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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • 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 workflow

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 o.......

Representative 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 .......

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

References

  1. Kilpatrick, L. E., Kilpatrick, E. L. Optimizing high-resolution mass spectrometry for the identification of low-abundance post-translational modifications of intact proteins. Journal of Proteome Research. 16 (9), 3255-3265 (2017).
  2. Scheffler, K., Viner, R., Damoc, E.

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Protein PrecipitationOrganic SolventsMass SpectrometrySample PreparationProteome PurificationAcetoneSodium ChlorideMethanolChloroformCentrifugationSDSPelletVortex

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