Organic Solvent-Based Protein Precipitation for Robust Proteome Purification Ahead of Mass Spectrometry

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 quantitation^1,2,3,4,5. 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 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....

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 recovery^31,32,33, 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 .......

Materials

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