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Microchamber integration unifies distinct separation modes for two-dimensional electrophoresis.

ABSTRACT: By combining isoelectric focusing (IEF) with subsequent gel electrophoresis, two-dimensional electrophoresis (2DE) affords more specific characterization of proteins than each constituent unit separation. In a new approach to integrating the two assay dimensions in a microscope slide-sized glass device, we introduce microfluidic 2DE using photopatterned polyacrylamide (PA) gel elements housed in a millimeter-scale, 20-?m-deep chamber. The microchamber minimizes information loss inherent to channel network architectures commonly used for microfluidic 2DE. To define the IEF axis along a "lane" at the top of the chamber, we used free solution carrier ampholytes and immobilized acrylamido buffers in the PA gels. This approach yielded high-resolution (0.1 pH unit) and rapid (<20 min) IEF. Next, protein transfer to the second dimension was accomplished by chemical mobilization perpendicular to the IEF axis. Mobilization drove focused proteins off the IEF lane and into a region for protein gel electrophoresis. Using fluorescently labeled proteins, we observed transfer-induced band broadening factors ~7.5-fold lower than those observed in microchannel networks. Both native polyacrylamide gel electrophoresis (PAGE) and pore-limit electrophoresis (PLE) were studied as the second assay dimension and completed in <15 min. PLE yields protein molecular mass information without the need for ionic surfactant or reducing agents, simplifying device design and operation. Microchamber-based 2DE unifies two independent separation dimensions in a single device with minimal transfer-associated information losses. Peak capacities for the total assay ranged from 256 to 35 with <1 h assay duration. The rapid microchamber 2DE assay has the potential to bridge an existing gap in targeted proteomics for protein biomarker validation and systems biology that may complement recent innovation in mass spectrometry.

SUBMITTER: Tentori AM

PROVIDER: S-EPMC3714212 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Microchamber integration unifies distinct separation modes for two-dimensional electrophoresis.

Tentori Augusto M AM Hughes Alex J AJ Herr Amy E AE

Analytical chemistry 20130424 9

By combining isoelectric focusing (IEF) with subsequent gel electrophoresis, two-dimensional electrophoresis (2DE) affords more specific characterization of proteins than each constituent unit separation. In a new approach to integrating the two assay dimensions in a microscope slide-sized glass device, we introduce microfluidic 2DE using photopatterned polyacrylamide (PA) gel elements housed in a millimeter-scale, 20-μm-deep chamber. The microchamber minimizes information loss inherent to chann ...[more]

PMID: 23565932

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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.

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Microchamber integration unifies distinct separation modes for two-dimensional electrophoresis.

Publications

Microchamber integration unifies distinct separation modes for two-dimensional electrophoresis.

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