Quantitative
mass spectrometry-based proteomics of complex biological
samples remains challenging in part due to the variability and charge
competition arising during electrospray ionization (ESI) of peptides
and the subsequent transfer and detection of ions. These issues preclude
direct quantification from signal intensity alone in the absence of
a standard. A deeper understanding of the governing principles of
peptide ionization and exploitation of the inherent ionization and
detection parameters of individual peptides is thus of great value.
Here, using the yeast proteome as a model system, we establish the
concept of peptide F-factor as a measure of detectability, closely
related to ionization efficiency. F-factor is calculated by normalizing
peptide precursor ion intensity by absolute abundance of the parent
protein. We investigated F-factor characteristics in different shotgun
proteomics experiments, including across multiple ESI-based LC–MS
platforms. We show that F-factors mirror previously observed physicochemical
predictors as peptide detectability but demonstrate a nonlinear relationship
between hydrophobicity and peptide detectability. Similarly, we use
F-factors to show how peptide ion coelution adversely affects detectability
and ionization. We suggest that F-factors have great utility for understanding
peptide detectability and gas-phase ion chemistry in complex peptide
mixtures, selection of surrogate peptides in targeted MS studies,
and for calibration of peptide ion signal in label-free workflows.
Data are available via ProteomeXchange with identifier PXD003472