Accurate Label-Free Protein Quantitation with High- and Low-Resolution Mass Spectrometers

Label-free
quantitation of proteins analyzed by tandem mass spectrometry
uses either integrated peak intensity from the parent-ion mass analysis
(MS1) or features from fragment-ion analysis (MS2), such as spectral
counts or summed fragment-ion intensity. We directly compared MS1
and MS2 quantitation by analyzing human protein standards diluted
into Escherichia coli extracts on an Orbitrap mass
spectrometer. We found that summed MS2 intensities were nearly as
accurate as integrated MS1 intensities, and both outperformed MS2
spectral counting in accuracy and linearity. We compared these results
to those obtained from two low-resolution ion-trap mass spectrometers;
summed MS2 intensities from LTQ and LTQ Velos instruments were similar
in accuracy to those from the Orbitrap. Data from all three instruments
are available via ProteomeXchange with identifier PXD000602. Abundance
measurements using MS1 or MS2 intensities had limitations, however.
While measured protein concentration was on average well-correlated
with the known concentration, there was considerable protein-to-protein
variation. Moreover, not all human proteins diluted to a mole fraction
of 10–3 or lower were detected, with a strong falloff
below 10–4 mole fraction. These results show that
MS1 and MS2 intensities are simple measures of protein abundance that
are on average accurate but should be limited to quantitation of proteins
of intermediate to higher fractional abundance.