Optimization and Modeling of Quadrupole Orbitrap Parameters for Sensitive Analysis toward Single-Cell Proteomics

Single-cell
proteomics represents a field of extremely sensitive
proteomic analysis, owing to the minute amount of yet complex proteins
in a single cell. Without amplification potential as of nucleic acids,
single-cell mass spectrometry (MS) analysis demands special instrumentation
running with optimized parameters to maximize the sensitivity and
throughput for comprehensive proteomic discovery. To facilitate such
analysis, we here investigated two factors critical to peptide sequencing
and protein detection in shotgun proteomics, i.e. precursor ion isolation
window (IW) and maximum precursor ion injection time (ITmax), on an
ultrahigh-field quadrupole Orbitrap (Q-Exactive HF). Counterintuitive
to the frequently used proteomic parameters for bulk samples (>100
ng), our experimental data and subsequent modeling suggested a universally
optimal IW of 4.0 Th for sample quantity ranging from 100 ng to 1
ng, and a sample-quantity dependent ITmax of more than 250 ms for
1-ng samples. Compared with the benchmark condition of IW = 2.0 Th
and ITmax = 50 ms, our optimization generated up to 300% increase
to the detected protein groups for 1-ng samples. The additionally
identified proteins allowed deeper penetration of proteome for better
revealing crucial cellular functions such as signaling and cell adhesion.
We hope this effort can prompt single-cell and trace proteomic analysis
and enable a rational selection of MS parameters.