Zehan Hu (Planegg / DE), Vincenzo Romaniello (Planegg / DE), Torsten Pirch (Planegg / DE), Claudia Martelli (Faellanden / CH), Irina Maerki (Faellanden / CH), Barbara Steigenberger (Martinsried / DE), Katrin Hartinger (Planegg / DE), Nils Kulak (Planegg / DE)
Introduction
LC-MS is a powerful tool in proteomics, helping to decode biological processes, discover new biomarkers, and understand the function of new drugs. For comprehensive insights, analysis of large sample numbers is crucial. However, one of the main bottlenecks remains the lack of a robust and rapid workflow for sample preparation. To overcome the above challenges, we present a unique in-tip stationary phase-based proteomics workflow integrated in a fully automated, yet compact liquid handling platform, the Automated ProteinPrep 96 (APP96TM), to process up to 96 samples/day. Notably, our approach does not require a centrifuge, positive pressure, or disposable tips.
Methods
The automated workflow is based on the PreOmics iST chemistry and includes all LC-MS sample preparation steps in a 96-well format: lysis, reduction and alkylation, digestion, and peptide clean-up. Plasma samples as well as human, insect and yeast cell pellets corresponding to a protein amount range between 2 and 50 µg were processed according to PreOmics iST protocol. 300 ng of peptides was separated with a 6 (for plasma samples)- or 30 (for others)-min gradient on the nanoElute 2 system and analyzed usingdia-PASEF® schemes on the timsTOF HT (Bruker). DIA search was performed using MaxQuant or Spectronaut 17/18 (Biognosys) using directDIA mode.
Results
Automated sample preparation on the APP96 provided equal or higher protein and precursor identifications for all tested sample types (plasma samples, mammalian, insect, and yeast cells) compared to manual sample preparation. In experiments with HeLa cells, more than 6800 protein groups and 110´000 peptides were identified with an impressive 98% overlap of protein groups between manual and automated workflows. Notably the introduction of newly designed POPTip achieved significant reduction of methionine oxidation due to reduced air contact during clean-up. Moreover, excellent intra-day (manual: 8-10 %CV vs. automated: 5-8 %CV) and inter-day (manual: 23 %CV vs. automated: 17 %CV) repeatability in protein quantity could be obtained with the automated workflow, outperforming the manual approach.
Conclusions
The APP96 platform combines robust, flexible, and fully automated sample preparation for LC-MS sample analysis with minimal environmental impact. Based on the high-quality and reliable data obtained, the APP96 enables confident upscaling of proteomics sample preparation, ideal suited large cohort studies.