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J Proteome Res. 2018 Oct 5;17(10):3586-3592. doi: 10.1021/acs.jproteome.8b00269. Epub 2018 Sep 13.

Probing the Sensitivity of the Orbitrap Lumos Mass Spectrometer Using a Standard Reference Protein in a Complex Background.

Author information

1
Stowers Institute for Medical Research , Kansas City , Missouri 64110 , United States.
2
Department of Pathology and Laboratory Medicine , University of Kansas Medical Center , Kansas City , Kansas 66160 , United States.

Abstract

The use of mass spectrometry as a tool to detect proteins of biological interest has become a cornerstone of proteomics. The popularity of mass spectrometry-based methods has increased along with instrument improvements in detection and speed. The Orbitrap Fusion Lumos mass spectrometer has recently been shown to have better fragmentation and detection than its predecessors. Here, we determined the sensitivity of the Lumos using the NIST monoclonal antibody reference material at various concentrations to detect its peptides in a background of S. cerevisiae whole cell lysate, which was kept at a constant concentration. The data collected by data-dependent acquisition showed that the spiked protein could be detected at 10 pg by an average of 4 peptides in 250 ng of whole cell lysate when the instrument was operated by detecting the peptide masses in the Orbitrap and the fragment masses in the ion trap (FTIT mode). In contrast, when the peptides and fragments were both detected in the Orbitrap on either the Lumos or Q-Exactive Plus (FTFT mode), the lowest concentration of NIST monoclonal antibody detected was 50 pg. The Lumos can detect a single protein at a level 2500 times lower than the whole cell background and the combination of detecting ions in the Orbitrap and ion trap can improve the identification of low abundance proteins. Furthermore, the total number of proteins identified from decreasing starting amounts of whole cell extracts was determined. The Lumos, when operated in FTIT mode, was able to identify twice as many proteins compared to the Q-Exactive+ at 5 ng of whole cell lysate. Similar numbers of proteins were identified on both platforms at higher concentrations of starting material. Therefore, the Lumos mass spectrometer is especially useful for detecting proteins of low abundance in complex backgrounds or samples that have limited starting material.

KEYWORDS:

Orbitrap Lumos; Proteomics; data-dependent acquisition; limit of detection; mass spectrometry; sensitivity

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