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 data-independent acquisition (DIA) 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 proteomes per instrument per day. This has shifted the bottleneck towards sample preparation creating a need for simple and efficient workflows. This publication from the Olsen group at the Novo Nordisk Foundation Center for Protein Research, describes the development of One-Tip, a simplified and streamlined workflow utilizing the Evotip to address this challenge.

The Evotip serves as a sample storage and introduction device for the Evosep One, and by integrating cell lysis and digestion on the Evotip directly, this conceptually represents the simplest one-pot workflow for proteomics, as no further sample handling is needed. In brief, the One-Tip methodology only requires two pipetting steps within the Evotip: one for the lysis and digestion buffer, and the other for the cell suspension in PBS. Followed by a one-hour digestion in a water-filled Evotip box and standard Evotip preparation, the One-Tip samples are ready for LC-MS/MS analysis after just 70 minutes of sample preparation time. Furthermore, they have applied this to an optimized narrow-window data-independent acquisition analysis using the Thermo Scientific Astral mass spectrometer. Coupled with the Whisper 40 SPD method, they benchmarked the sensitivity and reproducibility of the One-Tip methodology by increasing the number of HeLa cells from 20 to 3,000 cells.

An impressive depth was achieved from just 20 cells, with more than 46,000 peptides corresponding to 5,000 protein groups, whereas close to complete proteome coverage was obtained from ~1,000 cells with 120,000 peptides leading to more than 9,000 proteins. The One-Tip workflow demonstrated high reproducibility and quantification precision, with a remarkable coefficient of variation of 5-8% in samples with more than 100 cells.

To illustrate versatility and sensitivity, they investigated the development of mouse pre-implantation embyos at the single-cell level from oocyte and zygote to 4-cell stage. The zona pellucida, the extracellular glycoproteinaceous coat of oocytes, zygotes, 2-cell and 4-cell stages, was chemically removed and single cells were dissociated under a microscope and dispensed individually into Evotips, pre-loaded with lysis and digestion buffer. In total, 6216 protein groups were identified with a median of 5428, 5417, 5259 and 4779, for each stage, respectively. This yields more than ~2300 proteins compared to a recent study. Where they employed a sophisticated sample preparation workflow and four times longer LC gradient than used in this study, demonstrating that the One-Tip workflow is faster, more sensitive and more reproducible.

In conclusion, the One-Tip methodology allows for a simple, loss-less and robust one-pot sample preparation strategy from 1000’s of cells down to, in this study, 20 cells. However, the loss-less nature of the strategy is speculated to be proven very efficient even at the single cell- and potentially sub-cellular level as these technologies are further developed. Additionally, the simplicity of the workflow makes it easy to automate on liquid handlings robots and to become a new cornerstone for sensitive and high throughput proteomics.

Read more about the work here