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

Early events of Bacillus anthracis germination identified by time-course quantitative proteomics

Germination of Bacillus anthracis spores involves rehydration of the spore interior and rapid degradation of several of the protective layers, including the spore coat. Here, we examine the temporal changes that occur during B. anthracis spore germination using an isobaric tagging system. Over the course of 17 min from the onset of germination, the levels of at least 19 spore proteins significantly decrease. Included are acid-soluble proteins, several known and predicted coat proteins, and proteins of unknown function. Over half of these proteins are small (less than 100 amino acids) and would have been undetectable by conventional gel-based analysis. We also identified 20 proteins, whose levels modestly increased at the later time points when metabolism has likely resumed. Taken together, our data show that isobaric labeling of complex mixtures is particularly effective for temporal studies. Furthermore, we describe a rigorous statistical approach to define relevant changes that takes into account the nature of data obtained from multidimensional protein identification technology coupled with the use of isobaric tags. This study provides an expanded list of the proteins that may be involved in germination of the B. anthracis spore and their relative levels during germination.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/55849/1/5199_ftp.pd

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

Cord blood screening for hemoglobin disorders by high-performance liquid chromatography,

Ion-exchange high-performance liquid chromatography was employed as a screening method for abnormal hemoglobins in the newborn period. Samples of cord blood collected in EDTA tubes were used for this analysis. Hemolysates were injected onto 4.1 x 100-mm Synchropak ion-exchange columns using an automatic injector. Hemoglobin separation was carried out by means of a sodium acetate gradient. A total of 415 samples was analyzed. Hemoglobins A, F, and Bart's, as well as C or S when present, were separately eluted and quantitated using a 35-min gradient program. Four individuals with sickle cell disease, 26 with S or C trait, one with SC disease, and two others with alpha-chain variants were diagnosed with this method. The proportion of Bart's hemoglobin was greater than 1% in 33 individuals. The elution pattern was highly reproducible. The potential for complete automation and the ease with which quality control can be assured make this technique well suited for the detection of abnormal hemoglobins in the newborn period.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/23944/1/0000191.pd

Evidence for impaired t cell dna methylation in systemic lupus erythematosus and rheumatoid arthritis

Procainamide and hydralazine inhibit T cell DNA methylation and induce autoreactivity in cloned CD4+ T cells. These drugs also induce an autoimmune syndrome, suggesting a possible relationship between DNA hypomethylation, T cell autoreactivity, and certain autoimmune diseases. To test this relationship, DNA methylation was studied in T cells from patients with rheumatoid arthritis and patients with systemic lupus erythematosus, and was found to be impaired. These results support a relationship between DNA hypomethylation and some forms of autoimmune disease.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/37783/1/1780331109_ftp.pd

Detection of human somatic cell structural gene mutations by two-dimensional electrophoresis

The feasibility of detecting human somatic structural gene mutations by two dimensional electrophoresis has been investigated. A lymphoblastoid cell line was grown as a mass culture in the presence of ethylnitrosourea, after which cells were regrown as single cell clones. A total of 257 polypeptide spots were analyzed in gels derived from 186 clones. Four structural mutations were detected by visual analysis of the gels. Computer analysis of gels corresponding to the mutant clones was also undertaken. At a spot size threshold of 200 spots to be matched using a computer algorithm, all four mutant polypeptides were detected. These results indicate the usefulness of the two-dimensional approach for mutagenesis studies at the protein level.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/38517/1/340020103_ftp.pd

Proteomic Interrogation of Androgen Action in Prostate Cancer Cells Reveals Roles of Aminoacyl tRNA Synthetases

Prostate cancer remains the most common malignancy among men in United States, and there is no remedy currently available for the advanced stage hormone-refractory cancer. This is partly due to the incomplete understanding of androgen-regulated proteins and their encoded functions. Whole-cell proteomes of androgen-starved and androgen-treated LNCaP cells were analyzed by semi-quantitative MudPIT ESI- ion trap MS/MS and quantitative iTRAQ MALDI- TOF MS/MS platforms, with identification of more than 1300 high-confidence proteins. An enrichment-based pathway mapping of the androgen-regulated proteomic data sets revealed a significant dysregulation of aminoacyl tRNA synthetases, indicating an increase in protein biosynthesis- a hallmark during prostate cancer progression. This observation is supported by immunoblot and transcript data from LNCaP cells, and prostate cancer tissue. Thus, data derived from multiple proteomics platforms and transcript data coupled with informatics analysis provides a deeper insight into the functional consequences of androgen action in prostate cancer

The concept of transport capacity in geomorphology

The notion of sediment-transport capacity has been engrained in geomorphological and related literature for over 50 years, although its earliest roots date back explicitly to Gilbert in fluvial geomorphology in the 1870s and implicitly to eighteenth to nineteenth century developments in engineering. Despite cross fertilization between different process domains, there seem to have been independent inventions of the idea in aeolian geomorphology by Bagnold in the 1930s and in hillslope studies by Ellison in the 1940s. Here we review the invention and development of the idea of transport capacity in the fluvial, aeolian, coastal, hillslope, débris flow, and glacial process domains. As these various developments have occurred, different definitions have been used, which makes it both a difficult concept to test, and one that may lead to poor communications between those working in different domains of geomorphology. We argue that the original relation between the power of a flow and its ability to transport sediment can be challenged for three reasons. First, as sediment becomes entrained in a flow, the nature of the flow changes and so it is unreasonable to link the capacity of the water or wind only to the ability of the fluid to move sediment. Secondly, environmental sediment transport is complicated, and the range of processes involved in most movements means that simple relationships are unlikely to hold, not least because the movement of sediment often changes the substrate, which in turn affects the flow conditions. Thirdly, the inherently stochastic nature of sediment transport means that any capacity relationships do not scale either in time or in space. Consequently, new theories of sediment transport are needed to improve understanding and prediction and to guide measurement and management of all geomorphic systems