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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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Effects of acid and APols precipitation on the recovery of proteins from serum-free spent medium. Serum-free cell spent medium was precipitated by 5 % FA only, or followed by APols (1 mg/mL), or by acid + APols
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Fig4: Effects of acid and APols precipitation on the recovery of proteins from serum-free spent medium. Serum-free cell spent medium was precipitated by 5 % FA only, or followed by APols (1 mg/mL), or by acid + APols

Mentions: The next step was trying to figure out whether APols and/or the acidification of the samples were the main causes for the precipitation of proteins. Acidification to pH 3.0 is well known to lead to protein precipitation by the formation of insoluble salts between the acid anions, in this case formate, and the positively charged amino groups of the protein molecules. Also, it is known that the hydration layer of proteins is reduced at low pH. Overall, precipitation caused by acidification is particularly efficient for high protein concentrations (Salt et al. 1982; Retz and Steele 1977; Polson et al. 2003). In order to discern the roles that acid and APols play in protein precipitation, a set of protein precipitation experiments was performed with a serum-free spent medium, which only contains less than 0.01 mg/ml of hydrophilic secreted proteins. The spent media were first subjected to acid precipitation by adding 5 % FA to lower the pH of the solution. The resulting pellet was spun down. APols were then added to the supernatant to precipitate the remaining proteins. In the meantime, the “combined” acid plus APols precipitation strategy, which has been used in this work, was also performed for comparison. The amount of acid added and the final pHs were identical for the three experiments. As shown in Fig. 4, the acid only precipitation could hardly produce any protein bands from the spent medium. The starting material was already 50 times more than the other two scenarios, see experimental section for more details. However, after acid precipitation, the addition of APols to the supernatant led to the co-precipitation of proteins. Based on these results, we conclude that both acid and APols are important for the process of protein precipitation. The acid has at least two functions in the precipitation process: firstly, it reduces the solubility of proteins as described above, and secondly, it protonates APols which then become more hydrophobic. We postulate that for sample containing high concentrations of proteins acidification can initialize the nucleation process, and lead to protein aggregation and precipitation. However, for samples with lower concentration of proteins the APols would initialize and accelerate the aggregation process and leads to the co-precipitation of proteins.Fig. 4


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)

Effects of acid and APols precipitation on the recovery of proteins from serum-free spent medium. Serum-free cell spent medium was precipitated by 5 % FA only, or followed by APols (1 mg/mL), or by acid + APols
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4196042&req=5

Fig4: Effects of acid and APols precipitation on the recovery of proteins from serum-free spent medium. Serum-free cell spent medium was precipitated by 5 % FA only, or followed by APols (1 mg/mL), or by acid + APols
Mentions: The next step was trying to figure out whether APols and/or the acidification of the samples were the main causes for the precipitation of proteins. Acidification to pH 3.0 is well known to lead to protein precipitation by the formation of insoluble salts between the acid anions, in this case formate, and the positively charged amino groups of the protein molecules. Also, it is known that the hydration layer of proteins is reduced at low pH. Overall, precipitation caused by acidification is particularly efficient for high protein concentrations (Salt et al. 1982; Retz and Steele 1977; Polson et al. 2003). In order to discern the roles that acid and APols play in protein precipitation, a set of protein precipitation experiments was performed with a serum-free spent medium, which only contains less than 0.01 mg/ml of hydrophilic secreted proteins. The spent media were first subjected to acid precipitation by adding 5 % FA to lower the pH of the solution. The resulting pellet was spun down. APols were then added to the supernatant to precipitate the remaining proteins. In the meantime, the “combined” acid plus APols precipitation strategy, which has been used in this work, was also performed for comparison. The amount of acid added and the final pHs were identical for the three experiments. As shown in Fig. 4, the acid only precipitation could hardly produce any protein bands from the spent medium. The starting material was already 50 times more than the other two scenarios, see experimental section for more details. However, after acid precipitation, the addition of APols to the supernatant led to the co-precipitation of proteins. Based on these results, we conclude that both acid and APols are important for the process of protein precipitation. The acid has at least two functions in the precipitation process: firstly, it reduces the solubility of proteins as described above, and secondly, it protonates APols which then become more hydrophobic. We postulate that for sample containing high concentrations of proteins acidification can initialize the nucleation process, and lead to protein aggregation and precipitation. However, for samples with lower concentration of proteins the APols would initialize and accelerate the aggregation process and leads to the co-precipitation of proteins.Fig. 4

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