Toward the Development of a Novel Newborn Screening Modality: In-Depth Nontargeted Proteome Analysis of Dried Blood Spots with a Robotic Pipeline Using Low-Cost Iron Powders

We developed a simple protein extraction method for dried blood spots (DBS) that potentially meets the throughput required for newborn screening (NBS) and optimizes nontargeted proteomic analysis in combination with liquid chromatography coupled mass spectrometry in the data-independent-acquisition mode (DIA–LC–MS/MS). The developed pipeline, termed Non-targeted Analysis of Non-specifically DBS-Absorbed proteins (NANDA), successfully addressed the following three challenges: (1) processing of 96 3.2 mm DBS punches in parallel using low-cost iron powders with a robotic system, (2) identifying more than 5,000 proteins using DIA–LC–MS/MS, and (3) improving DIA–LC–MS/MS throughput to 45 samples/day with minimal compromise in protein coverage depth. The results imply that this pipeline can open new venues for conducting NBS using nontargeted quantitative proteome profiling, which has been a missing modality in NBS.