Untargeted label-free quantitative proteomics has become an increasingly powerful methodology that can provide vital quantitative information, either relative or absolute, on large parts of the expressed proteome. Contrarily to label-based methodologies, label-free quantitation is applicable to any kind of sample and, in principle, has no limitation on the number of samples that can be compared. These advantages make label-free quantitative proteomics the method of choice for analysis of clinical samples, primary cells and tissues.

In this study we set out to identify novel substrates for the family of Membrane-associated RING-CH (MARCH) E3 ubiquitin ligases in vivo. Regulation of cell surface receptors is essential for the maintenance of the cell proteostasis, and ubiquitination at the plasma membrane has been observed to be a fundamental post-translational mechanism regulating a wide variety of surface proteins, including immune receptors. The MARCH ligases are a subfamily of the RING E3 ligases originally identified in viruses to be involved in immune evasion strategies. Numerous in vertebrate studies suggest that the MARCH ligases’ main role is to regulate trafficking, surface expression and turnover of immune receptors, however most of their substrates remain unknown.

In order to identify novel surface targets of the MARCH ligases we developed a plasma-membrane enrichment strategy from primary antigen presenting cells (DCs), purified from wild-type and MARCH-deficient mouse spleens. Quantitative analysis of LC-MS/MS data was performed using MaxQuant for peptides/proteins identification and for feature detection, while methods for the quantitative analysis and statistics were developed in house. Through this approach we were able identify potential novel targets for most of the MARCH ligases as well as to confirm previously characterised ones. With this work we have established a robust methodology to systematically identify potential targets of ubiquitin ligases in vivo.