Optimized Fast and Sensitive Acquisition Methods for Shotgun Proteomics on a Quadrupole Orbitrap Mass Spectrometer

Advances in proteomics are continually driven by the
introduction
of new mass spectrometric instrumentation with improved performances.
The recently introduced quadrupole Orbitrap (Q Exactive) tandem mass
spectrometer allows fast acquisition of high-resolution higher-energy
collisional dissociation (HCD) tandem mass spectra due to the parallel
mode of operation, where the generation, filling, and storage of fragment
ions can be performed while simultaneously measuring another ion packet
in the Orbitrap mass analyzer. In this study, data-dependent acquisition
methods for “fast” or “sensitive” scanning
were optimized and assessed by comparing stable isotope labeled yeast
proteome coverage. We discovered that speed was the most important
parameter for sample loads above 125 ng, where a 95 ms HCD scanning
method allowed for identification and quantification of more than
2000 yeast proteins from 1 h of analysis time. At sample loads below
125 ng, a 156 ms HCD acquisition method improved the sensitivity,
mass accuracy, and quality of data and enabled us to identify 30%
more proteins and peptides than the faster scanning method. A similar
effect was observed when the LC gradient was extended to 2 or 3 h
for the analysis of complex mammalian whole cell lysates. Using a
3 h LC gradient, the sensitive method enabled identification
of more than 4000 proteins from 1 μg of tryptic HeLa digest,
which was almost 200 more identifications compared to the faster scanning
method. Our results demonstrate that peptide identification on a quadrupole
Orbitrap is dependent on sample amounts, acquisition speed, and data
quality, which emphasizes the need for acquisition methods tailored
for different sample loads and analytical preferences.