metabolomic Changes in Blood Samples of Lung Cancer Patients Identified by Gas Chromatography Time-of-Flight Mass Spectrometry.

Lung cancer is a leading cause of cancer deaths worldwide. Metabolic alterations in tumor cells coupled with systemic indicators of the host response to tumor development have the potential to yield blood profiles with clinical utility for diagnosis and monitoring of treatment. We report results from two separate studies using gas chromatography time-of-flight mass spectrometry (GC-TOF MS) to profile metabolites in human blood samples that significantly differ from non-small cell lung cancer (NSCLC) adenocarcinoma and other lung cancer cases. Metabolomic analysis of blood samples from the two studies yielded a total of 437 metabolites, of which 148 were identified as known compounds and 289 identified as unknown compounds. Differential analysis identified 15 known metabolites in one study and 18 in a second study that were statistically different (p-values <0.05). Levels of maltose, palmitic acid, glycerol, ethanolamine, glutamic acid, and lactic acid were increased in cancer samples while amino acids tryptophan, lysine and histidine decreased. Many of the metabolites were found to be significantly different in both studies, suggesting that metabolomics appears to be robust enough to find systemic changes from lung cancer, thus showing the potential of this type of analysis for lung cancer detection

Separation of transferrin types in human plasma by anion-exchange high-performance liquid chromatography

An anion-exchange high-performance liquid chromatographic procedure was developed for the separation of transferrin in human plasma. The procedure allowed the four molecular forms of transferrin, which differ with respect to bound iron, to be separated from each other and from other plasma proteins. Tansferrin variants, including B and D types, could also be identified using anion-exchange high-performance liquid chromatography. The approach followed for optimizing the separation of transferrin included identification of the peaks in the chromatogram by two-dimensional polyacrylamide gel electrophoresis. This approach could be extended to other proteins in plasma or biological fluids in order to optimize their separation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25127/1/0000560.pd

Identification of proteins from two-dimensional gel electrophoresis of human erythroleukemia cells using capillary high performance liquid chromatography/electrospray-ion trap-reflectron time-of-flight mass spectrometry with two-dimensional topographic map analysis of in-gel tryptic digest products

Protein spots from two-dimensional (2-D) gel electrophoresis of a human erythroleukemia cell line have been identified by analysis of the in-gel tryptic digests using capillary high performance liquid chromatography (HPLC) separation with on-line detection using electrospray ionization mass spectrometry (ESI-MS). This is performed using an electrospray/ion trap storage/reflectron time-of-flight mass spectrometer system (ESI-IT-reTOFMS). A 2-D topographic mapping display developed to process the on-line data acquired with this TOF system has been used to obtain mass identification of each peptide, even though the capillary HPLC only provides limited separation capability of the tryptic peptide mixtures studied herein. Using this method, a substantial fraction of the protein sequence can be covered and identified using the tryptic map. It is demonstrated that by entering the cell species, the approximate MW and pI range as determined by 2-D gel electrophoresis, and the tryptic peptide map into the database a unique match for identification of the protein generally results. It is also demonstrated that a much improved coverage of the protein sequence is obtained by this method relative to matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Copyright © 1999 John Wiley & Sons, Ltd.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/35077/1/732_ftp.pd

Proteomic risk markers for coronary heart disease and stroke: validation and mediation of randomized trial hormone therapy effects on these diseases

Background: We previously reported mass spectrometry-based proteomic discovery research to identify novel plasma proteins related to the risk of coronary heart disease (CHD) and stroke, and to identify proteins with concentrations affected by the use of postmenopausal hormone therapy. Here we report CHD and stroke risk validation studies for highly ranked proteins, and consider the extent to which protein concentration changes relate to disease risk or provide an explanation for hormone therapy effects on these outcomes. Methods: Five proteins potentially associated with CHD (beta-2 microglobulin (B2M), alpha-1-acid glycoprotein 1 (ORM1), thrombospondin-1(THBS1), complement factor D pre-protein (CFD), and insulin-like growth factor binding protein 1 (IGFBP1)) and five potentially associated with stroke (B2M, IGFBP2, IGFBP4, IGFBP6, and hemopexin (HPX)) had high discovery phase significance level ranking and an available ELISA assay, and were included in case-control validation studies within the Women’s Health Initiative (WHI) hormone therapy trials. Protein concentrations, at baseline and 1 year following randomization, were assessed for 358 CHD cases and 362 stroke cases, along with corresponding disease-free controls. Disease association, and mediation of estrogen-alone and estrogen plus progestin effects on CHD and stroke risk, were assessed using logistic regression. Results: B2M, THBS1, and CFD were confirmed (P <0.05) as novel CHD risk markers, and B2M, IGFBP2, and IGFBP4 were confirmed as novel stroke disease risk markers, while the assay for HPX proved to be unreliable. The change from baseline to 1 year in B2M was associated (P <0.05) with subsequent stroke risk, and trended similarly with subsequent CHD risk. Change from baseline to 1 year in IGFBP1 was also associated with CHD risk, and this change provided evidence of hormone therapy effect mediation. Conclusions: Plasma B2M is confirmed to be an informative risk marker for both CHD and stroke. The B2M increase experienced by women during the first year of hormone therapy trial participation conveys cardiovascular disease risk. The increase in IGFBP1 similarly conveys CHD risk, and the magnitude of the IGFBP1 increase following hormone therapy may be a mediator of hormone therapy effects. Plasma THBS1 and CFD are confirmed as CHD risk markers, and plasma IGFBP4 and IGFBP2 are confirmed as stroke risk markers. Clinical trials registration ClinicalTrials.gov identifier: NCT0000061

Overexpression of CD70 and overstimulation of IgG synthesis by lupus T cells and T cells treated with DNA methylation inhibitors

Objective Generalized DNA hypomethylation contributes to altered T cell function and gene expression in systemic lupus erythematosus (SLE). Some of the overexpressed genes participate in the disease process, but the full repertoire of genes affected is unknown. Methylation-sensitive T cell genes were identified by treating T cells with the DNA methyltransferase inhibitor 5-azacytidine and comparing gene expression with oligonucleotide arrays. CD70, a costimulatory ligand for B cell CD27, was one gene that reproducibly increased. We then determined whether CD70 is overexpressed on T cells treated with other DNA methylation inhibitors and on SLE T cells, and determined its functional significance. Methods Oligonucleotide arrays, real-time reverse transcription–polymerase chain reaction, and flow cytometry were used to compare CD70 expression in T cells treated with 2 DNA methyltransferase inhibitors (5-azacytidine and procainamide) and 3 ERK pathway inhibitors known to decrease DNA methyltransferase expression (U0126, PD98059, and hydralazine). The consequences of CD70 overexpression were tested by coculture of autologous T and B cells with and without anti-CD70 and measuring IgG production by enzyme-linked immunosorbent assay. The results were compared with those of T cells from lupus patients. Results SLE T cells and T cells treated with DNA methylation inhibitors overexpressed CD70 and overstimulated B cell IgG production. The increase in IgG synthesis was abrogated by anti-CD70. Conclusion SLE T cells and T cells treated with DNA methyltransferase inhibitors and ERK pathway inhibitors overexpress CD70. This increased B cell costimulation and subsequent immunoglobulin overproduction may contribute to drug-induced and idiopathic lupus.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/34311/1/20255_ftp.pd