Automated sample preparation with SP3 for low-input clinical proteomics

Molecular Systems Biology
Torsten MüllerJeroen Krijgsveld

Abstract

High-throughput and streamlined workflows are essential in clinical proteomics for standardized processing of samples 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. AutoSP3 performs unbiased protein purification and digestion, and delivers peptides that can be directly analyzed by LCMS, thereby significantly reducing hands-on time, reducing variability in protein quantification, and improving longitudinal reproducibility. We demonstrate the distinguishing ability of autoSP3 to process low-input samples, reproducibly quantifying 500-1,000 proteins from 100 to 1,000 cells. Furthermore, we applied this approach to a cohort of clinical FFPE pulmonary adenocarcinoma (ADC) samples and recapitulated their separation into known histological growth patterns. Finally, we integrated autoSP3 with AFA ultrasonication for the automated end-to-end sample preparation and LCMS analysis of 96 intact tissue samples. Collectively, this constitutes a generic, scalable, and cost-effective workflow with minimal manual inte...Continue Reading

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Citations

Aug 2, 2020·Nature Protocols·Katarzyna BuczakAlessandro Ori
Dec 5, 2020·Nature Communications·Karina Helena Morais CardozoValdemir Melechco Carvalho
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Jul 24, 2021·Analytical Chemistry·Zhichang YangLiangliang Sun
Aug 6, 2021·Journal of Proteome Research·Kendall MartinTujin Shi
Aug 13, 2021·Nature Communications·Geraldine NouaillesMartin Witzenrath
Jul 23, 2020·Journal of Proteome Research·Matthew Waas, Thomas Kislinger
Nov 5, 2021·Journal of Proteome Research·Stephan EckertBernhard Kuster

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Methods Mentioned

BETA
electrophoresis
PCR
DNA shearing
FACS
proteome
biopsy

Software Mentioned

autoSP3
R package tsne
MaxQuant
R package fgsea
GSEA
R
Andromeda
R package ReactomePA
AssayMap
SP3

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