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Increase in local protein concentration by field-inversion gel electrophoresis.

Tsai H, Low TY, Freeby S, Paulus A, Ramnarayanan K, Cheng CP, Leung HC - Proteome Sci (2007)

Bottom Line: Band intensities of proteins in FIGE with appropriate ratios of forward and backward pulse times were superior to CFE despite longer running times.These results revealed an increase in band intensity per defined gel volume.Native protein complexes ranging from 800 kDa to larger than 2000 kDa became apparent using FIGE compared with CFE.

View Article: PubMed Central - HTML - PubMed

Affiliation: Medical Proteomics and Bioanalysis Section, Genome Institute of Singapore, Singapore. tsaihh@gis.a-star.edu.sg.

ABSTRACT

Background: Proteins that migrate through cross-linked polyacrylamide gels (PAGs) under the influence of a constant electric field experience negative factors, such as diffusion and non-specific trapping in the gel matrix. These negative factors reduce protein concentrations within a defined gel volume with increasing migration distance and, therefore, decrease protein separation efficiency. Enhancement of protein separation efficiency was investigated by implementing pulsed field-inversion gel electrophoresis (FIGE).

Results: Separation of model protein species and large protein complexes was compared between FIGE and constant field electrophoresis (CFE) in different percentages of PAGs. Band intensities of proteins in FIGE with appropriate ratios of forward and backward pulse times were superior to CFE despite longer running times. These results revealed an increase in band intensity per defined gel volume. A biphasic protein relative mobility shift was observed in percentages of PAGs up to 14%. However, the effect of FIGE on protein separation was stochastic at higher PAG percentage. Rat liver lysates subjected to FIGE in the second-dimension separation of two-dimensional polyarcylamide gel electrophoresis (2D PAGE) showed a 20% increase in the number of discernible spots compared with CFE. Nine common spots from both FIGE and CFE were selected for peptide sequencing by mass spectrometry (MS), which revealed higher final ion scores of all nine protein spots from FIGE. Native protein complexes ranging from 800 kDa to larger than 2000 kDa became apparent using FIGE compared with CFE.

Conclusion: The present investigation suggests that FIGE under appropriate conditions improves protein separation efficiency during PAGE as a result of increased local protein concentration. FIGE can be implemented with minimal additional instrumentation in any laboratory setting. Despite the tradeoff of longer running times, FIGE can be a powerful protein separation tool.

No MeSH data available.


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Effects of FIGE on 2D PAGE analysis of rat liver lysates. Comparison of 2D PAGE images of rat liver lysate under FIGE (a) and CFE (b) conditions. Each gel represents 100 μg of rat liver lysate separated by isoelectric focusing (IEF) using a non-linear pH 3–10 IPG strip in the first dimension and a Criterion precast SDS-10–20% PAG in the second dimension at room temperature. Control denotes CFE and pulsed denotes FIGE with a ta/tr of 400/106 msec in the second-dimension separation. Gels were stained with Coomassie blue. Spots selected for LC-MS/MS analysis are denoted by numbers, and A denotes the internal calibrator (see also Table 3). The internal calibrator is the internal controls for equivalent sample loading in both control and pulsed conditions.
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Figure 4: Effects of FIGE on 2D PAGE analysis of rat liver lysates. Comparison of 2D PAGE images of rat liver lysate under FIGE (a) and CFE (b) conditions. Each gel represents 100 μg of rat liver lysate separated by isoelectric focusing (IEF) using a non-linear pH 3–10 IPG strip in the first dimension and a Criterion precast SDS-10–20% PAG in the second dimension at room temperature. Control denotes CFE and pulsed denotes FIGE with a ta/tr of 400/106 msec in the second-dimension separation. Gels were stained with Coomassie blue. Spots selected for LC-MS/MS analysis are denoted by numbers, and A denotes the internal calibrator (see also Table 3). The internal calibrator is the internal controls for equivalent sample loading in both control and pulsed conditions.

Mentions: We tested the applicability of FIGE in proteomics studies such as the 2D PAGE analysis of rat liver lysates with pulsing in the second dimension. The results were subsequently analyzed by image densitometry for spot number and intensity. Image analysis concluded that pulsing increased the overall number of spots by more than 20% as well as a general increase in spot intensity over CFE (Figure 4a and 4b). The total number of spots detected after CFE and FIGE were 477 and 611, respectively. In another set of experiments, gels were stained with Coomassie blue stain and the total number of detectable spots after CFE and FIGE were 190 and 240, respectively (data not shown). These results translated into a 26% increase in the total number of detectable spots.


Increase in local protein concentration by field-inversion gel electrophoresis.

Tsai H, Low TY, Freeby S, Paulus A, Ramnarayanan K, Cheng CP, Leung HC - Proteome Sci (2007)

Effects of FIGE on 2D PAGE analysis of rat liver lysates. Comparison of 2D PAGE images of rat liver lysate under FIGE (a) and CFE (b) conditions. Each gel represents 100 μg of rat liver lysate separated by isoelectric focusing (IEF) using a non-linear pH 3–10 IPG strip in the first dimension and a Criterion precast SDS-10–20% PAG in the second dimension at room temperature. Control denotes CFE and pulsed denotes FIGE with a ta/tr of 400/106 msec in the second-dimension separation. Gels were stained with Coomassie blue. Spots selected for LC-MS/MS analysis are denoted by numbers, and A denotes the internal calibrator (see also Table 3). The internal calibrator is the internal controls for equivalent sample loading in both control and pulsed conditions.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2211458&req=5

Figure 4: Effects of FIGE on 2D PAGE analysis of rat liver lysates. Comparison of 2D PAGE images of rat liver lysate under FIGE (a) and CFE (b) conditions. Each gel represents 100 μg of rat liver lysate separated by isoelectric focusing (IEF) using a non-linear pH 3–10 IPG strip in the first dimension and a Criterion precast SDS-10–20% PAG in the second dimension at room temperature. Control denotes CFE and pulsed denotes FIGE with a ta/tr of 400/106 msec in the second-dimension separation. Gels were stained with Coomassie blue. Spots selected for LC-MS/MS analysis are denoted by numbers, and A denotes the internal calibrator (see also Table 3). The internal calibrator is the internal controls for equivalent sample loading in both control and pulsed conditions.
Mentions: We tested the applicability of FIGE in proteomics studies such as the 2D PAGE analysis of rat liver lysates with pulsing in the second dimension. The results were subsequently analyzed by image densitometry for spot number and intensity. Image analysis concluded that pulsing increased the overall number of spots by more than 20% as well as a general increase in spot intensity over CFE (Figure 4a and 4b). The total number of spots detected after CFE and FIGE were 477 and 611, respectively. In another set of experiments, gels were stained with Coomassie blue stain and the total number of detectable spots after CFE and FIGE were 190 and 240, respectively (data not shown). These results translated into a 26% increase in the total number of detectable spots.

Bottom Line: Band intensities of proteins in FIGE with appropriate ratios of forward and backward pulse times were superior to CFE despite longer running times.These results revealed an increase in band intensity per defined gel volume.Native protein complexes ranging from 800 kDa to larger than 2000 kDa became apparent using FIGE compared with CFE.

View Article: PubMed Central - HTML - PubMed

Affiliation: Medical Proteomics and Bioanalysis Section, Genome Institute of Singapore, Singapore. tsaihh@gis.a-star.edu.sg.

ABSTRACT

Background: Proteins that migrate through cross-linked polyacrylamide gels (PAGs) under the influence of a constant electric field experience negative factors, such as diffusion and non-specific trapping in the gel matrix. These negative factors reduce protein concentrations within a defined gel volume with increasing migration distance and, therefore, decrease protein separation efficiency. Enhancement of protein separation efficiency was investigated by implementing pulsed field-inversion gel electrophoresis (FIGE).

Results: Separation of model protein species and large protein complexes was compared between FIGE and constant field electrophoresis (CFE) in different percentages of PAGs. Band intensities of proteins in FIGE with appropriate ratios of forward and backward pulse times were superior to CFE despite longer running times. These results revealed an increase in band intensity per defined gel volume. A biphasic protein relative mobility shift was observed in percentages of PAGs up to 14%. However, the effect of FIGE on protein separation was stochastic at higher PAG percentage. Rat liver lysates subjected to FIGE in the second-dimension separation of two-dimensional polyarcylamide gel electrophoresis (2D PAGE) showed a 20% increase in the number of discernible spots compared with CFE. Nine common spots from both FIGE and CFE were selected for peptide sequencing by mass spectrometry (MS), which revealed higher final ion scores of all nine protein spots from FIGE. Native protein complexes ranging from 800 kDa to larger than 2000 kDa became apparent using FIGE compared with CFE.

Conclusion: The present investigation suggests that FIGE under appropriate conditions improves protein separation efficiency during PAGE as a result of increased local protein concentration. FIGE can be implemented with minimal additional instrumentation in any laboratory setting. Despite the tradeoff of longer running times, FIGE can be a powerful protein separation tool.

No MeSH data available.


Related in: MedlinePlus