by Jessica Nickerson, Research Associate
Even the best mass spectrometer in the world can only detect the portion of the proteome that is successfully extracted and conserved across the sample prep workflow—so it is critical that the extraction buffer and denaturants that are employed are optimal to recover the entire biological proteome.
This applies especially to membrane proteins, known for their roles in cell-to-cell communication and as drug targets, making them extremely important analytes in clinical research and applications, as well as in the development of precision and personalized medicine advancements. While membrane proteins are some of the most targeted analytes in proteomics, they are also some of the most difficult to recover due to their hydrophobic character. Sodium dodecyl sulfate (SDS) is known for its efficacy in recovering hydrophobic proteins including membrane proteins while it also recovers the cytosolic fraction of the sample.
SDS also plays a prominent role in SDS PAGE and GELFrEE molecular weight-based separations, which are integral steps in many proteome workflows.
It is clear that SDS has its advantages in proteome sample prep, however, its major drawback is that it is a detriment to mass spectrometry, impairing chromatography and imparting ion suppression, interfering with the detection and quantitation of peptide or protein analytes.
If the advantages of SDS are to be fully capitalized on in the frontend of the proteome workflow, an effective and robust SDS depletion strategy is needed prior to analysis by mass spectrometry. Many of the common purification strategies force us to prioritize either quantitative sample recovery or optimal purity but fail to simultaneously achieve the two, however, complete recovery and purification are both necessary for optimal characterization by mass spectrometry.
Protein precipitation has evolved to provide quantitative proteome recovery in minutes [1, 2], but what’s more, is that it simultaneously depletes >99.8% of the SDS into the supernatant. Kachuk et al. have recommended a maximum residual SDS concentration of 10 ppm for optimal proteome characterization by mass spectrometry . The >99.8% SDS depletion efficiency of optimized protein precipitation, therefore, invites the use of up to 1% SDS (10 000 ppm) in the front end of the workflow, providing a robust and efficient means of depleting the surfactant without sacrificing sample recovery. In the initial precipitation step, most of the SDS partitions to the acetone-rich supernatant, while one additional wash of the protein pellet with a fresh aliquot of acetone brings the total SDS depletion to >99.8%. MS data is only as good as the sample we inject. So go ahead! Use the tried and true surfactant to get the most out of your sample with the confidence that our precipitation protocols will help get the most out of every LC-MS run.
 A. Crowell, M. Wall, & A.A. Doucette. Analytica Chimica Acta (2013), 796, 48-54
 J.L. Nickerson, & A.A. Doucette. J. Proteome Research (2020) 19(5), 2035-2042
 C. Kachuk, M. Faulkner, F. Liu and A.A. Doucette, J. Proteome Res., 15 (2016), pp. 2634-2642