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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

Experimental design. Experimental design of multidimensional fractionation using plasma from ALS patients involved in a phase II clinical trial with GA.
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Figure 1: Experimental design. Experimental design of multidimensional fractionation using plasma from ALS patients involved in a phase II clinical trial with GA.

Mentions: Patient serum/plasma represents clinical material that is easily obtained with fewer restrictions compared to other sample types, including cerebrospinal fluid or tissue biopsy; thus, it is one of the most commonly tested patient material from which diagnostic tests are performed. Collectively, protein concentrations span a very broad range (1012-fold) [20] in serum/plasma from which the differential concentration in individual protein concentrations presents potential targets for the discovery of clinically important biomarkers. However, the presence of very highly abundant proteins and the complexity of plasma proteins present formidable challenges. Twelve of the most abundant proteins comprise ~96% of the total protein mass from human plasma, with albumin comprising approximately 40–50% of protein. Presence of these abundant proteins in plasma samples masks differential levels of low to medium abundant ones. One strategy is to remove the most abundant proteins prior to profiling. To assess that approach, we used immunoaffinity chromatography with a column that is based on IgY technology to selectively remove 12 of the most abundant proteins in human serum/plasma (Figure 1, experimental design). One caveat of immunodepletion is that potential biomarkers that bind to albumin or highly abundant proteins may also be completely or partially depleted from serum samples through protein-protein interactions. However, this possibility can be evaluated with further analyses upon elution of the adherent protein fraction. Although the IgY-12 LC10 affinity column has the highest capacity of commercial immunoaffinity products currently available, only 250 μL of plasma sample can be processed during one chromatographic cycle. Flow through fractions (8–27 minutes) containing unbound proteins (Figure 2) were collected, pooled, concentrated, and submitted for analysis on the PF 2D platform. Protein yields from the flow through fractions were between 0.73 and 3.0 mg per mL of 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)

Experimental design. Experimental design of multidimensional fractionation using plasma from ALS patients involved in a phase II clinical trial with GA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Experimental design. Experimental design of multidimensional fractionation using plasma from ALS patients involved in a phase II clinical trial with GA.
Mentions: Patient serum/plasma represents clinical material that is easily obtained with fewer restrictions compared to other sample types, including cerebrospinal fluid or tissue biopsy; thus, it is one of the most commonly tested patient material from which diagnostic tests are performed. Collectively, protein concentrations span a very broad range (1012-fold) [20] in serum/plasma from which the differential concentration in individual protein concentrations presents potential targets for the discovery of clinically important biomarkers. However, the presence of very highly abundant proteins and the complexity of plasma proteins present formidable challenges. Twelve of the most abundant proteins comprise ~96% of the total protein mass from human plasma, with albumin comprising approximately 40–50% of protein. Presence of these abundant proteins in plasma samples masks differential levels of low to medium abundant ones. One strategy is to remove the most abundant proteins prior to profiling. To assess that approach, we used immunoaffinity chromatography with a column that is based on IgY technology to selectively remove 12 of the most abundant proteins in human serum/plasma (Figure 1, experimental design). One caveat of immunodepletion is that potential biomarkers that bind to albumin or highly abundant proteins may also be completely or partially depleted from serum samples through protein-protein interactions. However, this possibility can be evaluated with further analyses upon elution of the adherent protein fraction. Although the IgY-12 LC10 affinity column has the highest capacity of commercial immunoaffinity products currently available, only 250 μL of plasma sample can be processed during one chromatographic cycle. Flow through fractions (8–27 minutes) containing unbound proteins (Figure 2) were collected, pooled, concentrated, and submitted for analysis on the PF 2D platform. Protein yields from the flow through fractions were between 0.73 and 3.0 mg per mL of 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