Improving Proteome Coverage in Short LC Gradients

Thermo Scientific™ Orbitrap Exploris™ 480 mass spectrometer for high-throughput analyses using the Evosep One
By Team Evosep

In a collaboration with Thermo Fisher Scientific, the Olsen Group and Novo Nordisk Foundation Center for Protein Research have described the new Thermo Scientific™ Orbitrap Exploris™ 480 mass spectrometer for high-throughput analyses with short gradients using the Evosep One.

The new instrument combines a compact and robust quadrupole-orbitrap mass spectrometer equipped with a front-end High Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) Interface. With an automated sample preparation workflow they evaluated the performance in data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes in combination with FAIMS. They demonstrate that different compensation voltages (CVs) for FAIMS are optimal for DDA and DIA, respectively.

Combining DIA with FAIMS using single CVs, the instrument surpasses 2500 peptides identified per minute. This enables quantification of >5000 proteins with short online gradients allowing acquisition of 60 samples per day.

They identified one of the key applications for FAIMS by analyzing 5 ng of a HeLa digest from which >1000 proteins were reproducibly identified using DIA-FAIMS with 5 minute Evosep One separation (200 samples per day). FAIMS is capable of effectively removing the singly-charged background ions from ambient air and the electrospray buffer, which otherwise take up a lot of capacity when minute amounts of peptide mixtures are analyzed.

They also recorded an organ-wide map of proteome expression across 12 rat tissues quantified by tandem mass tags and label-free quantification using DIA with FAIMS to a depth of >10,000 proteins. They also present a streamlined and automated workflow for parallel preparation for up to 96 phosphopeptide samples, which they analyze on the Evosep One allowing for 200 samples per day.

The versatility of applications presented in this pre-print demonstrates the powerful combination of the Thermo Scientific ™ Orbitrap Exploris™ 480 mass spectrometer and the Evosep One.

A Compact Quadrupole-Orbitrap Mass Spectrometer with FAIMS Interface Improves Proteome Coverage in Short LC Gradients was published by Prof. Jesper Olsen and colleauges in 

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