3 research outputs found
Evaluation of Parameters for Confident Phosphorylation Site Localization Using an Orbitrap Fusion Tribrid Mass Spectrometer
Confident identification of sites
of protein phosphorylation by
mass spectrometry (MS) is essential to advance understanding of phosphorylation-mediated
signaling events. However, the development of novel instrumentation
requires that methods for MS data acquisition and its interrogation
be evaluated and optimized for high-throughput phosphoproteomics.
Here we compare and contrast eight MS acquisition methods on the novel
tribrid Orbitrap Fusion MS platform using both a synthetic phosphopeptide
library and a complex phosphopeptide-enriched cell lysate. In addition
to evaluating multiple fragmentation regimes (HCD, EThcD, and neutral-loss-triggered
ETĀ(ca/hc)ĀD) and analyzers for MS/MS (orbitrap (OT) versus ion trap
(IT)), we also compare two commonly used bioinformatics platforms,
Andromeda with PTM-score, and MASCOT with <i>ptmRS</i> for
confident phosphopeptide identification and, crucially, phosphosite
localization. Our findings demonstrate that optimal phosphosite identification
is achieved using HCD fragmentation and high-resolution orbitrap-based
MS/MS analysis, employing MASCOT/<i>ptmRS</i> for data interrogation.
Although EThcD is optimal for confident site localization for a given
PSM, the increased duty cycle compared with HCD compromises the numbers
of phosphosites identified. Finally, our data highlight that a charge-state-dependent
fragmentation regime and a multiple algorithm search strategy are
likely to be of benefit for confident large-scale phosphosite localization
QconCAT Standard for Calibration of Ion Mobility-Mass Spectrometry Systems
Ion mobility-mass spectrometry (IM-MS) is a useful technique
for
determining information about analyte ion conformation in addition
to mass/charge ratio. The physical principles that govern the mobility
of an ion through a gas in the presence of a uniform electric field
are well understood, enabling rotationally averaged collision cross
sections (Ī©) to be directly calculated from measured drift times
under well-defined experimental conditions. However, such āfirst
principleā calculations are not straightforward for Traveling
Wave (T-Wave) mobility separations due to the range of factors that
influence ion motion through the mobility cell. If collision cross
section information is required from T-Wave mobility separations,
then calibration of the instruments using known standards is essential
for each set of experimental conditions. To facilitate such calibration,
we have designed and generated an artificial protein based on the
QconCAT technology, QCAL-IM, which upon proteolysis can be used as
a universal ion mobility calibration standard. This single unique
standard enables empirical calculation of peptide ion collision cross
sections from the drift time on a T-Wave mobility instrument
Analysis of Intrinsic Peptide Detectability via Integrated Label-Free and SRM-Based Absolute Quantitative Proteomics
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 LCāMS
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