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A fully automated FAIMS-DIA mass spectrometry-based proteomic pipeline

ABSTRACT: 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 streamlining a fully automated sample preparation platform, 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 showed single compensation voltage (CV) at -35V not only yields deepest proteome coverage but also best correlates with DIA without FAIMS. Our in-depth comparison of direct-DIA database search engines showed Spectronaut outperforms others, providing highest quantifiable proteins. Next, we apply three common DIA strategies in characterizing human induced pluripotent stem cell (iPSC)-derived neurons and show single-shot MS using single CV(-35V)-FAIMS-DIA results in >9,000 quantifiable proteins with < 10% missing values, as well as superior reproducibility and accuracy compared to other existing DIA methods.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Stem Cell

SUBMITTER: Yue Andy Qi

LAB HEAD: Yue Andy Qi

PROVIDER: PXD029902 | Pride | 2023-08-22

REPOSITORIES: Pride

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