From Cells to Peptides: “One-Stop” Integrated Proteomic Processing Using Amphipols

In proteomics, detergents and chaotropes
are indispensable for
proteome analysis, not only for protein extraction, but also for protein
digestion. To increase the protein extraction efficiency, detergents
are usually added in the lysis buffer to extract membrane proteins
out of membrane structure and to maintain protein in solutions. In
general, these detergents need to be removed prior to protein digestion,
usually by precipitation or ultrafiltration. Digestion often takes
place in the presence of chaotropic reagents, such as urea, which
often need to be removed prior to mass spectrometry. The addition
and removal of detergents and chaotropes require multiple steps that
are time-consuming and can cause sample losses. Amphipols (APols)
are a different class of detergents that have physical and solubilization
properties that are distinct from conventional detergents. They have
primarily been used in protein structure analysis for membrane protein
trapping and stabilization. Here, we demonstrate a simple and rapid
protocol for total and membrane proteome preparation using APols.
We demonstrate that APols added for cell lysis help maintain the proteome
in solution, are compatible with protein digestion using trypsin,
and can readily be removed prior to mass spectrometry by a one-step
acidification and centrifugation. This protocol is much faster, can
be performed in a single tube, and can readily replace the conventional
detergent/chaotrope approaches for total and membrane proteome analysis.