Detergents are needed to extract hydrophobic proteins and maintain their solubility in solution. However, the presence of detergents in peptide samples severely inhibits ionization in mass spectrometry (MS) analysis and reduces chromatographic resolution in LC-MS. Therefore, the detergent must be removed to sensitively detect peptides by MS. This unit describes a rapid protocol in which ethyl acetate (ethyl acetate sds) extraction is used to remove octylglycosides from protease digesters without loss of peptides. The procedure can also be used to reduce the interference of sodium dodecyl sulfate, Nonidet P-40, or Triton X-100 on peptide samples for MS analysis.

Since OG can be completely extracted with ethyl acetate without detectable peptide loss, it is desirable to exchange OG for other existing detergents in the protein solution prior to digestion. Unit 4.8 describes how to change detergent. After exchange, the proteins in OG are denatured with 6M GnHCl, then reduced, carboxymethylated, and the denatured agents removed by ultrafiltration or dialysis of 0.5%OG prior to digestion. As an alternative to the denaturation treatment of GnHCl, an organic solvent compatible with the proteases used (e.g., 5-20% acetonitrile for trypsin) is included in the digestive mixture to improve the efficiency of digestion.

A solution of peptides extracted by water-saturated ethyl acetate can be extracted immediately after digestion or from stored samples. Acidification of protein digestives is not necessary. The peptide solution should be between 50 and 100μl in a 1.5ml microtube. The larger volume can be divided into two tubes or reduced to 100μl with SpeedVac. However, the salt concentration in the peptide solution should be kept at less than 200mM to prevent possible loss of the peptide to the organic phase during extraction.