Proteomics is the large-scale study of proteins, including their structures, functions, and interactions. The first step in any proteomic study is the extraction and solubilization of proteins from the biological sample. The goal of protein extraction is to obtain a representative and reproducible sample of proteins from the original source.
The choice of extraction method depends on the sample type, the protein of interest, and the downstream applications. For example, different methods are used for extracting proteins from animal tissues, plant tissues, bacteria, and yeast. Commonly used methods for protein extraction include sonication, grinding, homogenization, and chemical lysis.
Once the proteins are extracted, they must be solubilized to make them compatible with downstream analysis techniques such as mass spectrometry. Solubilization involves breaking down protein-protein interactions and exposing the hydrophobic regions of the proteins to a polar solvent. The choice of solubilization method depends on the properties of the protein, including its size, charge, and hydrophobicity.
Commonly used solubilization methods include the use of detergents, chaotropic agents, and organic solvents. Detergents can solubilize membrane proteins, while chaotropic agents such as urea and guanidine hydrochloride can solubilize denatured proteins. Organic solvents such as acetonitrile and methanol can solubilize hydrophobic proteins.
The protein extraction and solubilization steps are critical for the success of any proteomic study. Optimizing these steps can improve the yield, purity, and reproducibility of the sample, leading to more accurate and meaningful results.
1. Sample Generation
1.1 Lysis/Extraction/Rehydration Solution
1.1.1 Universal solubilization cocktail for mammalian tissues and cells
The typical lysis solution (17) consists of the following:
- 5 M Urea.
- 2 M Thiourea.
- 0.25% (v/v) CHAPS (Sigma).
- 0.25% (v/v) Tween-20 (Bio-Rad).
- 0.25% (v/v) sulfobetaine (SB) 3-10 (Sigma).
- 0.25% (w/v) carrier ampholytes (1:1:1:1 mixture of Bio-Lyte 3-10 [Bio-Rad], Servalyte 3-10 [Serva], Ampholine 3.5-9.5 [Amersham Biosciences], and Resolyte 4-8 [BDH]).
- 2 mM Tributylphosphine (TBP).
- 10% Isopropanol.
- 12.5% (v/v) water-saturated isobutanol.
- 5% (v/v) glycerol (Bio-Rad).
- 1 mM Sodium vanadate (phosphatase inhibitor, Sigma).
- 1X complete protease inhibitor cocktail (Boehringer-Mannheim).
This lysis solution can be stored tightly sealed at –80°C for several weeks. Care must be taken with respect to the potential evaporation of the alcohol over prolonged time periods. It may be necessary to change the detergent mixture and level for a given application. In some cases, 0.25–0.5% Triton X-100 yields better results than Tween-20, or increased levels of CHAPS solubilize more proteins. If TBP cannot be used, 100 mM dithiothreitol or 5% 2-mercaptoethanol can be used, but the results are not as good. The ampholyte level may also need to be increased if high amounts of sample are applied to the gel strip. In general, it is best to keep the detergent (0.75–2%) and ampholyte (0.25–1.5%) levels as low as possible for optimal resolution.
1.1.2 General solubilization cocktail for plant seeds
The typical plant extraction/solubilization solution consists of the following:
- 6 M Urea.
- 2 M Thiourea.
- 0.5% (v/v) CHAPS (Sigma).
- 0.25% (v/v) Triton X-100 (Bio-Rad).
- 0.25% (v/v) SB 3-10 (Sigma).
- 0.35% (w/v) carrier ampholytes (1:1:1:1 mixture of Bio-Lyte 3-10 [Bio-Rad], Servalyte 3-10 [Serva], Ampholine 3.5-9.5 [Amersham Biosciences] and Resolyte 4-8 [BDH]).
- 2 mM Tributylphosphine (TBP).
- 16% Isopropanol.
- 5% (v/v) glycerol (Bio-Rad).
- 1X complete protease inhibitor cocktail (Boehringer-Mannheim).
This lysis solution can be stored tightly sealed at –80°C for several weeks. As noted above, care must be taken with respect to the potential evaporation of the alcohol over prolonged time periods. As noted above, this cocktail may need to be modified empirically for specific plant tissue types.
1.2 Cell Lysates
Cells such as those of the U937 human monocytic cell line are typically solubilized directly in lysis/rehydration solution at a level of approx 20,000 cells/μL or approx 1.5 mg protein/mL. Much smaller cells (e.g., monocytes and splenocytes) are solubilized at approx 80,000 cells/μL, which represents approx 1.28 mg protein/mL). Following extraction for 30 min at room temperature on a Nutator mixer, the samples are clarified by centrifugation in a microcentrifuge at 15,300g for 5 min. The presence of alcohol appears to precipitate the nucleic acid out of solution, while the chaotropes-detergents mixture solubilizes and releases the RNA- and DNA-binding proteins.
1.3. Serum
Rat serum (typically around 55 mg protein/mL, but ranges from approx 40–100 mg protein/mL) is either analyzed by dilution directly into lysis/rehydration solution or is first depleted with an affinity column to remove albumin, IgGs, and so on. The depleted serum sample is then concentrated back to the starting protein level and diluted into lysis/rehydration solution.
1.4. Tissue
Tissue is frozen in liquid nitrogen and pulverized in liquid nitrogen with a BioPulverizer (sizes to accommodate from 10 mg to 10 g of tissue). The pulverized tissue is extracted at a level of approx 2–3 mg of tissue/mL of lysis/rehydration solution for 30 min at room temperature. The extract is clarified by centrifugation, and the supernatant is either analyzed immediately or stored at –80°C. Fresh bone samples can be pulverized and processed in a similar manner using the MicroCryoPulverizer.
1.5. Urine
Urine (typically less than 1.5 mg protein/mL in a 24-h collection) is concentrated using membrane filtration devices (Centricon 3, Amicon) or lyophilized prior to dilution into the lysis/rehydration solution at a final concentration of approx 1–2 mg protein/mL.
2. Focusing Parameters
Because the solubility of proteins is also dependent upon the amount of salts (and the resulting loss of water as joule heating occurs), the isoelectric focusing steps are slowly ramped up at the start of each electrophoretic run. The rehydrated immobilized pH gradient (IPG) strips are typically focused in Bio-Rad Protean IEF units using the following protocols. In general, the total number of volt-hours should be 50,000– 75,000. Narrower range pH gradients require longer focusing times than broad pH ranges.
1. pH 3.0–10, Linear or Nonlinear, 18-cm-Long IPG Gel Strips, "Normal Samples." 150-V rapid ramp for 1 h; 250-V rapid ramp for 1 h; 400-V rapid ramp for 4 hs; 10,000-V linear ramp for 14 h; 10,000-V rapid ramp as a hold step for additional volt-hours. The typical total volt-hours for focusing are 60,000–75,000.
2. pH 3.0–10, Linear or Nonlinear, 18-cm-Long IPG Gel Strips, Samples Containing Salts up to 150 mM. 50-V rapid ramp for 4 h; 150-V rapid ramp for 1 h; 250-V rapid ramp for 1 h; 400-V rapid ramp for 4 h; 10,000-V linear ramp for 12 h; 10,000-V rapid ramp as a hold step for additional volt-hours. The typical total volt-hours for focusing are 60,000–75,000.
3. pH 3.0–10, Linear or Nonlinear, 11-cm-Long IPG Gel Strips "Normal Samples." 150-V rapid ramp for 1 h; 250-V rapid ramp for 1 h; 400-V rapid ramp for 4 hs; 8,000-V linear ramp for 14 h; 8,000-V rapid ramp as a hold step for additional volt-hours. The typical total volt-hours for focusing are 55,000–60,000.
4. pH 3.0–10, Linear or Nonlinear, 11-cm-Long IPG Gel Strips, Samples Containing Salts up to 150 mM. 50-V rapid ramp for 4 h; 150-V rapid ramp for 1 h; 250-V rapid ramp for 1 h; 400-V rapid ramp for 4 h; 5,000-V linear ramp for 14 h; 5,000-V rapid ramp as a hold step for additional volt-hours. The typical total volt-hours for focusing are 55,000–60,000.
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Reference
- Walker, J. M. (Ed.). (2005). The proteomics protocols handbook. Humana press.