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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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Histogram of the protein abundance observed by different protein concentrating techniques. The PaxDb, protein abundance across organisms (Wang et al. 2012), a database of absolute protein abundance was used to assign abundance to each identified protein. (Red) a collection of proteins from the PaxDb illustrating the normal intensity distribution in the whole proteome, (Green) the secretome observed by APols precipitation, (Black) the secretome observed by drying down and classical in-solution digestion, (Purple) (Boersema et al. 2013) a similar size secretome analysis, and (Blue) results from total cell lysate analysis. The X-axis represents the protein abundance in ppm, whereas the Y-axis represents the relative density (Color figure online)
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Fig5: Histogram of the protein abundance observed by different protein concentrating techniques. The PaxDb, protein abundance across organisms (Wang et al. 2012), a database of absolute protein abundance was used to assign abundance to each identified protein. (Red) a collection of proteins from the PaxDb illustrating the normal intensity distribution in the whole proteome, (Green) the secretome observed by APols precipitation, (Black) the secretome observed by drying down and classical in-solution digestion, (Purple) (Boersema et al. 2013) a similar size secretome analysis, and (Blue) results from total cell lysate analysis. The X-axis represents the protein abundance in ppm, whereas the Y-axis represents the relative density (Color figure online)

Mentions: Interestingly, the APols approach for protein co-precipitation is much more efficient than acid precipitation for samples with low concentration of proteins. Therefore this approach appears well suited for diluted biological samples. Typically, the conventional approaches for protein precipitation such as acetone precipitation are less efficient and often need large volume (up to 5 times more volume). An alternative to protein precipitation is ultrafiltration, which is time-consuming when dealing with larger sample volume, and has a low sample recovery rate and high-molecular weight preference, especially when proteins are not denatured. Our approach based on the precipitation of APols is simple and efficient (Fig. 4). To further demonstrate the performance of the APols protein co-precipitation, we used serum-free spent medium, which has an even lower level of secreted proteins. HEK 293T cells were exposed to serum-free medium for 24 h before collection. APols-aided precipitation and protein digestion were performed and the peptides were analyzed by HPLC–ESI–MS/MS. All the manipulations including protein co-precipitation, resolubilization, reduction, alkylation, and digestion were performed in one tube using the APols one-stop approach. 1103 protein groups with 1 % FDR were identified (Table S1). The abundance distribution (Wang et al. 2012) profile of the proteins identified from APols-aided protein precipitation does not show any bias toward protein abundance preference (Fig. 5). It has exactly the same profile as the conventional and presumably non-biased drying down method, by which all secreted proteins were recovered by from the serum-free medium followed by in-solution digestion.Fig. 5


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)

Histogram of the protein abundance observed by different protein concentrating techniques. The PaxDb, protein abundance across organisms (Wang et al. 2012), a database of absolute protein abundance was used to assign abundance to each identified protein. (Red) a collection of proteins from the PaxDb illustrating the normal intensity distribution in the whole proteome, (Green) the secretome observed by APols precipitation, (Black) the secretome observed by drying down and classical in-solution digestion, (Purple) (Boersema et al. 2013) a similar size secretome analysis, and (Blue) results from total cell lysate analysis. The X-axis represents the protein abundance in ppm, whereas the Y-axis represents the relative density (Color figure online)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4196042&req=5

Fig5: Histogram of the protein abundance observed by different protein concentrating techniques. The PaxDb, protein abundance across organisms (Wang et al. 2012), a database of absolute protein abundance was used to assign abundance to each identified protein. (Red) a collection of proteins from the PaxDb illustrating the normal intensity distribution in the whole proteome, (Green) the secretome observed by APols precipitation, (Black) the secretome observed by drying down and classical in-solution digestion, (Purple) (Boersema et al. 2013) a similar size secretome analysis, and (Blue) results from total cell lysate analysis. The X-axis represents the protein abundance in ppm, whereas the Y-axis represents the relative density (Color figure online)
Mentions: Interestingly, the APols approach for protein co-precipitation is much more efficient than acid precipitation for samples with low concentration of proteins. Therefore this approach appears well suited for diluted biological samples. Typically, the conventional approaches for protein precipitation such as acetone precipitation are less efficient and often need large volume (up to 5 times more volume). An alternative to protein precipitation is ultrafiltration, which is time-consuming when dealing with larger sample volume, and has a low sample recovery rate and high-molecular weight preference, especially when proteins are not denatured. Our approach based on the precipitation of APols is simple and efficient (Fig. 4). To further demonstrate the performance of the APols protein co-precipitation, we used serum-free spent medium, which has an even lower level of secreted proteins. HEK 293T cells were exposed to serum-free medium for 24 h before collection. APols-aided precipitation and protein digestion were performed and the peptides were analyzed by HPLC–ESI–MS/MS. All the manipulations including protein co-precipitation, resolubilization, reduction, alkylation, and digestion were performed in one tube using the APols one-stop approach. 1103 protein groups with 1 % FDR were identified (Table S1). The abundance distribution (Wang et al. 2012) profile of the proteins identified from APols-aided protein precipitation does not show any bias toward protein abundance preference (Fig. 5). It has exactly the same profile as the conventional and presumably non-biased drying down method, by which all secreted proteins were recovered by from the serum-free medium followed by in-solution digestion.Fig. 5

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