Optimal Replication and the Importance of Experimental Design for Gel-Based Quantitative Proteomics

Hunt, S. M. N.; Thomas, Mervyn; Sebastian, Lucille; Pedersen, Susanne K.; Harcourt, Rebecca; Sloane, and Andrew J.; Wilkins, Marc R.

doi:10.1021/pr049758y

Cited by 117 publications

(85 citation statements)

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“…Moreover, we performed a power analysis to asses the number of sample replicates that need to be analyzed in order to confidently discover differentially expressed proteins. The accepted power threshold is ≥0.8 [36]. We considered differentially expressed spots those having a q-value ≤0.05 and a power ≥0.8.…”

Section: Image Analysis and Statisticsmentioning

confidence: 99%

Proteomic analysis of A2780/S ovarian cancer cell response to the cytotoxic organogold(III) compound Aubipyc

Gamberi

Massai

Magherini

et al. 2014

Journal of Proteomics

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Aubipy c is an organogold(III) compound endowed with encouraging anti-proliferative properties in vitro that is being evaluated pre-clinically as a prospective anticancer agent. A classical proteomic approach is exploited here to elucidate the mechanisms of its biological actions in A2780 human ovarian cancer cells. Based on 2-D gel electrophoresis separation and subsequent mass spectrometry identification, a considerable number of differentially expressed proteins were highlighted in A2780 cancer cells treated with Aubipy c . Bioinformatic analysis of the groups of up-regulated and down-regulated proteins pointed out that Aubipy c primarily perturbs mitochondrial processes and the glycolytic pathway. Notably, some major alterations in the glycolytic pathway were validated through Western blot and metabolic investigations. Biological significanceThis is the first proteomic analysis regarding Aubipy c cytotoxicity in A2780/S ovarian cancer cell line. Aubipy c is a promising gold(III) compound which manifests an appreciable cytotoxicity toward the cell line A2780, being able to overcome resistance to platinum. The proteomic study revealed for Aubipy c different cellular alterations with respect to cisplatin as well as to other gold compound such as auranofin. Remarkably, the bioinformatic analysis of proteomic data pointed out that Aubipy c treatment affected, directly or indirectly, several glycolytic enzymes. These data suggest a new mechanism of action for this gold drug and might have an impact on the use of gold-based drug in cancer treatment.

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Section: Image Analysis and Statisticsmentioning

confidence: 99%

Proteomic analysis of A2780/S ovarian cancer cell response to the cytotoxic organogold(III) compound Aubipyc

Gamberi

Massai

Magherini

et al. 2014

Journal of Proteomics

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“…The reproducibility of proteomic techniques used, as assayed by regression analysis, co-efficient of variation or other variance estimation techniques [18], is typically not reported. Power analyses, which can be used to infer the number of samples that should be analysed to discover a statistically significant result [18][19][20], are rarely undertaken. Weak experimental designs, particularly in a field where technical challenges remain in the production of high quality data, can make it difficult or impossible to determine if differences reported between two or more samples are likely to reflect variation in a biological system or are solely analytically derived.…”

Section: Experimental Designmentioning

confidence: 99%

Guidelines for the next 10 years of proteomics

Wilkins¹,

Appel²,

Eyk³

et al. 2006

Proteomics

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In the last ten years, the field of proteomics has expanded at a rapid rate. A range of exciting new technology has been developed and enthusiastically applied to an enormous variety of biological questions. However, the degree of stringency required in proteomic data generation and analysis appears to have been underestimated. As a result, there are likely to be numerous published findings that are of questionable quality, requiring further confirmation and/or validation. This manuscript outlines a number of key issues in proteomic research, including those associated with experimental design, differential display and biomarker discovery, protein identification and analytical incompleteness. In an effort to set a standard that reflects current thinking on the necessary and desirable characteristics of publishable manuscripts in the field, a minimal set of guidelines for proteomics research is then described. These guidelines will serve as a set of criteria which editors of PROTEOMICS will use for assessment of future submissions to the Journal.

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“…The number and type of replicates required is influenced by the reproducibility, the optimal sample size and the advantages and limitations of pooling biological samples in proteomic study have been recently addressed [81][82][83][84][85][86].…”

Section: -De Todaymentioning

confidence: 99%

Two‐dimensional gel electrophoresis and mass spectrometry for biomarker discovery

Penque

2009

Proteomics Clinical Apps

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The proteome project, initiated in 1995, was made possible by 2-DE combined with MS. The project main objective was and remains the identification of all proteins expressed by a cell, tissue or organism in a given time and condition. Following this objective, the global profiling of proteins in health versus pathological state by the 2-DE/MS-based proteomic approach has contributed to the elucidation of the basic mechanisms of disease by discovering candidate disease biomarkers and disease targets for new drug development. This review will briefly summarize the historical evolution of 2-DE up to today, and review 2-DE/MS technology and its specific methods of study of immunoresponse (immunoproteomics), PTM of proteins, complex proteinprotein interactions (interactome), the proteome of cell membrane and intracellular proteome turnover in disease biomarker discovery. 2-DE historical evolutionGlobal profiling of proteins in healthy versus pathological states is a strategy to discover biomarkers for early diagnostics, disease progression, treatment response and novel targets for therapeutic drugs development. The idea of making an inventory of all the proteins in a cell, tissue or organism with normal or abnormal physiology, was (re)initiated in the middle of the 1990s with the proteome project [1]. Since then, many technologies to make the analysis of the proteome a reality have been united.The perfect technological 'marriage' that made the proteome project feasible was between high resolution 2-DE for separation of large numbers of proteins and MS for identification and characterization of the proteins displayed on this gel [2]. However, before this happy union occured, science and technology devoted to protein study had a long journey until 2-DE and MS combination was possible (Fig. 1). It started in the last century, in 1937, when Tiselius [3] introduced electrophoresis technique as a modification of Reiner's early concept (1927) [4] to analyse a complex mixture of proteins such as serum proteins. Only 20 years later, in 1959, electrophoresis on a gel support formed by crosslinked polymerization of Cyanogum 41, a commercial name for two organic monomers, acrylamide and the crosslinking agent, N,N1-methylenebisacrylamide, was shown to be useful and was named PAGE by Raymond and Weintraub [5]. The adaptation of polyacrylamide gel to IEF in a stable pH gradient, developed by Svensson in 1962 [6], was possible by the end of the 1960s [7][8][9]. About this time, the 2-D by combining IEF to separate undenatured proteins according to their charge and gradient SDS-PAGE for a second separation, according to their molecular mass, was for the first time introduced by Kenrick and Margolis (1970) [10]. Only in 1975, was IEF in denaturing conditions, as known today, used to follow the 2-D PAGE and it was described in three independent works [11][12][13]. The most powerful demonstraCorrespondence: Dr. Deborah Penque, Laboratório de Proteó-mica, Departamento de Genética, Instituto Nacional de Saúde, Dr. Ricardo Jorge, I.P., A...

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Optimal Replication and the Importance of Experimental Design for Gel-Based Quantitative Proteomics

Cited by 117 publications

References 20 publications

Proteomic analysis of A2780/S ovarian cancer cell response to the cytotoxic organogold(III) compound Aubipyc

Proteomic analysis of A2780/S ovarian cancer cell response to the cytotoxic organogold(III) compound Aubipyc

Guidelines for the next 10 years of proteomics

Two‐dimensional gel electrophoresis and mass spectrometry for biomarker discovery

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