Maximizing recovery of water-soluble proteins through acetone precipitation

Crowell, Andrew M.J.; Wall, Mark J.; Doucette, Alan A.

doi:10.1016/j.aca.2013.08.005

Cited by 133 publications

(118 citation statements)

References 21 publications

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“…The surfactants were later removed by an acetone precipitation procedure, an approach that was found to achieve an effective removal of surfactants by previous studies. 25,26 We surmised that this is probably because surfactant greatly facilitates more effective (i) reduction, alkylation and denaturing of proteins in a highly complex mixture and (ii) removal of nonprotein matrix components (some of which may compromise digestion and LC/MS analysis). Here we extensively investigated the effects of surfactant on the peptide recovery of a therapeutic mAb in various matrices.…”

Section: * S Supporting Informationmentioning

confidence: 99%

Surfactant-Aided Precipitation/on-Pellet-Digestion (SOD) Procedure Provides Robust and Rapid Sample Preparation for Reproducible, Accurate and Sensitive LC/MS Quantification of Therapeutic Protein in Plasma and Tissues

Zhang

Johnson

et al. 2015

Anal. Chem.

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For targeted protein quantification by liquid chromatography mass spectrometry (LC/MS), an optimal approach for efficient, robust and hi-throughput sample preparation is critical, but often remains elusive. Here we describe a straightforward surfactant-aided-precipitation/on-pellet-digestion (SOD) strategy that provides effective sample cleanup and enables high and constant peptide yields in various matrices, allowing reproducible, accurate and sensitive protein quantification. This strategy was developed using quantification of monocolnocal antibody in tissues and plasma as the model system. Surfactant treatment before precipitation substantially increased peptide recovery and reproducibility from plasma/tissue, likely because surfactant permits extensive denaturation/reduction/alkylation of proteins and inactivation of endogenous protease inhibitors, and facilitates removal of matrix components. The subsequent precipitation procedure effectively eliminates the surfactant and nonprotein matrix components, and the thorough denaturation by both surfactant and precipitation enabled very rapid on-pellet-digestion (45 min at 37 °C) with high peptide recovery. The performance of SOD was systematically compared against in-solution-digestion, in-gel-digestion and filter-aided-sample-preparation (FASP) in plasma/tissues, and then examined in a full pharmacokinetic study in rats. SOD achieved the best peptide recovery (∼21.0-700% higher than the other three methods across various matrices), reproducibility (3.75-10.9%) and sensitivity (28-30 ng/g across plasma and tissue matrices), and its performance was independent of matrix types. Finally, in validation and pharmacokinetic studies in rats, SOD outperformed other methods and provided highly accurate and precise quantification in all plasma samples without using stable isotope labeled (SIL)-protein internal standard (I.S.). In summary, the SOD method has proven to be highly robust, efficient and rapid, making it readily adaptable to large-scale clinical and pharmaceutical quantification of biomarkers or biotherapeutics.

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Section: * S Supporting Informationmentioning

confidence: 99%

Surfactant-Aided Precipitation/on-Pellet-Digestion (SOD) Procedure Provides Robust and Rapid Sample Preparation for Reproducible, Accurate and Sensitive LC/MS Quantification of Therapeutic Protein in Plasma and Tissues

Zhang

Johnson

et al. 2015

Anal. Chem.

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“…Th e isoelectric points (pI) of hemoglobin, CAT, SOD, and CA are 6.8, 5.4, 4.95, and 5.9 respectively. As we can see, only the pI of hemoglobin is close to the pH of the solution at 7.4; thus hemoglobin tends to aggregate when adding organic solvents that have reduced dielectric strength (Crowell et al 2013). In spite of its low pI value, CAT also aggregates after ethanol -chloroform treatment, possibly due to its large size and the ion pairing eff ect.…”

Section: Discussionmentioning

confidence: 90%

Extraction of superoxide dismutase, catalase, and carbonic anhydrase from stroma-free red blood cell hemolysate for the preparation of the nanobiotechnological complex of polyhemoglobin–superoxide dismutase–catalase–carbonic anhydrase

Guo

Gynn

Chang

2015

Artificial Cells, Nanomedicine, and Biotechnology

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We report a novel method to simultaneously extract superoxide dismutase (SOD), catalase (CAT), and carbonic anhydrase (CA) from the same sample of red blood cells (RBCs). This avoids the need to use expensive commercial enzymes, thus enabling a cost-effective process for large-scale production of a nanobiotechnological polyHb-SOD-CAT-CA complex, with enhancement of all three red blood cell functions. An optimal concentration of phosphate buffer for ethanol-chloroform treatment results in good recovery of CAT, SOD, and CA after extraction. Different concentrations of the enzymes can be used to enhance the activity of polyHb-SOD-CAT-CA to 2, 4, or 6 times that of RBC.

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“…The ion-pairing effect contributes to PROSPR's precipitation efficiency. Moreover, organic solvent with a salt improves protein removal [87].…”

Section: Protein Organic Solvent Precipitationmentioning

confidence: 99%

Methods to isolate extracellular vesicles for diagnosis

Kang

Kim

Park

2017

Micro and Nano Syst Lett

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Extracellular vesicles (EVs) are small membrane-bound bodies that are released into extracellular space by diverse cells, and are found in body fluids like blood, urine and saliva. EVs contain RNA, DNA and proteins, which can be biomarkers for diagnosis. EVs can be obtained by minimally-invasive biopsy, so they are useful in disease diagnosis. High yield and purity contribute to precise diagnosis of disease, but damaged EVs and impurities can cause confused results. However, EV isolation methods have different yields and purities. Furthermore, the isolation method that is most suitable to maximize EV recovery efficiency depends on the experimental conditions. This review focuses on merits and demerits of several types of EV isolation methods, and provides examples of how to diagnose disease by exploiting information obtained by analysis of EVs.

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Maximizing recovery of water-soluble proteins through acetone precipitation

Cited by 133 publications

References 21 publications

Surfactant-Aided Precipitation/on-Pellet-Digestion (SOD) Procedure Provides Robust and Rapid Sample Preparation for Reproducible, Accurate and Sensitive LC/MS Quantification of Therapeutic Protein in Plasma and Tissues

Surfactant-Aided Precipitation/on-Pellet-Digestion (SOD) Procedure Provides Robust and Rapid Sample Preparation for Reproducible, Accurate and Sensitive LC/MS Quantification of Therapeutic Protein in Plasma and Tissues

Extraction of superoxide dismutase, catalase, and carbonic anhydrase from stroma-free red blood cell hemolysate for the preparation of the nanobiotechnological complex of polyhemoglobin–superoxide dismutase–catalase–carbonic anhydrase

Methods to isolate extracellular vesicles for diagnosis

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