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Multidimensional protein fractionation using ProteomeLab PF 2D for profiling amyotrophic lateral sclerosis immunity: A preliminary report.

Schlautman JD, Rozek W, Stetler R, Mosley RL, Gendelman HE, Ciborowski P - Proteome Sci (2008)

Bottom Line: Analysis of differences in the resulting two-dimensional maps of fractions obtained from the PF 2D and the ability to identify proteins from these fractions allowed sensitivity threshold measurements.Masked proteins in the PF 2D fractions are discussed.We offer some insight into the strengths and limitations of this emerging proteomic platform.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, USA. jschlautman@unmc.edu

ABSTRACT

Background: The ProteomeLab PF 2D platform is a relatively new approach to global protein profiling. Herein, it was used for investigation of plasma proteome changes in amyotrophic lateral sclerosis (ALS) patients before and during immunization with glatiramer acetate (GA) in a clinical trial.

Results: The experimental design included immunoaffinity depletion of 12 most abundant proteins from plasma samples with the ProteomeLab IgY-12 LC10 column kit as first dimension separation, also referred to as immuno-partitioning. Second and third dimension separations of the enriched proteome were performed on the PF 2D platform utilizing 2D isoelectric focusing and RP-HPLC with the resulting fractions collected for analysis. 1D gel electrophoresis was added as a fourth dimension when sufficient protein was available. Protein identification from collected fractions was performed using nano-LC-MS/MS approach. Analysis of differences in the resulting two-dimensional maps of fractions obtained from the PF 2D and the ability to identify proteins from these fractions allowed sensitivity threshold measurements. Masked proteins in the PF 2D fractions are discussed.

Conclusion: We offer some insight into the strengths and limitations of this emerging proteomic platform.

No MeSH data available.


Related in: MedlinePlus

Results of profiling analysis. A. PF 2D two-dimensional heat maps of a representative set of samples obtained from one individual before and after GA immunization. PF 2D first dimension separation is based on isoelectric point (pI). PF 2D second dimension separation utilizes reverse phase HPLC fractionation. B. Comparison of two aligned peaks from analyses shown in (A) displaying a quantitative difference in protein contents measured by peak area (volume). Colour scheme ranges from purple (low absorbance) to red (high absorbance). The difference between absorbencies is shown in the middle as either a red or green band, representing the sample with greater absorbance at a specific peak.
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Figure 3: Results of profiling analysis. A. PF 2D two-dimensional heat maps of a representative set of samples obtained from one individual before and after GA immunization. PF 2D first dimension separation is based on isoelectric point (pI). PF 2D second dimension separation utilizes reverse phase HPLC fractionation. B. Comparison of two aligned peaks from analyses shown in (A) displaying a quantitative difference in protein contents measured by peak area (volume). Colour scheme ranges from purple (low absorbance) to red (high absorbance). The difference between absorbencies is shown in the middle as either a red or green band, representing the sample with greater absorbance at a specific peak.

Mentions: The proteomic profiling platform ProteomeLabâ„¢ PF 2D offers 2-dimensional fractionation in which intact proteins are first separated by chromatofocusing proteins by pI and separated in the second dimension by their hydrophobic properties. The pH profiles from the chromatofocusing absorbencies were obtained from first dimension separation at 280 nm and thirty fractions were selected from each sample to submit for second dimension separation by hydrophobic chromatography. Second dimension absorbance profiles were compiled and displayed as a two-dimensional map using a feature of Mapping Tools software. The map displays pI fractions as lanes with the colour intensity of each band (absorbance at 214 nm) corresponding to protein bands located at their retention time of the second dimension separation (Figure 3A and 3B, Table 1).


Multidimensional protein fractionation using ProteomeLab PF 2D for profiling amyotrophic lateral sclerosis immunity: A preliminary report.

Schlautman JD, Rozek W, Stetler R, Mosley RL, Gendelman HE, Ciborowski P - Proteome Sci (2008)

Results of profiling analysis. A. PF 2D two-dimensional heat maps of a representative set of samples obtained from one individual before and after GA immunization. PF 2D first dimension separation is based on isoelectric point (pI). PF 2D second dimension separation utilizes reverse phase HPLC fractionation. B. Comparison of two aligned peaks from analyses shown in (A) displaying a quantitative difference in protein contents measured by peak area (volume). Colour scheme ranges from purple (low absorbance) to red (high absorbance). The difference between absorbencies is shown in the middle as either a red or green band, representing the sample with greater absorbance at a specific peak.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2543004&req=5

Figure 3: Results of profiling analysis. A. PF 2D two-dimensional heat maps of a representative set of samples obtained from one individual before and after GA immunization. PF 2D first dimension separation is based on isoelectric point (pI). PF 2D second dimension separation utilizes reverse phase HPLC fractionation. B. Comparison of two aligned peaks from analyses shown in (A) displaying a quantitative difference in protein contents measured by peak area (volume). Colour scheme ranges from purple (low absorbance) to red (high absorbance). The difference between absorbencies is shown in the middle as either a red or green band, representing the sample with greater absorbance at a specific peak.
Mentions: The proteomic profiling platform ProteomeLabâ„¢ PF 2D offers 2-dimensional fractionation in which intact proteins are first separated by chromatofocusing proteins by pI and separated in the second dimension by their hydrophobic properties. The pH profiles from the chromatofocusing absorbencies were obtained from first dimension separation at 280 nm and thirty fractions were selected from each sample to submit for second dimension separation by hydrophobic chromatography. Second dimension absorbance profiles were compiled and displayed as a two-dimensional map using a feature of Mapping Tools software. The map displays pI fractions as lanes with the colour intensity of each band (absorbance at 214 nm) corresponding to protein bands located at their retention time of the second dimension separation (Figure 3A and 3B, Table 1).

Bottom Line: Analysis of differences in the resulting two-dimensional maps of fractions obtained from the PF 2D and the ability to identify proteins from these fractions allowed sensitivity threshold measurements.Masked proteins in the PF 2D fractions are discussed.We offer some insight into the strengths and limitations of this emerging proteomic platform.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, USA. jschlautman@unmc.edu

ABSTRACT

Background: The ProteomeLab PF 2D platform is a relatively new approach to global protein profiling. Herein, it was used for investigation of plasma proteome changes in amyotrophic lateral sclerosis (ALS) patients before and during immunization with glatiramer acetate (GA) in a clinical trial.

Results: The experimental design included immunoaffinity depletion of 12 most abundant proteins from plasma samples with the ProteomeLab IgY-12 LC10 column kit as first dimension separation, also referred to as immuno-partitioning. Second and third dimension separations of the enriched proteome were performed on the PF 2D platform utilizing 2D isoelectric focusing and RP-HPLC with the resulting fractions collected for analysis. 1D gel electrophoresis was added as a fourth dimension when sufficient protein was available. Protein identification from collected fractions was performed using nano-LC-MS/MS approach. Analysis of differences in the resulting two-dimensional maps of fractions obtained from the PF 2D and the ability to identify proteins from these fractions allowed sensitivity threshold measurements. Masked proteins in the PF 2D fractions are discussed.

Conclusion: We offer some insight into the strengths and limitations of this emerging proteomic platform.

No MeSH data available.


Related in: MedlinePlus