Automated sample preparation with SP 3 for low‐input clinical proteomics

Abstract

High-throughput and streamlined workflows are essential in clinical proteomics for standardized processing of samples originating from a variety of sources, including fresh frozen tissue, FFPE tissue, or blood. To reach this goal, we have implemented single-pot solid-phase-enhanced sample preparation (SP3) on a liquid handling robot for automated processing (autoSP3) of tissue lysates in a 96-well format, performing unbiased protein purification and digestion, and delivering peptides that can be directly analyzed by LCMS. AutoSP3 eliminates hands-on time and minimizes the risk of error, reduces variability in protein quantification and improves longitudinal performance and reproducibility. We demonstrate the distinguishing ability of autoSP3 to process low-input samples, reproducibly quantifying 500-1000 proteins from 100-1000 cells (<100 ng protein). Furthermore, we applied this approach to a cohort of clinical FFPE pulmonary adenocarcinoma (ADC) samples, and recapitulate their separation into known histological growth patterns based on proteome profiles. Collectively, autoSP3 provides a generic, scalable, and cost-effective pipeline for routine and standardized proteomic sample processing that should enable reproducible proteomics in a broad range of clinical and non-clinical applications. * Abbreviations : ABC : Ammonium bicarbonate ACN : Acetonitrile ADC : Adenocarcinoma BCA : Bicinchoninic acid assay CAA : Chloroacetamide CV : Coefficient of variation DMEM : Dulbecco’s modified Eagle’s medium DTT : Dithiothreitol EDTA : Ethylenediaminetetraacetic acid EtOH : Ethanol FA : Formic acid FBS : Fetal Bovine serum FFPE : Formalin-fixed and paraffin-embedded FWHM : Full width half maximum H&E : staining Hematoxylin and Eosin staining iBAQ : Intensity-based absolute Quantification LCMS : Liquid Chromatography coupled to Mass Spectrometry LFQ : Label-free quantification MS : Mass spectrometry MS/MS : Tandem mass spectrometry NSCLC : Non-small Cell Lung Cancer PBS : Phosphate-buffered saline PCR : Polymerase chain reaction PIC : Protease-inhibitor cocktail QE HF : Q-Exactive High Field Mass Spectrometer RSLC : Rapid Separation Liquid Chromatography SDS : Sodium dodecylsulfate SP3 : Single-pot solid-phase-enhanced sample preparation TCEP : Tris(2-carboxyethyl)phosphine TFA : Trifluoroacetic acid TMT : Tandem mass-tag t-SNE : t-distributed stochastic neighbor embedding WHO : World Health Organization