Preparative Denaturing Isoelectric Focusing for Enhancing Sensitivity of Proteomic Studies

Serna-Sanz, Antonio; Rairdan, Greg; Peck, Scott C.

doi:10.1385/1-59259-966-4:99

Cited by 3 publications

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“…For some samples, a preparative step before gel electrophoresis may even be required. 45 Thus, the established methods of proteomics are multidimensional analytical systems, involving two or three sample preparation dimensions prior to MS detection. Other alternative sample preparative methods are also being investigated such as microwave-assisted methods.…”

Section: Resultsmentioning

confidence: 99%

“…The complexity of the samples studied, however, requires the use of preparative steps, which can be performed by either liquid chromatography (LC) or gel electrophoresis. For some samples, a preparative step before gel electrophoresis may even be required . Thus, the established methods of proteomics are multidimensional analytical systems, involving two or three sample preparation dimensions prior to MS detection.…”

Section: Resultsmentioning

confidence: 99%

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Ion-Transfer Voltammetric Behavior of Protein Digests at Liquid|Liquid Interfaces

et al. 2009

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The development of new methods for the detection of proteins and peptides is of widespread importance. In this work, the electrochemical behavior of peptide mixtures resulting from proteolytic digestion of proteins was investigated at the polarized liquid|liquid interface (or the interface between two immiscible electrolyte solutions, ITIES). The influence of pepsin digestion on three proteins (hemoglobin, lysozyme, and cytochrome c) was studied, and it was revealed that resulting cyclic voltammograms of the three protein digests were different due to the unique peptide mixtures for a given protein. Differential pulse stripping voltammetry of protein digests enabled the detection of digested proteins at concentrations ranging between 0.55 and 4.22 microM. A limit of detection of 0.55 microM of the initial concentration of protein was achieved, demonstrating the analytical possibilities of such an electrochemical method. These results show that ion-transfer voltammetry offers the opportunity to study and develop label-free detection of peptides resulting from enzymatic digestions of proteins and may thus have a role in development of new proteomic technologies.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Ion-Transfer Voltammetric Behavior of Protein Digests at Liquid|Liquid Interfaces

et al. 2009

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“…29 After phenol back extraction, protein content was measured by BCA. 30 Protein separation by liquid IEF was carried out as described by Serna et al 31 Briefly, 50 mg (for acidic subproteome) or 20 mg (for differential expression analysis) of purified protein was resuspended in 50 mL of focusing buffer (7 M urea, 2 M thiourea, 1% CHAPS, 5% ampholytes and 12.5% glycerol). Proteins in focusing buffer were loaded into a large Rotofor chamber and focused for four hours at constant power (10 W).…”

Section: Preparative Denaturing Ief (Rotofor)mentioning

confidence: 99%

Quantitative Proteome and Acidic Subproteome Profiling of Candida albicans Yeast-to-Hypha Transition

et al. 2010

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Candida albicans yeast-to-hypha morphological transition is involved in the virulence strategy of this opportunistic fungal pathogen. Changes in relative abundance of the Candida proteome related to this process were analyzed using different two-dimensional differential in-gel electrophoresis (2D-DIGE)-based approaches. First, a comparative analysis of yeast and hyphal cytoplasmic proteins allowed the detection of 106 protein spots with significant variation in abundance. Sixty-one of them, corresponding to 46 proteins, were identified. As most of the differentially abundant proteins had an acidic isoelectric point, a large-scale prefractionation approach to analyze the acidic C. albicans subproteome was carried out. Ninety acidic C. albicans proteins were identified by either gel-based or nongel-based approaches. Additionally, different workflows combining preparative isoelectric focusing, Cy labeling, and narrow pH gradient 2-DE gels were tested to analyze the differences in relative protein abundance between yeast and hyphal acidic subproteomes. It was possible to identify 21 differentially abundant acidic proteins; 10 of them were not identified in the previous 2D-DIGE gels. Functional and network interaction analyses of the 56 differentially abundant proteins identified by both approaches rendered an integrated view of metabolic and cellular process reorganization during the yeast-to-hypha transition. With these results, we propose a model of metabolic reorganization.

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“…For comparison, the detection threshold of proteins is 1-10 ng when using silver nitrate (Lilley, Razzaq, & Dupree, 2002;Tonge et al, 2001) and approximately 500 pg when employing CyDye DIGE fluors . These limitations may be (partially) overcome by pre-fractionation of samples (Barnea, Sorkin, Ziv, Beer, & Admon, 2005;Lopez et al, 2000;Serna-Sanz, Rairdan, & Peck, 2007;Tang et al, 2005;Yuan & Desiderio, 2005).…”

Section: Potential Problems and Limitations Of Proteome Analysis Of Bmentioning

confidence: 99%

Genomics, Proteomics, and the Nervous System

Clelland¹

2011

Advances in Neurobiology

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except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights.Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com) v Preface Mammalian central and peripheral nervous systems are highly complex at the structural, genetic and molecular levels, composed of multiple cell types and tissue structures. Thousands of genes, regulated at the genomic level via sequence variation or epigenetic regulation, are expressed at the RNA level and translated into proteins required to develop and maintain these cells and tissues, and along with small regulatory RNA molecules, lipids, and small molecule neurotransmitters, these gene products constitute the physical substrate for learning, memory, emotion, sensory perception, and consciousness itself. The potential for malfunction of this large number of complex biological systems is great, leading to the many behavioral and cognitive deficits observed in human psychiatric and neurological disorders, such as schizophrenia, autism and Alzheimer's disease.This Volume of Advances of Neurobiology discusses research designed to increase our understanding of the nervous system and its structures and activities, through the utilization of genomic and proteomic technologies, addressing facets including development and epigenetic regulation, functions in learning and memory, and changes associated with neurological and psychiatric disorders. Specifically, the development of high-throughput genomic and proteomic analysis technologies, including microarray and high-throughput DNA sequencing technology, as well as integrated protein separation and mass spectrometry analysis systems, have created the opportunity for researchers to collect datasets that include measurements for all or most of the RNA species or the complement of proteins, within a particular biological sample. These high dimensional datasets are being generated for different nervous system cells and tissues, such as laser-capture microdissected neurons, or samples of postmortem pre-frontal cortical tissue. Different approaches have then been utilized to extract pertinent information, and these range from comparisons of postmortem cells and/or tissues using samples collected from subjects with and without disease states, for example patients with Alzheimer's disease compared to control subjects, in order to discover differences between the samples that reflect aspects of the disease pathology, and that can then be investigated further to determine their role(s) in disease development. In addition, and in disorders such as autism, genome-wide expre...

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Preparative Denaturing Isoelectric Focusing for Enhancing Sensitivity of Proteomic Studies

Cited by 3 publications

References 0 publications

Ion-Transfer Voltammetric Behavior of Protein Digests at Liquid|Liquid Interfaces

Ion-Transfer Voltammetric Behavior of Protein Digests at Liquid|Liquid Interfaces

Quantitative Proteome and Acidic Subproteome Profiling of Candida albicans Yeast-to-Hypha Transition

Genomics, Proteomics, and the Nervous System

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