a beginner’s guide to Evosep 

If you are new to our technology or just getting started this event is for you. We will introduce the basics of the Evosep One technology and include hot-from-the-lab data from our users. Nicolai Bache, Head of Applications at Evosep, will host the event, bringing you up to speed with the latest additions to the Evosep One technology.

The Evosep One is a plug-and-plug standardized platform designed for robustness and high throughput. It has been designed specifically to address and eliminate the prevalent challenges associated with throughput and robustness of nano-flow LC-MS workflows while maintaining sufficient sensitivity and resolving power for clinical applications.

The Evosep One technology is centered around the Evotip and integrates sample preparation with LC-MS. The Evotip is essentially a disposable trap column in a pipette tip format with a small plug of C18 stationary phase at the bottom of the tip. The Evotips are used to desalt and clean up the samples prior to LC-MS analysis, however, the traditional subsequent steps of eluting, drying down, re-suspending the samples from tips are completely omitted and instead the tips are loaded directly into Evosep One for analysis.

The system uses four low-pressure syringe pumps in parallel to elute samples from the Evotip with a chromatographic gradient. The resulting gradient with the pre-separated sample is moved into a storage loop, that is then switched in-line with a single high-pressure pump and an analytical separation column for focusing. With this simplified workflow on pre-set gradients, the Evosep One offers a throughput from 30 to 300 samples per day.

SPEAKERS

CETSA MS Profiling for a Comparative Assessment of FDA-Approved Antivirals Repurposed for COVID-19 Therapy Identifies TRIP13 as a Remdesivir Off-Target

Talk by Alexey Chernobrovkin,  Senior Research Scientist, Pelago Bioscience

Cellular thermal shift assay (CETSA) coupled with high-resolution mass-spectrometry (MS) is a powerful method to delineate direct and indirect interactions between small molecules and protein targets in intact cells. Biologically active compounds can induce changes in thermal stability, in their primary binding partners, and in proteins that in turn interact with the direct targets .

This study aims to assess these host cell targets for a panel of FDA-approved antiviral compounds including remdesivir in noninfected cells.

Assessing Parkinson’s Disease-linked LRRK2 kinase activity by multiplexed targeted mass spectrometry 

Talk by Raja Nirujogi, Post Doc, University of Dundee

Mutations that increase the protein kinase activity of LRRK2 are one of the most common causes of familial Parkinson’s disease. LRRK2 phosphorylates a subset of Rab GTPases within their Switch-II motif, impacting interaction with effectors. We describe and validate a new, multiplexed targeted mass spectrometry assay assisted with EvoSep One LC system to quantify endogenous levels of LRRK2-phosphorylated Rab substrates (Rab1, Rab3, Rab8, Rab10, Rab35 and Rab43) as well as total levels of Rabs, LRRK2 and LRRK2-phosphorylated at the Ser910 and Ser935 biomarker sites.

Exploiting this assay, we quantify for the first time the relative levels of each of the pRab proteins in different cells (mouse embryonic fibroblasts, human neutrophils) and mouse tissues (brain, kidney, lung and spleen). We define how these components are impacted by Parkinson’s pathogenic mutations (LRRK2[R1441C] and VPS35[D620N]) and LRRK2 inhibitors. We find that the VPS35[D620N], but not LRRK2[R1441C] mutation, enhances Rab1 phosphorylation in a manner blocked by administration of an LRRK2 inhibitor, providing the first evidence that endogenous Rab1 is a physiological substrate for LRRK2. We exploit this assay to demonstrate that in Parkinson’s patients with VPS35[D620N] mutations, phosphorylation of multiple Rab proteins (Rab1, Rab3, Rab8, Rab10 and Rab43) is elevated.

We highlight the benefits of this assay over immunoblotting approaches currently deployed to assess LRRK2 Rab signalling pathway.