In this study, mass spectrometry proteomics was used to investigate whether there are qualitative or quantitative differences in the mammalian plasma membrane proteins extracted with SMA compared to a detergent control. For this, cell surface proteins of 3T3L1 fibroblasts were biotinylated and extracted using either styrene maleic acid (SMA) or detergent. Following affinity pull-down of biotinylated proteins with NeutrAvidin beads, samples were analysed by nanoLC-MS. Here, we report for the first time, a global proteomics protocol for detection of a mammalian cell ‘SMALPome’, membrane proteins incorporated into SMA nanodiscs.