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 from a variety of sources, including fresh-frozen tissue, FFPE tissue, or blood. To reach this goal, we have implemented single-pot solid-phaseenhanced 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 intervention, enabling reproducible tissue proteomics in a broad range of clinical and non-clinical applications.

A Critical Review of Bottom-Up Proteomics: The Good, the Bad, and the Future of This Field

“…Suspension trapping (STrap) is a method that uses tips packed with a quartz or glass filter and a hydrophobic C18 layer to perform sample clean-up and protein digestion in one step [66]. Single-Pot Solid-Phase-enhanced Sample Preparation (SP3) uses paramagnetic beads for the same purpose [67] and can be easily automated using a liquid handling robot [68]. These methods can be easily automated and integrated.…”

Establishing a mass spectrometry-based system for rapid detection of SARS-CoV-2 in large clinical sample cohorts

“…SP3 is based on the binding of proteins to paramagnetic beads in the presence of an organic solvent, followed by extensive washings and digestion 38 . Recently, Müller and colleagues described an automated SP3 implemented on an Agilent Bravo platform, which was applied to cell culture and formalin-fixed paraffin-embedded tissue sections 39 . Their method required 3.5 h to process 96 samples until reaching the digestion step, which required an additional 4 h. With our strategy, all steps can be completed in the robotic platform and the resulting microplate is ready to be processed by TFC-MS/MS.…”

A rigorous evaluation of optimal peptide targets for MS-based clinical diagnostics of Coronavirus Disease 2019 (COVID-19)

“…Most conventional bottom-up proteomics experiments utilize trypsin digestions which occur overnight with incubation at 37°C, meaning a single sample would have to be processed and analyzed over the course of two days; this would have to be significantly reduced as the conventional 24-48 hour complete turnaround of RT-qPCR assays is being decreased through the use of strategies such as direct RT-qPCR 12 , RT-LAMP 13 , and CRISPR-based amplification strategies 14, 58, 59 . The turnaround time of clinical proteomics can potentially be decreased for individual samples using modified or alternative protein digestion enzymes with higher rates of reactivity 60 ; in addition, automation of clinical proteomics technology can provide reproducible, robust analyses of patient samples 61, 62 .…”