Rapid and Quantitative Protein Precipitation for Proteome Analysis by Mass Spectrometry

Abstract

Protein precipitation is a common front-end preparation strategy for proteome analysis, as well as other applications (e.g., protein depletion for small molecule analysis, bulk commercial preparation of protein). Highly variable conditions used to precipitate proteins, ranging in solvent type, strength, time, and temperature, reflect inconsistent and low recovery. As a consequence, incomplete proteome coverage diminishes the utility of precipitation for proteome sample preparation ahead of mass spectrometry. We herein investigate and optimize the conditions affecting protein recovery through precipitation using acetone at a defined ionic strength. By increasing the salt concentration and incubation temperature with 80% acetone, we show that rapid (2 min) precipitation provides consistently high protein recovery (98 ± 1%) of complex proteome extracts. Rapid precipitation is also applicable to isolate dilute proteins starting as low as 1 μg mL–1. Furthermore, analysis of the protein pellet by bottom-up mass spectrometry (MS) reveals unbiased recovery of all proteins with respect to molecular weight, isoelectric point (pI), and hydrophobicity. Our robust strategy to isolate proteins maximizes recovery and throughput, exploiting the analytical advantages of precipitation over alternative techniques. Data are available via ProteomeXchange with identifier PXD015674.

Thioesterase superfamily member 1 undergoes stimulus-coupled conformational reorganization to regulate metabolism in mice

“…Protein concentrations were estimated with a Bradford protein assay prior to acetone precipitation, done as described 27 . Pellets were resuspended in 8 M urea (Proteomics grade, PlusOne; GE Healthcare, Chicago IL), 5 mM TCEP (Bond-breaker; Thermo Fisher Scientific, Waltham, MA), 50 mM triethyl ammonium bicarbonate (TEAB, Sigma-Aldrich) and then alkylated with 100 mM iodoacetamide to a final concentration of 9 mM.…”

Sample preparation and fractionation techniques for intact proteins for mass spectrometric analysis

“…Lately, Nickerson et al. [220] has shown in their study that a unique combination of high salt and elevated temperature significantly improves the precipitation efficiency of proteins in 80% acetone. They achieved quantitative recovery (98 ± 1%) by rapid (2 min) precipitation of a complex proteome mixture at room temperature.…”

Brain Acetyl-CoA Production and Phosphorylation of Cytoskeletal Proteins Are Targets of CYP46A1 Activity Modulation and Altered Sterol Flux

“…The pellet after the second centrifugation was resuspended in 0.5 ml of Syn-PER™ reagent, aliquoted, flash frozen in liquid nitrogen and stored until use at −80 °C. For omics analyses, proteins were extracted from synaptosomal fractions with trifluoroacetic acid [25], precipitated with acetone [26] and reconstituted in 1% SDS containing 50 mM Tris-HCl, pH 8.0, 5 mM Tris(2-carboxyethyl)phosphine hydrochloride and 20 mM chloroacetamide. Solutions were heated to 95 °C for 10 min, cooled, probe-sonicated and centrifuged for clarification.…”