Membrane proteins (MPs) are involved in almost all cellular processes and comprise approximately 30% of the proteome, which makes them an ideal choice for 60% of drug targets. Although the study and characterization of MPs" structure, function, and interactions are clearly essential, they represent a challenge for current analytical methods, owing mostly to low production yields and the need for membrane mimetics to solubilize the proteins before analysis. Cross-linking mass spectrometry (XL-MS) has grown dramatically into a routinely utilized strategy for characterizing protein higher-order structure and mapping protein-protein interaction networks on a proteome-wide scale. However, the XL-MS analysis of membrane proteins is still a significant challenge due to their hydrophobic properties as well as the lipid-rich environment. In this work, we optimized sample preparation and detection of crosslinked peptides, including disulfide mapping for membrane proteins in SMALPs or detergents.

Different amine-reactive crosslinkers, including DSS, DSSO and tBuPhoX were used to crosslink membrane proteins in SMALPs or detergents. Cross-linked samples were reduced, alkylated, and digested with trypsin or pepsin. For disulfide mapping, proteins were hydrolyzed with microwave-assisted acid hydrolysis (MAAH) and desalted using C18 stage tips. All data were acquired using the Thermo Scientific™ Vanquish™ Neo system connected to an EASY-Spray™ PepMap™ RSLC C18 column (0.075 mm x 250 mm, (Thermo Fisher Scientific)) at 250 nL/min flow rate and a Thermo Scientific™ Orbitrap™ Ascend Structural Biology Tribrid™ mass spectrometer coupled with a FAIMS Pro Duo interface. Data were analyzed using Thermo Scientific™ Proteome Discoverer™ 3.1 software and XlinkX 3.1 node.

Our structural understanding of membrane proteins is very limited, largely due to their instability outside of the native lipid environment. They are usually extracted or solubilized in detergents or styrene maleic acid (SMA) lipid particles (SMALPs). It has been a challenge to develop XL sample preparation methods for membrane proteins that are compatible with mass spectrometry. In this study, we developed end-to-end XL-MS/S-S workflows for membrane proteins using different amino-reactive crosslinkers. In the optimized workflow, crosslinking was performed in detergents or SMALPs to keep their native conformations, followed by buffer exchange to 1% DDM or 0.1% RapiGest. Trypsin digestion was carried out using 0.1% RapiGest . Pepsin digestion was carried out in 1% formic acid . MAAH was performed in the original sample buffer with 25% TFA. With this new sample preparation methods, we were able to achieve a sequence coverage of more than 90% of the membrane proteins in SMALPs or detergents. More importantly, we identified crosslinked peptides and provide critical information for downstream structural analysis of GPCRs, HIV proteins, and bacterial enzymes by X-Ray and cryo-EM.