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Evaluating the performance of new approaches to spot quantification and differential expression in 2-dimensional gel electrophoresis studies.

ABSTRACT: Spot detection and quantification for 2-DE are challenging and important tasks to fully extract the proteomic information from these data. Traditional analytical methods have significant weaknesses, including spot mismatching and missing data, which require time-consuming manual editing to correct, dramatically decreasing throughput and compromising the objectivity and reproducibility of the analysis. To address this issue, we developed Pinnacle, a novel, quick, automatic, noncommercial method that borrows strength across gels in spot detection and has been shown to yield more precise spot quantifications than traditional methods. New commercial software, notably SameSpots, has also recently been developed as an improvement over traditional workflows. In this paper, we briefly describe Pinnacle and compare its performance to SameSpots in spot detection, spot quantification precision, and differential expression. Our analysis is performed in a rigorous fashion that, unlike other comparisons in the literature, summarizes performance across all spots detected on the gels, and we manually optimize SameSpots results while simply running Pinnacle with standard settings and no manual editing. While both methods showed marked improvement over a commercially available traditional method PG240, Pinnacle consistently yielded spot quantifications with greater validity and reliability, avoided spot delineation problems, and detected more differentially expressed proteins than SameSpots, and represents a significant noncommercial alternative for 2-DE processing.

SUBMITTER: Morris JS

PROVIDER: S-EPMC2802214 | biostudies-other | 2010 Jan

REPOSITORIES: biostudies-other

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Evaluating the performance of new approaches to spot quantification and differential expression in 2-dimensional gel electrophoresis studies.

Morris Jeffrey S JS Clark Brittan N BN Wei Wei W Gutstein Howard B HB

Journal of proteome research 20100101 1

Spot detection and quantification for 2-DE are challenging and important tasks to fully extract the proteomic information from these data. Traditional analytical methods have significant weaknesses, including spot mismatching and missing data, which require time-consuming manual editing to correct, dramatically decreasing throughput and compromising the objectivity and reproducibility of the analysis. To address this issue, we developed Pinnacle, a novel, quick, automatic, noncommercial method t ...[more]

PMID: 19919108

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Project description:BackgroundProteome analysis is frequently applied in identifying the proteins or biomarkers in knee synovial fluids (SF) that are associated with osteoarthritis and other arthritic disorders. The 2-dimensional gel electrophoresis (2-DE) is the technique of choice in these studies. Disease biomarkers usually appear in low concentrations and may be masked by high abundant proteins. Therefore, the main aim of this study was to find the most suitable sample preparation method that can optimize the expression of proteins on 2-DE gels that can be used to develop a reference proteome picture for non-osteoarthritic knee synovial fluid samples. Proteome pictures obtained from osteoarthritic knee synovial fluids can then be compared with the reference proteome pictures obtained in this study to assist us in identifying the disease biomarkers more correctly.ResultsThe proteomic tool of 2-DE with immobilized pH gradients was applied in this study. A total of 12 2-DE gel images were constructed from SF samples that were free of osteoarthritis. In these samples, 3 were not treated with any sample preparation methods, 3 were treated with acetone, 3 were treated with 2-DE Clean-Up Kit, and 3 were treated with the combination of acetone and 2-D Clean-Up Kit prior to 2-DE analysis. Gel images were analyzed using the PDQuest Basic 8.0.1 Analytical software. Protein spots that were of interest were excised from the gels and sent for identification by mass spectrometry. Total SF total protein concentration was calculated to be 21.98 ± 0.86 mg/mL. The untreated SF samples were detected to have 456 ± 33 protein spots on 2-DE gel images. Acetone treated SF samples were detected to have 320 ± 28 protein spots, 2-D Clean-Up Kit treated SF samples were detected to have 413 ± 31 protein spots, and the combined treatment method of acetone and 2-D Clean-Up Kit was detected to have 278 ± 26 protein spots 2-DE gel images. SF samples treated with 2-D Clean-Up Kit revealed clearer presentation of the isoforms and increased intensities of the less abundant proteins of haptoglobin, apolipoprotein A-IV, prostaglandin-D synthase, alpha-1B-glycoprotein, and alpha-2-HS-glycoprotein on 2-DE gel images as compared with untreated SF samples and SF samples treated with acetone.ConclusionsThe acetone precipitation method and the combined treatment effect of acetone and 2-DE Clean-Up Kit are not preferred in preparing SF samples for 2-DE analysis as both protein intensities and numbers decrease significantly. On the other hand, 2-D Clean-Up Kit treated SF samples revealed clearer isoforms and higher intensities for the less abundant proteins of haptoglobin, apolipoprotein A-IV, prostaglandin-D synthase, alpha-1B-glycoprotein, and alpha-2-HS-glycoprotein on 2-DE gels. As a result, it is recommended that SF samples should be treated with protein clean up products such as 2-D Clean-Up Kit first before conducting proteomic research in searching for the relevant biomarkers associated with knee osteoarthritis.

Dataset Information

Evaluating the performance of new approaches to spot quantification and differential expression in 2-dimensional gel electrophoresis studies.

Publications

Evaluating the performance of new approaches to spot quantification and differential expression in 2-dimensional gel electrophoresis studies.

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