A complete and automated end-to-end sample preparation strategy for high-throughput and standardized proteomics with high sensitivity

Proteomics is going through a significant paradigm shift and is rapidly evolving towards higher throughput and robust easier-to-use technologies. The use of short LC gradients combined with fast and sensitive mass spectrometers has been a key aspect in this development. The Evosep One is designed for high-throughput and high-volume applications, where standardized methods with increasing robustness enable routine analysis of hundreds of samples per instrument per day. This has moved the bottleneck towards sample preparation, which requires standardized and fully automated end-to-end workflows.

We have developed an automated end-to-end workflow for proteomics sample preparation, designed for fast and robust protein digestion capable of processing up to 500 samples on one AssayMap Bravo in a single workday. The workflow results in ready-to-use samples for LC-MS as tryptic peptides are loaded directly onto Evotips in a complete hands-free manner, starting from protein lysate. It is optimized for speed and is completed within just 90 minutes. To illustrate the robustness and reproducibility, 500 samples were prepared in one day and analyzed with the corresponding high-throughput 500 samples per day method in a randomized order.

At this level of throughput, the cost per sample is an important consideration. Therefore, we scaled down the use of consumables and designed the workflow to digest only what is needed for one LC-MS injection to minimize enzyme usage. To examine the sensitivity of our sample preparation workflow, we scaled down to even lower starting input materials and observed significant sensitivity down to just 1 ng protein starting amount.