Simple Sodium Dodecyl Sulfate-Assisted Sample Preparation Method for LC-MS-Based Proteomics Applications

Abstract

Sodium dodecyl sulfate (SDS) is one of the most popular laboratory reagents used for biological sample extraction; however, the presence of this reagent in samples challenges LC-MS-based proteomics analyses because it can interfer with reversed-phase LC separations and electrospray ionization. This study reports a simple SDS-assisted proteomics sample preparation method facilitated by a novel peptide-level SDS removal step. In an initial demonstration, SDS was effectively (>99.9%) removed from peptide samples through ion substitution-mediated DS- precipitation using potassium chloride (KCl), and excellent peptide recovery (>95%) was observed for <20 μg peptides. Further experiments demonstrated the compatibility of this protocol with LC-MS/MS analyses. The resulting proteome coverage obtained for both mammalian tissues and bacterial samples was comparable to or better than that obtained for the same sample types prepared using standard proteomics preparation methods and analyzed using LC-MS/MS. These results suggest the SDS-assisted protocol is a practical, simple, and broadly applicable proteomics sample processing method, which can be particularly useful when dealing with samples difficult to solubilize by other methods.

Discovery and Targeted Proteomics on Cutaneous Biopsies Infected by Borrelia to Investigate Lyme Disease*

“…Moreover, the major drawback of SDS is its incompatibility with MS due to high ion suppression (42,43). To solve this problem, SDS removal using ion exchange chromatography (44), dialysis (45), ethyl acetate extraction (46), protein precipitation, use of a spin column removal kit (47), SDS precipitation (48), and filteraided sample preparation have been tested (24). Alternatively, detergents with properties similar to SDS but that are MScompatible have been used.…”

Amicon-adapted enhanced FASP: an in-solution digestion-based alternative sample preparation method to FASP

“…Lysates were solubilized in 6 M urea, reduced for 60 min at 37°C with 10 mM DTT and alkylated by 30 mM iodoacetamide for 30 min. One hundred-microgram whole cell lysate aliquots were next diluted six-fold in ABC buffer and digested in increasing concentrations of SDS 5 . Half of samples were afterward incubated for 12h at 37°C in the presence of (1) trypsin (enzyme-to-protein ratio 1:100 w/w) or (2) trypsin/Lys-C Mix (ratio 1:25 w/w) for proteolysis.…”

“…The precipitation protocol is based on Zhou et al 5 and Zhou et al 24 , with minor modifications. Briefly, samples were mixed with an equal volume of 4 M KCl, acidified with 2% FA and peptides were collected in the supernatant after centrifugation at 15,700 g for 15 min to pellet the potassium dodecyl sulfate (KDS) and deoxycholate precipitates.…”

“…In fact, sodium dodecyl sulfate (SDS) is a powerful anionic detergent and one of the most widely used reagents for solubilisation and denaturation of proteins 4 . By binding amino acids through hydrophobic and ionic interactions, SDS alters proteins' spatial conformational structure and inhibits proteases activity, thus enabling long-term conservation of structurally preserved proteins 5 . In spite of all its advantages, SDS significantly suppresses analyte ion signals in electrospray ionization (ESI)-MS, alters the chromatographic separation of peptides during liquid chromatography (LC) 4,6 , and strongly inhibits trypsin activity 7 , even at very low concentrations, which considerably limits protein identification.…”

“…Removal of SDS SDS is not easily removed from proteins to which it binds strongly through ionic and hydrophobic interactions 5 . In FASP, usage of a washing buffer composed solely of urea has been assumed to be sufficient to completely deplete the SDS from up to 30 µl of a small protein extract (≤250 µg) 9,13,14 .…”