Assessment of membrane peptide loss in common bottom-up proteomic workflows leads to an improved membrane-inclusive sample preparation protocol

Analysis of membrane proteins in bottom-up proteomics often relies on characterisation of their water-soluble regions. Identification coverage of transmembrane domains through widely used methods remains poor. Membrane spanning regions are difficult to analyse due to lack of trypsin cleavage sites and abundance of nonpolar amino acids. Even though membrane proteins are key drug targets, are crucial to cell transport and communication, their analysis remains a challenging task.

To develop a more membrane inclusive method tailored to the analysis of membrane peptides, their losses were closely assessed during sample treatment. Three common sample preparation protocols were compared – eFASP(1), SP3(2) and SP4(3) with respect to membrane peptide identifications. Additional fractions generated during sample treatment were collected and analysed regarding membrane peptide loss. These include molecular-weight-cut-off filter washes employed in eFASP, or bead and test tube washes in SP3 and SP4 protocols, respectively. All these washes contained valuable source of membrane peptides overlooked by standard sample processing protocols.

Further method modifications comprise improved elution of hydrophobic peptides during liquid chromatography by using isopropanol/acetonitrile mixture in 0.1% formic acid as a strong eluent and dissolution of samples prior to analysis in 30% ACN.

Addressing challenges in hydrophobic peptide analysis in a newly modified sample preparation method enabled us to analyse underrepresented membrane regions more comprehensively in proteomic experiments.

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