Thermo Fisher Research and Development - 2024_Stellar_Instrument_Platform

Hybrid Quadrupole Mass Filter – Radial Ejection Linear Ion Trap and Intelligent Data Acquisition Enable Highly Multiplex Targeted Proteomics

Targeted mass spectrometry (MS) methods are powerful tools for selective and sensitive analysis of peptides identified by global discovery experiments. Selected reaction monitoring (SRM) is currently the most widely accepted MS method in the clinic, due to its reliability and analytical performance. However, due to limited throughput and the difficulty in setting up and analyzing large scale assays, SRM and parallel reaction monitoring (PRM) are typically used only for very refined assays of on the order of 100 targets or less. Here we introduce a new MS platform with a quadrupole mass filter, collision cell, linear ion trap architecture that has increased acquisition rates compared to the analogous hardware found in the OrbitrapTM TribridTM series instruments. The platform can target more analytes than existing SRM and PRM instruments – in the range of 5000 to 8000 peptides per hour. This capability for high multiplexing is enabled by acquisition rates of 70-100 Hz for peptide applications, and the incorporation of real-time chromatogram alignment that adjusts for retention time drift and enables narrow time scheduled acquisition windows. Finally, we describe a Skyline external software tool that implements the building of targeted methods based on data independent acquisition chromatogram libraries or unscheduled analysis of heavy labeled standards. We show that the platform delivers ~10x lower LOQs than traditional SRM analysis for a highly multiplex assay and also demonstrate how analytical figures of merit change while varying method duration with a constant number of analytes, or by keeping a constant time duration while varying the number of analytes.

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Characterization of Stellar MS
Data License: CC BY 4.0 | ProteomeXchange: PXD052734 | doi: https://doi.org/10.6069/588k-5w72
  • Organism: Homo sapiens, Gallus gallus, Escherichia coli
  • Instrument: Stellar
  • SpikeIn: Yes
  • Keywords: Stellar, PRM, Targeted, matrix-matched, E. coli, HeLa, chicken, plasma, label-free, PQ500, absolute, quantitation
  • Lab head: Philip Remes Submitter: Philip Remes
Abstract
A set of plasma peptides were targeted with selected reaction monitoring (SRM) on an Altis Plus and also with parallel reaction monitoring (PRM) on a Stellar. The maximum concurrency of the assay was 624 transitions for the Altis, but only 128 precursors for the Stellar. The quantitative performance of the assays was characterized with a matrix-matched dilution curve of human into chicken plasma, where the Stellar had LOQs and LODs that were on average 12x and 2x better than the Altis. Targeted assays were created for absolute quantitation of the PQ500 light and heavy peptides for a 60 samples-per-day (SPD) and 100 SPD throughputs. The quantitative performance of these assays was characterized using dilution curves of the heavy standards spiked into 300 ng of human plasma with the Stellar mass spectrometer. Both assays gave good results, with the 60 SPD assay having LOQs about 1.6x better on average than the 100 SPD assay. Targeted assays were created for label-free quantitation of E. coli spiked into 200 ng of HeLa. Two 60 samples-per-day methods were created, one with 397 peptides, and another with 1302 peptides. These assays were based off of a gas-phase-fractionation (GPF) data independent acquisition (DIA) experiment using the Thermo Fisher Scientific Stellar mass spectrometer. The quantitative performance of these assays was characterized over the range of E. coli amounts from 100 ng to 0.78 ng. Both size assays gave good results, with the n=397 assay having LOQs about 1.2x better on average than the n=1302 assay.
Sample Description
PQ500 sample from Biognosys https://biognosys.com/content/uploads/2022/03/Biognosys_PQ500_Manual.pdf E. coli digest from Waters https://www.waters.com/nextgen/us/en/shop/standards--reagents/186003196-massprep-e-coli-digest-standard.html HeLa digest from Pierce https://www.thermofisher.com/order/catalog/product/88328
Created on 5/30/24, 7:07 PM