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

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.