A Fully Automated FAIMS-DIA Mass Spectrometry-Based Proteomic Pipeline

Summary: Here, we present a standardized, “off-the-shelf” proteomics pipeline working in a single 96-well plate to achieve deep coverage of cellular proteomes with high throughput and scalability. This integrated pipeline streamlines a fully automated sample preparation platform, a data-independent acquisition (DIA) coupled with high-field asymmetric waveform ion mobility spectrometer (FAIMS) interface, and an optimized library-free DIA database search strategy. Our systematic evaluation of FAIMS-DIA showing single compensation voltage (CV) at −35 V not only yields the deepest proteome coverage but also best correlates with DIA without FAIMS. Our in-depth comparison of direct-DIA database search engines shows that Spectronaut outperforms others, providing the highest quantifiable proteins. Next, we apply three common DIA strategies in characterizing human induced pluripotent stem cell (iPSC)-derived neurons and show single-shot mass spectrometry (MS) using single-CV (−35 V)-FAIMS-DIA results in >9,000 quantifiable proteins with <10% missing values, as well as superior reproducibility and accuracy compared with other existing DIA methods. Motivation: Although mass spectrometry (MS)-based quantitative proteomics has been significantly advanced in the past decade, reproducibility and robustness are roadblocks preventing this technique from being widely applied to large biomedical research communities. We aim to streamline and automate major components of MS-based proteomics, providing an extensively optimized and validated platform for large-scale studies in modern proteomic laboratories and contract research organizations.