A Soft Preparative Method for Membrane Proteome Analysis Using Frit Inlet Asymmetrical Flow Field-Flow Fractionation: Application in a Prostatic Cancer Cell Line

Abstract

Membrane proteins participate in a number of important biological functions such as signal transduction, molecular transport, and cell-cell interactions. However, due to the nature of membrane proteins, the development of a preparative method that produces a sufficient yield of purified membrane proteins from the cell remains a challenge. In the present study, frit inlet asymmetrical flow field-flow fractionation (FI-AFIFFF) was employed to fractionate membrane fragments containing membrane proteins from free cytoplasmic proteins of prostatic cancer cell (DU145 cell) lysates. The isolated membrane proteins were then digested and analyzed by nanoflow liquid chromatography/tandem mass spectrometry (nLC-ESI-MS-MS). Since fractionation of the cell lysate mixtures containing membrane fragments and cytoplasmic proteins could be achieved based on the differences of their sizes in FI-AFIFFF, membrane fragments were partially isolated from the cytoplasmic proteins and collected. The performance of FI-AFIFFF for prefractionation of the membrane proteome was examined by comparing the number of membrane proteins that were identified with the number identified using an ultracentrifugation method. The application of FI-AFIFFF to membrane proteomics produced an increased yield of purified membrane proteins with fewer cytoplasmic proteins compared to a conventional ultracentrifugation method.