Effective Removal of Nonionic Detergents in Protein Mass Spectrometry, Hydrogen/Deuterium Exchange, and Proteomics

Abstract

Detergents are frequently used for protein isolation and solubilization. Their presence is crucial in membrane protein protocols or in lipid raft proteomics. However, they are usually poorly compatible with mass spectrometry. Several different sample preparation protocols are routinely used, but they are either laborious or suffer from sample losses. Here, we describe our alternative method for nonionic detergent removal. It is based on selective detergent extraction after capture of the sample on a reversed phase cartridge. The extraction is performed by chlorinated solvents and works well for polyoxyethylene based nonionic detergents, but also for polymers like polyethylene and propylene glycol. Detergent removal can be also carried out on the protein level but a special care must be taken with hydrophobic proteins. In such cases, it is preferable to perform detergent removal after proteolysis which digests the protein to peptides and reduces the hydrophobicity. The method can easily be automated and is compatible with hydrogen/deuterium exchange coupled to mass spectrometry.

Site-directed mutagenesis combined with oxidative methionine labeling for probing structural transitions of a membrane protein by mass spectrometry

“…It is therefore not surprising that membrane protein covalent labeling has become a fairly routine approach, whereas only a handful of HDX studies in this area have appeared over the past few years [28]. Luckily, however, several very recent studies indicate that rapid progress is now being made in the area of membrane protein HDX/MS [75][76][77].…”

Yeast ADP/ATP Carrier Isoform 2

“…Samples were desalted, and detergent and lipids were removed, as previously described (20). Peptides were separated on a reversed phase C18 column (1 ϫ 100 mm, Jupiter; Phenomenex), as previously described (20).…”

“…The HPLC system was connected directly to the spectrometer's ESI source through a T-piece splitting device. The settings used were as previously described (20). Data were processed using DataAnalysis 3.0 (Bruker), and MASCOT was used to match MS/MS spectra to a single-protein data base containing the ScAnc2p sequence.…”

“…1B, lane 2). The detergent used is relevant for further HDX-MS studies, as shown in Rey et al (20). Two different acidic proteases were tested for in-solution digestion of ScAnc2p as a CATR complex.…”

“…Because of its more efficient digestion of ScAnc2p, pepsin was used in subsequent experiments. Our previous studies of soluble and membrane-bound proteins showed immobilized proteases to offer several advantages in HDX-MS experiments (6,10,20). In addition to higher protease stability and the absence of auto-digestion, immobilized proteases provide a much higher local protein concentration.…”