Summary
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