Fully automated workflow for integrated sample digestion and Evotip loading enabling high-throughput clinical proteomics

Protein identification and quantification is an important tool for biomarker discovery. With the increased sensitivity and speed of modern mass spectrometers, sample-preparation remains a bottleneck for studying large cohorts. To address this issue, we prepared and evaluated a simple and efficient workflow on the Opentrons OT-2 (OT-2) robot that combines sample digestion, cleanup and Evotip loading in a fully automated manner, allowing the processing of up to 192 samples in 6 hours. Our results demonstrate a highly sensitive workflow yielding both reproducibility and stability even at low sample inputs. The workflow is optimized for minimal sample starting amount to reduce the costs for reagents needed for sample preparation, which is critical when analyzing large biological cohorts. Building on the digesting workflow, we incorporated an automated phosphopeptide enrichment step using magnetic Ti-IMAC beads. This allows for a fully automated proteome and phosphoproteome sample preparation in a single step with high sensitivity. Using the integrated workflow, we evaluated the effects of cancer immune therapy on the plasma proteome in metastatic melanoma patients. ### Competing Interest Statement Dorte B. Bekker-Jensen, Joel Mario Vej-Nielsen, Magnus Huusfeldt, and Nicolai Bache are employees of Evosep Biosystems, manufacturer of instrumentation used in this work. Other authors declare no competing interests.