Refinement of paramagnetic bead–based digestion protocol for automatic sample preparation using an artificial neural network

Despite technological advances in the proteomics field, sample preparation still represents the main bottleneck in mass spectrometry (MS) analysis. Bead-based protein aggregation techniques have recently emerged as an efficient, reproducible, and high-throughput alternative for protein extraction and digestion. Here, a refined paramagnetic bead–based digestion protocol is described for Opentrons® OT-2 platform (OT-2) as a versatile, reproducible, and affordable alternative for the automatic sample preparation for MS analysis. For this purpose, an artificial neural network (ANN) was applied to maximize the number of peptides without missed cleavages identified in HeLa extract by combining factors such as the quantity (µg) of trypsin/Lys-C and beads (MagReSyn® Amine), % (w/v) SDS, % (v/v) acetonitrile (ACN), and time of digestion (h). ANN model predicted the optimal conditions for the digestion of 50 µg of HeLa extract, pointing to the use of 2.5% (w/v) SDS and 300 µg of beads for sample preparation and long-term digestion (16h) with 0.15 µg Lys-C and 2.5 µg trypsin (≈ 1:17 ratio). Based on the results of the ANN model, the manual protocol was automated in OT-2. The performance of the automatic protocol was evaluated with different sample types, including human plasma, rat bile, Arabidopsis thaliana leaves, Escherichia coli cells, and mouse tissue cortex, showing great reproducibility and low sample-to-sample variability in all cases. Notwithstanding, we must highlight the performance of this method in the preparation of a challenging biological fluid as bile, a proximal fluid that is rich in bile salts, bilirubin, cholesterol, and fatty acids, among other MS interferents. Compared to other protocols described in the literature for the extraction of digestion of bile proteins, our method allowed to identify exclusively 9.91% (x proteins), thus contributing to improving the coverage of the bile proteome.