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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.
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.
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
1. Material considerations and sample pre-preparation
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.......
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 .......
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.
....Name | Company | Catalog Number | Comments |
Acetone | Fisher Scientific | AC177170010 | ≤0.002 % aldehyde |
Acetonitrile | Fisher Scientific | A998-4 | HPLC grade |
Ammonium Bicarbonate | Millipore Sigma | A6141-1KG | solid |
Beta mercaptoethanol | Millipore Sigma | M3148-25ML | Molecular biology grade |
Bromophenol blue | Millipore Sigma | B8026-5G | Bromophenol blue sodium salt |
Chloroform | Fisher Scientific | C298-400 | Chloroform |
Formic Acid | Honeywell | 56302 | Eluent additive for LC-MS |
Fusion Lumos Mass Spectrometer | ThermoFisher Scientific | for analysis of standard protein mixture | |
Glycerol | Millipore Sigma | 356352-1L-M | For molecular biology, > 99% |
Isopropanol | Fisher Scientific | A4641 | HPLC grade |
Methanol | Fisher Scientific | A452SK-4 | HPLC grade |
Microcentrifuge | Fisher Scientific | 75-400-102 | up to 21,000 xg |
Microcentrifuge Tube (1.5 mL) | Fisher Scientific | 05-408-130 | tapered bottom |
Microcentrifuge Tube        (2 mL) | Fisher Scientific | 02-681-321 | rounded bottom |
Micropipette Tips        (0.1-10 μL) | Fisher Scientific | 21-197-28 | Universal pipet tip, non-sterile |
Micropipette Tips        (1-200 μL) | Fisher Scientific | 07-200-302 | Universal pipet tip, non-sterile |
Micropipette Tips       (200-1000 μL) | Fisher Scientific | 07-200-303 | Universal pipet tip, non-sterile |
Micropipettes | Fisher Scientific | 13-710-903 | Micropipet Trio pack |
Pepsin | Millipore Sigma | P0525000 | Lyophilized powder,          >3200 units/ mg |
ProTrap XG | Proteoform Scientific | PXG-0002 | 50 complete units per box |
Sodium Chloride | Millipore Sigma | S9888-1KG | ACS reagent, >99 % |
Sodium Dodecyl Sulfate | ThermoFisher Scientific | 28312 | powdered solid |
timsTOF Pro Mass Spectrometer | Bruker | for analysis of liver proteome extract | |
Trifluoroacetic Acid | ThermoFisher Scientific | L06374.AP | 99% |
Tris | Fisher Scientific | BP152-500 | Molecular biology grade |
Trypsin | Millipore Sigma | 9002-07-7 | From bovine pancreas, TPCK-treated |
Urea | Bio-Rad | 1610731 | solid |
Water (deionized) | Sartorius Arium Mini Water Purification System | 76307-662 | Type 1 ultrapure (18.2 MΩ cm) |
Zinc Sulfate | Millipore Sigma | 307491-100G | solid |
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