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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

The present protocol describes solvent-based protein precipitation under controlled conditions for robust and rapid recovery and purification of proteome samples prior to mass spectrometry.

Abstract

While multiple advances in mass spectrometry (MS) instruments have improved qualitative and quantitative proteome analysis, more reliable front-end approaches to isolate, enrich, and process proteins ahead of MS are critical for successful proteome characterization. Low, inconsistent protein recovery and residual impurities such as surfactants are detrimental to MS analysis. Protein precipitation is often considered unreliable, time-consuming, and technically challenging to perform compared to other sample preparation strategies. These concerns are overcome by employing optimal protein precipitation protocols. For acetone precipitation, the combination of specific salts, temperature control, solvent composition, and precipitation time is critical, while the efficiency of chloroform/methanol/water precipitation depends on proper pipetting and vial manipulation. Alternatively, these precipitation protocols are streamlined and semi-automated within a disposable spin cartridge. The expected outcomes of solvent-based protein precipitation in the conventional format and using a disposable, two-stage filtration and extraction cartridge are illustrated in this work. This includes the detailed characterization of proteomic mixtures by bottom-up LC-MS/MS analysis. The superior performance of SDS-based workflows is also demonstrated relative to non-contaminated protein.

Introduction

Proteome analysis by mass spectrometry has become increasingly rigorous, owing to the enhanced sensitivity, resolution, scan speed, and versatility of modern MS instruments. MS advances contribute to greater protein identification efficiency and more precise quantitation1,2,3,4,5. With improved MS instrumentation, researchers demand a correspondingly consistent front-end sample preparation strategy capable of quantitative recovery of high-purity proteins in minimal time across all stages of the workflow6,7,8,9,10,11. To accurately reflect the proteome status of a biological system, proteins must be isolated from the native sample matrix in an efficient and unbiased fashion. To this end, including a denaturing surfactant, such as sodium dodecyl sulfate (SDS), ensures efficient protein extraction and solubilization12. However, SDS strongly interferes with electrospray ionization, causing severe MS signal suppression if not properly eliminated13.

Various SDS depletion strategies are available for subsequent proteome analysis, such as the retention of proteins above a molecular weight cutoff filter contained within disposable spin cartridges14,15,16. The filter-aided sample preparation method (FASP) is favored as it effectively depletes SDS below 10 ppm, facilitating optimal MS. However, protein recovery with FASP is variable, which prompted the exploration of other techniques. Chromatographic approaches that selectively capture protein (or surfactant) have evolved into various convenient cartridges or bead-based formats17,18,19,20,21. Given these simple and (ideally) consistent strategies to protein purification, the classical approach of protein precipitation with organic solvents is often overlooked as a promising approach to protein isolation. While solvent precipitation is shown to deplete SDS below critical levels successfully, protein recovery has been a longstanding concern of this approach. Multiple groups have observed a protein recovery bias, with unacceptably low precipitation yields as a function of protein concentration, molecular weight, and hydrophobicity22,23. Due to the diversity of precipitation protocols reported in the literature, standardized precipitation conditions were developed. In 2013, Crowell et al. first reported the dependence of ionic strength on the precipitation efficiency of proteins in 80% acetone24. For all proteins examined, the addition of up to 30 mM sodium chloride was shown to be essential to maximize yields (up to 100% recovery). More recently, Nickerson et al. showed that the combination of even higher ionic strength (up to 100 mM) with elevated temperature (20 °C) during acetone precipitation gave near quantitative recovery in 2-5 min25. A slight drop in the recovery of low molecular weight (LMW) proteins was observed. Therefore, a subsequent report by Baghalabadi et al. demonstrated the successful recovery of LMW proteins and peptides (≤5 kDa) by combining specific salts, particularly zinc sulfate, with a higher level of organic solvent (97% acetone)26.

While refining the precipitation protocol lends a more reliable protein purification strategy for MS-based proteomics, the success of conventional precipitation relies heavily on user technique. A primary goal of this work is to present a robust precipitation strategy that facilitates the isolation of the protein pellet from the contaminating supernatant. A disposable filtration cartridge was developed to eliminate pipetting by isolating aggregated protein above a porous PTFE membrane filter27. MS-interfering components in the supernatant are effectively removed in a short, low-speed centrifugation step. The disposable filter cartridge also offers an interchangeable SPE cartridge, which facilitates subsequent sample clean-up following resolubilization and optional protein digestion, ahead of mass spectrometry.

A series of recommended proteome precipitation workflows are presented here, including modified acetone and chloroform/methanol/water28 protocols, in a conventional (vial-based) and a semi-automated format in a disposable two-state filtration and extraction cartridge. The resulting protein recoveries and SDS depletion efficiencies are highlighted, together with bottom-up LC-MS/MS proteome coverage, to demonstrate the expected outcome from each protocol. The practical benefits and drawbacks associated with each approach are discussed.

Protocol

1. Material considerations and sample pre-preparation

  1. Use only high purity solvents (acetone, chloroform, methanol) (>99.5%) and chemicals, free of excess moisture.
  2. Prepare sodium chloride and zinc sulfate solutions (1 M) in water.
    NOTE: Salt solutions can be stored indefinitely at room temperature, as long as they are free of contaminant or microbial growth.
  3. Use the smallest polypropylene (PP) microcentrifuge vial sufficient to retain the required volume of sample and solvents to induce precipitation.
  4. Ensure that the SDS concentration in the sample to be precipitated is no greater than 2% (w/v). If SDS is higher, dilute the sample with water.
  5. Ensure a protein concentration between 0.01 and 10 g/L for optimal precipitation efficiency.
    NOTE: The optimal mass for precipitation ranges between 1-100 µg of protein.
  6. Ensure all solvents and solutions are free of particulate matter before use. Perform either a filtration (<0.5 µm) or centrifugation step (10,000 x g for 1 min at room temperature) to remove undissolved particulates.
  7. If disulfide bond reduction and alkylation are required, conduct these steps prior to protein precipitation. Excess reducing and alkylating reagents will be removed through the precipitation process.
  8. Precipitate the proteins by selecting and performing one of the protocols (steps 2, 3, 4, or 5).

2. Rapid (vial-based) protein precipitation with acetone

  1. Pipet 90 µL of (particulate-free) protein or proteome solution into a PP microcentrifuge tube. Then, add 10 µL of 1 M aqueous NaCl.
    NOTE: If the ionic strength of the proteome extract already exceeds 100 mM, no additional salt is necessary.
  2. Pipet 400 µL of acetone into the sample. Cap the vial and tap the vial gently to combine the solvents. Vigorous mixing is not required.
    NOTE: The volume of protein, salt, and acetone can be increased so long as the relative ratio of each is maintained.
  3. Allow the vial to incubate at room temperature, undisturbed, for a minimum of 2 min.
    NOTE: Longer incubations, including those at reduced temperature (e.g., conventional acetone precipitation employs overnight precipitation in the freezer), may result in the formation of larger (visible) aggregated protein particulates (Figure 1A), which generally do not improve total protein recovery.
  4. Following incubation, place samples in a centrifuge, noting the orientation of the vial. Spin for a minimum of 2 min, at 10,000 x g or higher at room temperature.
  5. Uncap the vial and gently decant the supernatant by slowly inverting the vial to a waste container. Touch the inverted vial to a paper towel to draw residual solvent from the vial.
    CAUTION: Waste solvents should be retained and discarded as per appropriate protocols.
  6. For SDS-containing samples, dispense 400 µL of fresh acetone, being careful not to disturb the pellet.
    NOTE: Step 2.6 is optional.
    1. Immediately centrifuge the sample (10,000 x g or higher for 1 min at room temperature), placing the vial into the rotor in the same orientation as the initial spin. Decant the wash solvent as described in step 2.5.
  7. Allow the sample to fully dry with the cap open (~1 min). Recap the vial and proceed with pellet solubilization (step 6).

3. Precipitation of low molecular weight (LMW) peptides (ZnSO4 + acetone)

  1. Dispense 54 µL of proteome extract to a 2 mL PP vial, and then add 6 µL of 1 M ZnSO4.
    NOTE: Optimal recovery of LMW peptides (≤5 kDa) is obtained by adding acetone to a final 97% by volume. Assuming a 2 mL PP vial, the maximal initial sample volume is 54 µL.
  2. Add 1940 µL of acetone to a final 97% by volume. Swirl gently to mix, and let stand undisturbed on the benchtop for a minimum of 2 min.
  3. Centrifuge (10,000 x g for 1 min at room temperature) and remove the supernatant by inverting the vial, and then touching the vial to a paper towel.
  4. For SDS-containing samples, dispense 400 µL of fresh acetone, being careful not to disturb the pellet.
    NOTE: Step 3.4 is optional.
    1. Immediately centrifuge the sample as per step 3.3, placing the vial into the rotor in the same orientation as the initial spin. Decant the wash solvent as described in step 3.3.
  5. Re-solubilize the resulting dry pellet in an aqueous solvent with brief vortexing or sonication (~5 min).

4. Protein precipitation by chloroform/methanol/water (CMW)

  1. Dispense 100 µL of the protein or proteome solution into a PP vial. Add 400 µL of methanol, followed by 100 µL of chloroform. Cap the vial and vortex briefly to mix.
    NOTE: For CMW precipitation, 1.5 mL vials with narrow bottoms are preferred (Figure 2A).
    CAUTION: Chloroform solvent should be handled in an appropriate ventilation hood. All solvents that contact chloroform should be treated as halogenated waste when disposed of.
  2. Quickly dispense 300 µL of water directly into the center of the vial. Cap the vial. Allow the sample to sit on the benchtop undisturbed for 1 min.
    NOTE: The solution will immediately appear cloudy white. Avoid mixing the vial following the addition of water.
  3. Place the PP vial in a centrifuge and spin for a minimum of 5 min (10,000 x g or higher at room temperature).
    NOTE: Once centrifuged, two visible solvent layers will form (top layer = methanol/water; bottom = chloroform). A solid protein pellet forms at the solvent interface (Figure 2A).
  4. Using a large (1 mL) micropipette tip, and holding the vial at ~45°, remove ~700 µL of the solvent from the upper layer at a uniform rate.
  5. Use a smaller (200 µL) micropipette tip to continue removing the upper solvent layer from the ~45° tilted vial. Pipet in one continuous motion until the upper solvent layer forms a bead in the vial.
  6. Add 400 µL of fresh methanol to the sample vial, without disturbing the pellet, by dispensing the solvent down the side of the vial.
  7. Cap the vial. Combine the solvent layers by gently rocking the vial to swirl the solvents together.
    NOTE: It is essential to avoid disrupting the pellet. Do not vortex the vial.
  8. Noting the orientation of the vial in the rotor, centrifuge for a minimum of 10 min (10,000 x g at room temperature). The protein pellet adheres to the bottom of the vial (Figure 2B).
  9. Tip the vial at 45°, with the pellet facing down. Place the pipette tip along the upper edge of the vial and remove the supernatant with a 1 mL micropipette tip at a slow but continuous rate. Retain ~20 µL of solvent in the vial.
  10. Wash the protein pellet for SDS-containing samples by slowly dispensing 400 µL of fresh methanol. Do not vortex the vial.
    1. Proceed directly with centrifugation (10,000 x g for 2 min at temperature), placing the vial into the rotor with the same orientation as the initial spin.
  11. Remove the solvent, as per step 4.9. Allow the sample to air dry in a fumehood until the residual solvent evaporates.
  12. Consult the recommended resolubilization procedures in step 6.

5. Protein precipitation using a disposable filtration cartridge

NOTE: Each solvent-based precipitation protocol described in steps 2-5 can be performed in a two-stage filtration and extraction cartridge (see Table of Materials).

  1. With the plug attached to the upper filtration cartridge (Figure 3A), dispense the desired volume of the extracted proteome, salt, and solvent as outlined in one of the three options below.
    1. (Option 1) For protein precipitation with acetone, combine 90 µL of protein or proteome solution, 10 µL of 1 M aqueous NaCl, and 400 µL of acetone. Incubate for a minimum of 2 min on the benchtop.
      NOTE: A visible precipitate will develop for concentrated protein samples (1 g/L) (Figure 3B).
    2. (Option 2) For LMW peptide precipitation, combine 15 µL of the sample, 1.5 µL of 1 M ZnSO4, and 485 µL of acetone. Incubate for a minimum of 2 min on the benchtop.
      NOTE: A salt concentration of 90 mM in the aqueous sample will not impact recovery relative to the 100 mM recommended in step 3.
    3. (Option 3) For CMW precipitation, add 50 µL of proteome extract, 200 µL of methanol, and 50 µL of chloroform. Cap the vial and briefly vortex to combine.
      1. Quickly dispense 150 µL of water directly into the center of the vial. Incubate for 1 min on the benchtop.
  2. Centrifuge for 2 min at 2,500 x g at room temperature with the plug still attached to the filtration cartridge.
  3. Invert the cartridge, and then unscrew and remove the plug from the cartridge base.
  4. Place the filtration cartridge in a clean vial and return to the centrifuge. Spin for 3 min at 500 x g at room temperature. Discard the flow-through solvent from the lower vial.
    NOTE: If any solvent remains in the upper filtration cartridge, return to the centrifuge and perform an additional spin.
  5. Wash the protein pellet by adding 400 µL of acetone to the filtration cartridge (for CMW precipitation, step 5.1.3, add 400 µL of methanol).
  6. Centrifuge for 3 min at 500 x g at room temperature or until no solvent remains in the upper cartridge.
  7. Re-solubilize the precipitation pellet as described in step 6.

6. Resolubilization of protein pellet

  1. Wet the membrane at the base of the filtration cartridge by dispensing 2-5 µL of isopropanol directly to the membrane immediately before the resolubilization protocols described below.
  2. Follow one of the following resolubilization methods.
    1. (Option 1) Add a minimum of 20 µL of aqueous buffer containing ≥2% SDS to the filtration cartridge. Cap and vortex vigorously (~1 min). Alternatively, sonicate (>10 min) to disperse the protein pellet.
      1. Heat the sample at 95 °C for 5 min). Repeat the mixing step after heating.
        NOTE: Laemmli gel loading buffer can re-solubilize the protein pellet. However, SDS-containing samples are incompatible with trypsin digestion and reversed-phase LC and MS.
    2. (Option 2) Prepare a solution of 80% (v/v) formic acid in water. Prechill the acid solution (-20 °C), as well as the filtration cartridge containing precipitated protein.
      1. Dispense 50 µL of cold formic acid into the cartridge; cap and vortex for 30 s. Return to the freezer (-20 °C) for 10 min.
      2. Vortex the cartridge again for 30 s. Then, repeat the chilling and mixing cycle one more time (10 min, -20 °C, 30 s vortex).
      3. Add water to a final 500 µL, diluting the formic acid to 8%.
        NOTE: The cold formic acid protocol is incompatible with subsequent trypsin digestion but is compatible with LC-MS.
    3. (Option 3) Add 50 µL of freshly prepared 8 M urea in water to the filtration cartridge. Sonicate for 30 min.
      1. Allow the cartridge to incubate on the benchtop for 1 h (up to overnight).
      2. Dilute the 8 M urea a minimum 5-fold with water or appropriate buffer.
        NOTE: Once diluted, the urea solubilization protocol is compatible with subsequent trypsin digestion, as well as LC-MS.

7. Protein digestion

  1. For bottom-up MS analysis, subject the re-solubilized proteins to enzymatic digestion using one of the two methods mentioned below.
    1. (Option 1) For formic acid resolubilization, reduce the initial volume of 80% formic acid in step 6.2.2.1 to 25 µL. In step 6.2.2.3, use 375 µL of water to dilute the formic acid to 5% (v/v).
    2. Dispense pepsin into the cartridge at an approximate protein to enzyme ratio of 50:1. With a plug attached to the filtration cartridge, incubate the sample overnight at room temperature.
  2. (Option 2) For resolubilization in urea, ensure a pH between 8 and 8.3 with the inclusion of 100 mM of Tris or ammonium bicarbonate in step 6.2.3.2.
    1. Add trypsin at an approximate protein to enzyme mass ratio of 50:1. With a plug attached to the cartridge, incubate the sample overnight in a warm water bath at 37 °C.
    2. Terminate the digestion by acidifying the solution with 10% TFA to a final 1%.
  3. Recover the pepsin- or trypsin-digested protein by removing the plug from the base of the filter and centrifuging the cartridge contained within a clean vial (2 min, 5000 x g, room temperature).

8. SPE clean-up

NOTE: For additional sample desalting following digestion or solvent exchange, the sample can be subject to reversed-phase clean-up as described.

  1. Prime an SPE cartridge (see Table of Materials) by passing 300 µL of methanol (2 min, 400 x g) followed by 300 µL of 5% acetonitrile/0.1% of TFA (2 min, 400 x g).
  2. Connect the primed SPE cartridge to the base of the filtration cartridge containing re-solubilized or digested protein.
  3. Spin the protein through the SPE cartridge (5 min, 800 x g at room temperature). If solvent remains in the upper cartridge, return the cartridge to the centrifuge and repeat the spin.
    NOTE: Passing the sample through the SPE cartridge a second time may improve recovery.
  4. Add 300 µL of 5% acetonitrile/0.1% of TFA in water to the cartridge. To wash, flow through the SPE cartridge (2 min, 2000 x g). Discard the flow-through.
  5. For LMW proteins or digested peptides, elute the sample by flowing 300 µL of 50% acetonitrile/0.1% TFA (5 min, 2500 x g).
  6. For intact proteins, follow step 8.5 with an additional elution step using 300 µL of 75% acetonitrile/0.1% TFA. Combine the two resulting extracts.
    NOTE: Step 8.6 is optional.

Results

Figure 4 summarizes the expected SDS depletion following vial-based or cartridge-facilitated precipitation of proteins in a disposable filter cartridge using acetone. Conventional overnight incubation (-20 °C) in acetone is compared to the rapid acetone precipitation protocol at room temperature (step 2), as well as CMW precipitation (step 4). Residual SDS was quantified by the methylene blue active substances (MBAS) assay29. Briefly, 100 µL sample was combi...

Discussion

Optimal MS characterization is achieved when residual SDS is depleted below 10 ppm. While alternative approaches, such as FASP and on-bead digestion, offer quantitative SDS depletion with variable recovery31,32,33, the primary objective of precipitation is to maximize purity and yield simultaneously. This depends on effectively isolating the supernatant (containing the SDS) without disturbing the protein pellet. With vial-based ...

Disclosures

The Doucette laboratory conceived of and patented the ProTrap XG employed in this study. AAD is also a founding partner of Proteoform Scientific, which commercialized the sample preparation cartridge.

Acknowledgements

This work was funded by the Natural Sciences and Engineering Research Council of Canada. The authors thank Bioinformatics Solutions Inc. (Waterloo, Canada) and SPARC BioCentre (Molecular Analysis) at the Hospital for Sick Children (Toronto, Canada) for their contributions to the acquisition of MS data.

Materials

NameCompanyCatalog NumberComments
AcetoneFisher ScientificAC177170010≤0.002 % aldehyde
AcetonitrileFisher ScientificA998-4HPLC grade
Ammonium BicarbonateMillipore SigmaA6141-1KGsolid
Beta mercaptoethanolMillipore SigmaM3148-25MLMolecular biology grade
Bromophenol blueMillipore SigmaB8026-5GBromophenol blue sodium salt
ChloroformFisher ScientificC298-400Chloroform
Formic AcidHoneywell56302Eluent additive for LC-MS
Fusion Lumos Mass SpectrometerThermoFisher Scientificfor analysis of standard protein mixture
GlycerolMillipore Sigma356352-1L-MFor molecular biology, > 99%
IsopropanolFisher ScientificA4641HPLC grade
MethanolFisher ScientificA452SK-4HPLC grade
MicrocentrifugeFisher Scientific75-400-102up to 21,000 xg
Microcentrifuge Tube (1.5 mL)Fisher Scientific05-408-130tapered bottom
Microcentrifuge Tube         (2 mL)Fisher Scientific02-681-321rounded bottom
Micropipette Tips         (0.1-10 μL)Fisher Scientific21-197-28Universal pipet tip, non-sterile
Micropipette Tips         (1-200 μL)Fisher Scientific07-200-302Universal pipet tip, non-sterile
Micropipette Tips        (200-1000 μL)Fisher Scientific07-200-303Universal pipet tip, non-sterile
MicropipettesFisher Scientific13-710-903Micropipet Trio pack
PepsinMillipore SigmaP0525000Lyophilized powder,           >3200 units/ mg
ProTrap XGProteoform ScientificPXG-000250 complete units per box
Sodium ChlorideMillipore SigmaS9888-1KGACS reagent, >99 %
Sodium Dodecyl SulfateThermoFisher Scientific28312powdered solid
timsTOF Pro Mass SpectrometerBrukerfor analysis of liver proteome extract
Trifluoroacetic AcidThermoFisher ScientificL06374.AP99%
TrisFisher ScientificBP152-500Molecular biology grade
TrypsinMillipore Sigma9002-07-7From bovine pancreas, TPCK-treated
UreaBio-Rad1610731solid
Water (deionized)Sartorius Arium Mini Water Purification System76307-662Type 1 ultrapure (18.2 MΩ cm)
Zinc SulfateMillipore Sigma307491-100Gsolid

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