Chapters authored
Precipitation of Detergent-Containing Samples for Top-Down and Bottom-Up Proteomics
Prior to proteome analysis by mass spectrometry (MS), protein mixtures must first be subject to various sample preparation steps. The goal is to isolate proteins in high yield, and with high purity. Liquid chromatography (LC) separation is also integral to comprehensive proteome characterization, and so a key component of sample preparation is simply to solubilize the proteome in LC-MS compatible solvents. Hydrophobic proteins (membrane proteins) represent a greater challenge to maintain protein solubility during sample preparation. Sodium dodecyl sulfate (SDS) is a favored detergent to solubilize proteins, and also is used to impart mass-based fractionation (i.e., SDS PAGE, GELFrEE). However, SDS is incompatible with downstream LC-MS analysis. Fortunately, effective strategies for SDS removal do exist, which permits the use of this surfactant in proteomics workflows. Here we highlight an approach that is grounded in the classic technique—protein precipitation. The technique has been updated and has recently seen a revival as a strategy permitting high protein recovery, with exceptional purity. Moreover, with aid of simple disposable spin cartridges, protein precipitation can meet the needs of high throughput, automated, and reproducible proteome purification, enabling the analysis of SDS-containing samples in both top-down and bottom-up formats.
Part of the book: Proteomics Technologies and Applications