MSACL 2024 Industry Workshop Presentation *This Podium Presentation is occuring in the context of an Industry Workshop that starts at the time below. Its actual start time may be up to 40 minutes later, depending on order of presentation if there are multiple presentations planned by the workshop host. Workshop Host: Agilent Technologies Day: Tuesday March 19 Time: 12:15* Location: San Carlos 3 (Marriott > Mezzanine | Stairs from Lobby or SkyBridge from Conference Ctr)

An efficient and fully automated end-to-end sample preparation strategy using the AssayMAP Bravo for Evosep-MRM
	Nicolai Bache, PhD Evosep
Presenter Bio: Dr. Nicolai Bache, VP Proteomics Research, Vertical Markets at Evosep holds a PhD in mass spectrometry and proteomics from the University of Southern Denmark followed by a 2 year postdoc at the Children’s Medical Research Institute in Sydney. In 2010 he moved to proteomics tools and MedTech industry and has gained experience through several companies such as Proxeon Biosystems, Thermo Scientific and Bruker Daltonics before co-founding Evosep Biosystems in early 2016. He has had a clear mission throughout his career to enable and promote clinical proteomics. At Evosep Nicolai leads the technology and application development together with leading experts in the field and spearheads new innovations tailored for scalable and standardised proteomics required for clinical applications.

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.

In this context, automated loading of Evotips has emerged as a pivotal advancement in sample preparation workflows. The Evotip can now be loaded using an innovative and fully automated strategy with the Agilent AssayMAP Bravo. The liquid handler is equipped with 96 syringes, which enables very fast sample loading on the Evotip, as a full box of Evotips are loaded in approximately four minutes with optimal performance. By precisely layering liquids within the Evotip and utilizing positive air pressure, the fully automated loading strategy for Evotips is the central component of a modular strategy that allows for addition of functional modules such as digestion.
As an example of this, we have integrated protein aggregation capture (PAC) assisted digestion in an end-to-end standardized workflow using the Agilent AssayMAP Bravo enabling up to 96 samples in parallel starting from protein lysate to purified tryptic peptides on Evotips ready for LC-MS analysis.

The protocol is optimized for speed and simplicity, carefully considering efficient pipette tip usage, mixing steps and digestion times, resulting in a complete ‘one-touch’ protocol for digestion and Evotip loading in 90 minutes for 96 samples, when starting from just 1 ug protein lysate.
As peptides are loaded directly onto Evotips, the sample can be stored up to several weeks before LC/MS analysis with no further preparation required. This bypasses the need for sample evaporation and resuspension of the sample, making the use of additional pipette tips and sample vials obsolete, forming a resource and cost efficient automated end-to-end workflow for high productivity.

The workflow was benchmarked using Evosep-MRM with the Agilent 6495 C mass spectrometer and showed the needed reproducibility and sensitivity required for scalable and high-throughput proteomics.