by Victoria Miller, Senior Scientist
Sodium dodecyl sulphate (SDS) also known as sodium lauryl sulfate, is recognized as one of the best detergents for solubilizing protein. It is in use in countless protein labs throughout the world as a solubilizer and a denaturant. Most lab scientists are familiar with its role in SDS-PAGE, unravelling the proteins present and associating with the proteins through both electrostatic and hydrophobic interactions, imparting a negative charge proportional to the protein size. This ability of SDS is exploited in SDS-PAGE to allow separation based exclusively on size (using charge as a proxy). SDS binds to proteins at approximately a 1.4 to 1 ratio, resulting in a more or less consistent charge to mass ratio, and allowing the electric current applied to separate the proteins based on size.
SDS has been touted as the preferred detergent to extract proteins, especially the harder to extract membrane proteins that contain a higher-than-average number of hydrophobic amino acids, making these proteins more difficult to extract. SDS efficiently disturbs the plasma membrane of the cell and, if used at concentrations higher than its CMC (8mM or 0.23% w/v) will disrupt quaternary, tertiary and secondary structure.
However, having all this SDS around is not advantageous to proteomics work flow steps after extraction. SDS will interfere with trypsin’s cleavage of the isolated proteins as well as alter the interaction of the proteins with reversed phase resins, and cause ion suppression. Studies have revealed that the maximum SDS concentration desirable for a sample loaded onto a LC-MS/MS is 0.01 % or 100 ppm. Considering most lysis buffers that use SDS use it at concentrations far exceeding this upper limit at 1, 2 or even 5% SDS (10 000, 20 000 or 50 000 ppm), simple dilution is not going to be sufficient to remove SDS. We’ll leave how to get rid of SDS for another post, but once you have removed the SDS, how do you know you have a clean sample? Is there a test for this?
Our team appreciates all of the technological advances made in mass instrumentation over the past decades, but sometimes, a tried-and-true method has its place even in the most sophisticated lab. Nearly 30 years ago Arand et al published a method that could detect trace amounts of SDS in the presence of both protein and nucleic acids. This method was embraced by the environmental science field and is used extensively to monitor anionic detergents in wastewater. This assay, termed the methylene blue active substances (MBAS) assay, works on the simple premise that SDS will pair with the positively charged methylene blue and that product is chloroform extractable. The result is a colorimetric assay, readable on any visible spectrophotometer capable of reading 650 nm. As the SDS concentration decreases, the amount of methylene blue that partitions into the aqueous phase decreases, giving a blue, visual cue to the amount of SDS present in a sample. A trained eye can spot the difference between 10 and 5 ppm, and an untrained eye can distinguish between 50 and 5 ppm. In our QC lab, we use the MBAS and we are able to accurately and reproducibly read down to 0.00016 or 1.6 ppm SDS. We can accurately determine how much SDS remains in our LC-MS/MS sample, and have peace of mind that our peaks will be accurate.
Try it out, we would love to hear about your experiences with it and SDS. Reach out any time at firstname.lastname@example.org