Simultaneous profiling of phospho- and palmitoyl-proteome landscape in serum exosomes through paramagnetic separation

Protein post-translational modification (PTM) events in exosomes play a critical role in key biological processes and hold significance in the occurrence and development of diverse diseases. Thus, monitoring the state of PTMs in exosomes is highly appealing but technically challenging owing to the low abundance of exosomes and PTM-proteins, as well as the presence of diverse impurities in biofluids.

In this work, we developed a magnetic solid-phase extraction (MSPE) approach based on magnetic materials functionalized with Ti⁴⁺ and bis(vinylsulphonyl)methane (MagBVS-Ti⁴⁺) for the profiling of exosomal phospho- and palmitoyl-proteins. Coupled with LC-MS/MS, a total of 2264 phosphopeptides and 2450 palmitoyl-peptides were identified from human serum exosomes. Furthermore, a series of bioinformatics analyses were conducted, systematically revealing the functions of identified phospho- and palmitoyl-proteins.

This study establishes a MSPE-based workflow for the simultaneous profiling of phospho- and palmitoyl-proteome landscape in serum exosomes. The proposed approach provides novel insights into multi-PTM research and shows potential for the application in large-scale proteomic analysis for developing biomarkers and identifying potential therapeutic targets for diseases.