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APols-aided protein precipitation: a rapid method for concentrating proteins for proteomic analysis.

Ning Z, Hawley B, Seebun D, Figeys D - J. Membr. Biol. (2014)

Bottom Line: In contrast with tryptic peptides, a decrease in pH leads to the unbiased co-precipitation of APols with proteins, including globular hydrophilic proteins.We demonstrated that this precipitation is a combined effect of acid precipitation and the APols' protein interactions.Also, we have been able to demonstrate that APols-aided protein precipitation works well on diluted samples, such as secretome sample, and provides a rapid method for protein concentration.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Immunology and Microbiology, Ottawa Institute of Systems Biology, University of Ottawa, Ottawa, ON, Canada.

ABSTRACT
Amphipols (APols) are a newly designed and milder class of detergent. They have been used primarily in protein structure analysis for membrane protein trapping and stabilization. We have recently demonstrated that APols can be used as an alternative detergent for proteome extraction and digestion, to achieve a "One-stop" single-tube workflow for proteomics. In this workflow, APols are removed by precipitation after protein digestion without depleting the digested peptides. Here, we took further advantage of this precipitation characteristic of APols to concentrate proteins from diluted samples. In contrast with tryptic peptides, a decrease in pH leads to the unbiased co-precipitation of APols with proteins, including globular hydrophilic proteins. We demonstrated that this precipitation is a combined effect of acid precipitation and the APols' protein interactions. Also, we have been able to demonstrate that APols-aided protein precipitation works well on diluted samples, such as secretome sample, and provides a rapid method for protein concentration.

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Effect of APols concentration on protein precipitation efficiency. Aliquot of HEK293T cells was lysed using 200 µL 0.05, 0.2, 1, or 2 mg/mL APols in 50 mM ABC to achieve a concentration of 2.5 mg/mL, then precipitated and reconstituted. The same volumes of the total lysate, supernatant, and reconstituted precipitates were analyzed by SDS-PAGE
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Fig3: Effect of APols concentration on protein precipitation efficiency. Aliquot of HEK293T cells was lysed using 200 µL 0.05, 0.2, 1, or 2 mg/mL APols in 50 mM ABC to achieve a concentration of 2.5 mg/mL, then precipitated and reconstituted. The same volumes of the total lysate, supernatant, and reconstituted precipitates were analyzed by SDS-PAGE

Mentions: We then tested whether the concentration of APols had an effect on the precipitation of proteins. A dependence on concentration would likely indicate that APols predominantly bind to proteins and then help in their precipitation, whereas, an independence from concentration would indicate that the precipitation of APols predominantly leads to the co-precipitation of proteins. We lowered the APols concentration gradually in the presence of a constant protein concentration, to see whether the concentration of APols (or APols to protein ratio) would have an effect on the quantity of protein precipitated. Our results indicated no significant differences in protein precipitations using 2 mg/mL (14:1 APols:Protein mass ratio) down to 0.05 mg/mL (0.3:1 APols:protein ratio) of APols as shown in Fig. 3. Therefore, the amount of protein precipitated does not appear to be different even when the concentration of APols is approximately three times lower than the proteins. We believe this points to a co-precipitation phenomenon. This also means that less APols can be used for protein precipitation from complex samples, which is important when dealing with large volume and/or dilute protein samples.Fig. 3


APols-aided protein precipitation: a rapid method for concentrating proteins for proteomic analysis.

Ning Z, Hawley B, Seebun D, Figeys D - J. Membr. Biol. (2014)

Effect of APols concentration on protein precipitation efficiency. Aliquot of HEK293T cells was lysed using 200 µL 0.05, 0.2, 1, or 2 mg/mL APols in 50 mM ABC to achieve a concentration of 2.5 mg/mL, then precipitated and reconstituted. The same volumes of the total lysate, supernatant, and reconstituted precipitates were analyzed by SDS-PAGE
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4196042&req=5

Fig3: Effect of APols concentration on protein precipitation efficiency. Aliquot of HEK293T cells was lysed using 200 µL 0.05, 0.2, 1, or 2 mg/mL APols in 50 mM ABC to achieve a concentration of 2.5 mg/mL, then precipitated and reconstituted. The same volumes of the total lysate, supernatant, and reconstituted precipitates were analyzed by SDS-PAGE
Mentions: We then tested whether the concentration of APols had an effect on the precipitation of proteins. A dependence on concentration would likely indicate that APols predominantly bind to proteins and then help in their precipitation, whereas, an independence from concentration would indicate that the precipitation of APols predominantly leads to the co-precipitation of proteins. We lowered the APols concentration gradually in the presence of a constant protein concentration, to see whether the concentration of APols (or APols to protein ratio) would have an effect on the quantity of protein precipitated. Our results indicated no significant differences in protein precipitations using 2 mg/mL (14:1 APols:Protein mass ratio) down to 0.05 mg/mL (0.3:1 APols:protein ratio) of APols as shown in Fig. 3. Therefore, the amount of protein precipitated does not appear to be different even when the concentration of APols is approximately three times lower than the proteins. We believe this points to a co-precipitation phenomenon. This also means that less APols can be used for protein precipitation from complex samples, which is important when dealing with large volume and/or dilute protein samples.Fig. 3

Bottom Line: In contrast with tryptic peptides, a decrease in pH leads to the unbiased co-precipitation of APols with proteins, including globular hydrophilic proteins.We demonstrated that this precipitation is a combined effect of acid precipitation and the APols' protein interactions.Also, we have been able to demonstrate that APols-aided protein precipitation works well on diluted samples, such as secretome sample, and provides a rapid method for protein concentration.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Immunology and Microbiology, Ottawa Institute of Systems Biology, University of Ottawa, Ottawa, ON, Canada.

ABSTRACT
Amphipols (APols) are a newly designed and milder class of detergent. They have been used primarily in protein structure analysis for membrane protein trapping and stabilization. We have recently demonstrated that APols can be used as an alternative detergent for proteome extraction and digestion, to achieve a "One-stop" single-tube workflow for proteomics. In this workflow, APols are removed by precipitation after protein digestion without depleting the digested peptides. Here, we took further advantage of this precipitation characteristic of APols to concentrate proteins from diluted samples. In contrast with tryptic peptides, a decrease in pH leads to the unbiased co-precipitation of APols with proteins, including globular hydrophilic proteins. We demonstrated that this precipitation is a combined effect of acid precipitation and the APols' protein interactions. Also, we have been able to demonstrate that APols-aided protein precipitation works well on diluted samples, such as secretome sample, and provides a rapid method for protein concentration.

Show MeSH