Efficient Protein Digestion and Quantification

Protein samples were digested on-bead using multimode magnetic microparticles (MagReSyn^® HILIC, ReSyn Biosciences) in a KingFisher Duo™ system (Thermo Fisher Scientific), as previously described [32 (link), 33 (link)], with minor modifications. Briefly, magnetic hydrophilic affinity microparticles (20 µl, 200 µg) were equilibrated in 200 µl 100 mM NH₄Ac pH 4.5, 15% MeCN. The microparticles were transferred to a well containing the protein-binding buffer solution and mixed for 30 min at RT. The captured proteins were washed twice in 200 µl 95% MeCN and transferred to 200 µl 25 mM ammonium bicarbonate containing 1 µg sequencing grade modified trypsin (Promega, Madison, USA) and mixed for 4 h at 37 °C. Finally, beads were washed in 1% trifluoracetic acid to elute any remaining bound peptides. The resulting peptides (pool of digest and eluate) were vacuum dried, resuspended in 2% MeCN, 0.2% FA and quantified using the Pierce™ Peptide Quantification (Thermo Fisher Scientific) assay as per the manufacturer’s instructions.
A project specific system suitability-quality control (PQC) sample was prepared by pooling an equal volume of 16 urinary peptide samples. Additionally, a complex proteome digest was used as a general system suitability assessment. These PQC samples were injected at least once with each batch and analysed and processed together with the study samples.