Mag-Net™ Urine: Improved and complementary workflow for clinical urinary proteomics

Intro:

Urine is an ideal biofluid for clinical proteomics experiments due to its non-invasive ease of collection. Urinary microvesicles from both the urinary tract and systemic sources can provide a diverse source of information related to the health status of a patient. We present an automated, reproducible, and robust sample processing workflow for mass spectrometry-based urinary microvesicle analysis in clinical proteomics. This workflow has been adapted from the Mag-Net™ method (Wu et al. 2023) for deep plasma profiling through enrichment of membrane vesicles. The adapted workflow was compared with two published methods for urinary proteome profiling based on MagReSyn® HILIC microparticles, uHLC (Govender et al. 2023) and protein aggregation capture (PAC) (Batth et al. 2019).

Methods:

Combinations of existing and novel MagReSyn® beads were evaluated to achieve the greatest proteome coverage. Various capture methods were also tested. Captured vesicles were then lysed and the proteins were solubilised, reduced, and alkylated whilst bound on-bead. Following washes, using organic solvents, on-bead digest was performed through the addition of trypsin and LysC and 2 h incubation at 47°C.

Resulting peptides were loaded onto Evotips and analysed using an Evosep One (60SPD) coupled to a Bruker timsTOF HT (default short gradient diaPASEF method). Data were searched in Spectronaut 18 using DirectDIA+™. Protein and peptide data were filtered at 0.01 q-value and processed using STRING-db. Custom data analysis scripts were used to assess microvesicle enrichment.

Preliminary Data:

The automated Mag-Net™ urine workflow generates peptides ready for mass spectrometry analysis in 6 hours. A large overlap was observed between the workflows with workflow-specific unique peptides and proteins, as expected. The PAC urine workflow showed the highest protein group identifications. Approximately 30% of the detected proteins were unique to urine samples processed with Mag-Net™. Gene ontology-based cellular component analysis showed strong enrichment of vesicles, exosomes and membrane-bounded organelle associated proteins, alluding to positive capture of urinary microvesicles by the Mag-Net™ Urine workflow. Both Mag-Net™ and uHLC enriched for proteins associated with extracellular exosomes, while PAC primarily enriched for extracellular proteins. The specificity of the Mag-Net™ urine method can be further improved through systematic optimization of binding buffer composition, microsphere functional chemistry and microsphere to urine ratio, which is currently on-going.

Preliminary data suggest that the Mag-Net™ Urine workflow can be used alone or in conjunction with standard soluble protein urinary workflows, such as uHLC and PAC, to gain deeper insights into specific disease states in clinical proteomics studies.