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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

1D Electrophoresis. 1D electrophoresis (fourth dimension fractionation) showing differential expression of protein band above 62 kDa m.w. marker. Hemopexin was identified as the most prominent protein in this band by LCMS/MS sequencing. Equal amount of protein (based on absorbance at 214 nm) from each sample was loaded per lane. Gel was stained with SyproRuby.
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Figure 5: 1D Electrophoresis. 1D electrophoresis (fourth dimension fractionation) showing differential expression of protein band above 62 kDa m.w. marker. Hemopexin was identified as the most prominent protein in this band by LCMS/MS sequencing. Equal amount of protein (based on absorbance at 214 nm) from each sample was loaded per lane. Gel was stained with SyproRuby.

Mentions: One of the advantages of the PF 2D profiling platform is a possibility of fractionation in the fourth dimension. In our experimental design, IgY immunodepletion (partitioning) served as first dimension partitioning, isoelectric focusing provided second dimension analysis, and RP-HPLC yielded the third dimension. Several fractions after 3 dimensional analyses were selected for a fourth dimension, 1-dimensional electrophoresis (1DE), to evaluate whether the fraction isolated as a single peak consisted of only one protein. Frequently, fractions (covering a retention time of 0.67 min) with high peak height contain several proteins whose quantities are opposite to each other, thus masking differential expression. We selected one matching fraction from all six samples (pH 5.9 to 5.6, retention time 16.04 to 16.71 min.) with peak height ranging between 1.11 and 1.76 (Table 1). Analysis of these six matching fractions based on levels of absorbance after 3-dimensional fractionation did not demonstrate statistical differences between fractions, thus indicating that the total amount of protein in each fraction was the same. We expected and further confirmed by 1 DE that these fractions consisted of multiple, non-separated, proteins. Therefore, in a subsequent step, we analyzed by 1 DE equal amounts of each fraction containing equal amounts of protein based on absorbance at 214 nm. As expected, this analysis showed multiple bands in each fraction (Figure 5). A characteristic pattern showed increased intensity of a protein band with a molecular mass above 62 kDa in samples from immunized patients. It is possible that using narrower pH fractionation, e.g. 0.1 units instead of 0.3 units used in this study, would help to further separate proteins. This band was identified as hemopexin based on nano-LC-MS/MS analysis of peptides derived from tryptic digests and demonstrates that levels of this protein are increased in patients immunized with GA. The most extensively studied function of hemopexin is its binding heme, having the highest affinity of any known protein. Also, as a heme scavenger, hemopexin protects organisms from the oxidative damage that can be caused by free heme. Interestingly, histidine-rich glycoprotein precursor, whose function is not understood and which has been found in this study, is also a heme binding protein. Both proteins are made by liver and secreted to plasma.


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)

1D Electrophoresis. 1D electrophoresis (fourth dimension fractionation) showing differential expression of protein band above 62 kDa m.w. marker. Hemopexin was identified as the most prominent protein in this band by LCMS/MS sequencing. Equal amount of protein (based on absorbance at 214 nm) from each sample was loaded per lane. Gel was stained with SyproRuby.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: 1D Electrophoresis. 1D electrophoresis (fourth dimension fractionation) showing differential expression of protein band above 62 kDa m.w. marker. Hemopexin was identified as the most prominent protein in this band by LCMS/MS sequencing. Equal amount of protein (based on absorbance at 214 nm) from each sample was loaded per lane. Gel was stained with SyproRuby.
Mentions: One of the advantages of the PF 2D profiling platform is a possibility of fractionation in the fourth dimension. In our experimental design, IgY immunodepletion (partitioning) served as first dimension partitioning, isoelectric focusing provided second dimension analysis, and RP-HPLC yielded the third dimension. Several fractions after 3 dimensional analyses were selected for a fourth dimension, 1-dimensional electrophoresis (1DE), to evaluate whether the fraction isolated as a single peak consisted of only one protein. Frequently, fractions (covering a retention time of 0.67 min) with high peak height contain several proteins whose quantities are opposite to each other, thus masking differential expression. We selected one matching fraction from all six samples (pH 5.9 to 5.6, retention time 16.04 to 16.71 min.) with peak height ranging between 1.11 and 1.76 (Table 1). Analysis of these six matching fractions based on levels of absorbance after 3-dimensional fractionation did not demonstrate statistical differences between fractions, thus indicating that the total amount of protein in each fraction was the same. We expected and further confirmed by 1 DE that these fractions consisted of multiple, non-separated, proteins. Therefore, in a subsequent step, we analyzed by 1 DE equal amounts of each fraction containing equal amounts of protein based on absorbance at 214 nm. As expected, this analysis showed multiple bands in each fraction (Figure 5). A characteristic pattern showed increased intensity of a protein band with a molecular mass above 62 kDa in samples from immunized patients. It is possible that using narrower pH fractionation, e.g. 0.1 units instead of 0.3 units used in this study, would help to further separate proteins. This band was identified as hemopexin based on nano-LC-MS/MS analysis of peptides derived from tryptic digests and demonstrates that levels of this protein are increased in patients immunized with GA. The most extensively studied function of hemopexin is its binding heme, having the highest affinity of any known protein. Also, as a heme scavenger, hemopexin protects organisms from the oxidative damage that can be caused by free heme. Interestingly, histidine-rich glycoprotein precursor, whose function is not understood and which has been found in this study, is also a heme binding protein. Both proteins are made by liver and secreted to plasma.

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