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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: 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 contrast with tryptic peptides, a decrease in pH leads to the unbiased co-precipitation of APols with proteins, including globular hydrophilic proteins.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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Overlaps between the proteins identified in total lysate, APols precipitate and supernatant. The area of each circle is proportional to the actual number of protein identified. The diagram was plotted using eulerAPE (http://www.eulerdiagrams.org/eulerAPE/)
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Fig2: Overlaps between the proteins identified in total lysate, APols precipitate and supernatant. The area of each circle is proportional to the actual number of protein identified. The diagram was plotted using eulerAPE (http://www.eulerdiagrams.org/eulerAPE/)

Mentions: Here, we explored whether APols can be used for the enrichment of proteins by their co-precipitation upon protonation of APols. Our initial focus was the membrane proteome. We first tested whether APols could be used to precipitate membrane proteins from a complex mixture. A final concentration of 1 mg/mL APols was added to 1 mg proteins obtained from a total lysate from HEK 293T cells. The solution was then acidified to pH 3.0 and centrifuged. The supernatant was collected together with two washes of the APols precipitation. The proteins recovered from the supernatant, two washes, and the APols pellets were analyzed by gel electrophoresis (Fig. 1). To our surprise, the vast majority of the proteins appeared to be co-precipitated by APols, with limited protein amounts found in the supernatant and two washes. As well, it appeared that most of the proteins were recovered following reconstitution of the APols precipitation. The same precipitation was readily performed on hydrophilic proteins including BSA, lysozyme, and myoglobin solution, as well as spent cell medium (Figs. S1, S2). Mass spectrometric identification of the proteins co-precipitated by APols, the supernatant, and total HEK 293T cell lysate revealed that APols could precipitate most of the proteins in total lysate (Fig. 2, Fig. S3). The unexpected number of proteins identified from the supernatant fraction is probably from low-abundant proteins and smaller degraded/truncated protein fragments, which cannot be precipitated efficiently by acid. The identification result also showed no significant bias by APols co-precipitation in terms of protein hydrophobicity (GRAVY), molecular weight (MW), and isoelectric point (pI) (Fig. S4). Therefore, it appears that APols can co-precipitate with proteins when the pH is lowered regardless of protein’s hydrophobicity.Fig. 1


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)

Overlaps between the proteins identified in total lysate, APols precipitate and supernatant. The area of each circle is proportional to the actual number of protein identified. The diagram was plotted using eulerAPE (http://www.eulerdiagrams.org/eulerAPE/)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4196042&req=5

Fig2: Overlaps between the proteins identified in total lysate, APols precipitate and supernatant. The area of each circle is proportional to the actual number of protein identified. The diagram was plotted using eulerAPE (http://www.eulerdiagrams.org/eulerAPE/)
Mentions: Here, we explored whether APols can be used for the enrichment of proteins by their co-precipitation upon protonation of APols. Our initial focus was the membrane proteome. We first tested whether APols could be used to precipitate membrane proteins from a complex mixture. A final concentration of 1 mg/mL APols was added to 1 mg proteins obtained from a total lysate from HEK 293T cells. The solution was then acidified to pH 3.0 and centrifuged. The supernatant was collected together with two washes of the APols precipitation. The proteins recovered from the supernatant, two washes, and the APols pellets were analyzed by gel electrophoresis (Fig. 1). To our surprise, the vast majority of the proteins appeared to be co-precipitated by APols, with limited protein amounts found in the supernatant and two washes. As well, it appeared that most of the proteins were recovered following reconstitution of the APols precipitation. The same precipitation was readily performed on hydrophilic proteins including BSA, lysozyme, and myoglobin solution, as well as spent cell medium (Figs. S1, S2). Mass spectrometric identification of the proteins co-precipitated by APols, the supernatant, and total HEK 293T cell lysate revealed that APols could precipitate most of the proteins in total lysate (Fig. 2, Fig. S3). The unexpected number of proteins identified from the supernatant fraction is probably from low-abundant proteins and smaller degraded/truncated protein fragments, which cannot be precipitated efficiently by acid. The identification result also showed no significant bias by APols co-precipitation in terms of protein hydrophobicity (GRAVY), molecular weight (MW), and isoelectric point (pI) (Fig. S4). Therefore, it appears that APols can co-precipitate with proteins when the pH is lowered regardless of protein’s hydrophobicity.Fig. 1

Bottom Line: 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 contrast with tryptic peptides, a decrease in pH leads to the unbiased co-precipitation of APols with proteins, including globular hydrophilic proteins.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