Proteomics

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Rapid and quantitative precipitation of a yeast proteome

ABSTRACT: Protein precipitation has applications as a front-end preparation strategy for proteome analysis, as well as depleting proteins in serum for small molecule analysis or for commercial preparation of bulk protein. The highly variable conditions previously implemented to precipitate proteins are reflected by inconsistent and low recovery, as well as incomplete proteome coverage, diminishing the utility of precipitation for sample preparation. We herein investigate and optimize the conditions affecting protein recovery, using acetone at a defined ionic strength. We show rapid (2 min) precipitation at room temperature provides consistently high protein recovery (98 +/- 1%), with MS protein identifications equivalent to overnight, -20 °C incubations. Our robust strategy to isolate proteins maximizes recovery with minimal structural bias, exploiting the analytical advantages of precipitation over alternative techniques.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

TISSUE(S): Cell Culture

SUBMITTER: Jessica Nickerson  

LAB HEAD: Alan Austin Doucette

PROVIDER: PXD015674 | Pride | 2020-03-23

REPOSITORIES: Pride

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Publications

Rapid and Quantitative Protein Precipitation for Proteome Analysis by Mass Spectrometry.

Nickerson Jessica L JL   Doucette Alan A AA  

Journal of proteome research 20200331 5

Protein precipitation is a common front-end preparation strategy for proteome analysis, as well as other applications (<i>e.g</i>., 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 o  ...[more]

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