Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Hands-off: fully automated & TMT-compatible sample preparation on the PreON liquid handling platform employing the iST-NHS workflow (#781)

Garwin Pichler 1 , Fabian Hosp 1 , Nils A. Kulak 1
  1. PreOmics, Martinsried, BAVARIA, Germany

Sample preparation is an important component of the overall mass spectrometry-based proteomics workflow and remains to be a limiting factor for high-throughput analyses. Here, we present a fully automated end-to-end solution for standardized sample processing, including cell lysis, digestion, TMT labeling and peptide cleanup within 4 hours. In order to minimize sample loss and improve reproducibility, we sought to completely automate TMT sample processing from cell lysis to ready-to-measure labeled peptides. To facilitate this, we aimed to combine the NHS adaption of the iST technology (Kulak et al., 2014) with a newly developed automation platform called PreON.

The workflow described here integrates robotic handling with chemical labeling of peptides in a one-pot reaction device, including cell lysis, protein denaturation, reduction, alkylation, digestion as well as the peptide cleanup. The PreOn platform is a newly developed benchtop liquid handling system capable of processing up to 12 samples in parallel. It is integrated with a built-in centrifuge with evaporation feature, ultrasound liquid detection, deck load check and a drag-and-drop method selection for easy, fast and convenient operation. 

Here, we demonstrate full automation of sample processing without any manual interference from cell lysis to ready-to-measure peptides for various biological sample types such as multiple cancer cell lines, bacteria, yeast or human plasma, scaling from 1-100 µg of protein input material. Furthermore, we integrate chemical labeling such as iTRAQ or TMT in the whole workflow and thus permit multiplexing in a fully automated fashion. To this end, we have employed 11-plex TMT to successfully label several cell lines achieving >98% labeling efficiency and a reproducibility of R2 = 0.98 for biological replicates (CVs <10%). The combination of the one-pot iST workflow and the fully automated liquid handling enables a simplified, efficient and highly reproducible preparation of biological input materials and ranges.