Optimized Workflow for Multiplexed Phosphorylation Analysis of TMT-Labeled Peptides Using High-Field Asymmetric Waveform Ion Mobility Spectrometry

Phosphorylation is a post-translational modification
with a vital
role in cellular signaling. Isobaric labeling-based strategies, such
as tandem mass tags (TMT), can measure the relative phosphorylation
states of peptides in a multiplexed format. However, the low stoichiometry
of protein phosphorylation constrains the depth of phosphopeptide
analysis by mass spectrometry. As such, robust and sensitive workflows
are required. Here we evaluate and optimize high-Field Asymmetric
waveform Ion Mobility Spectrometry (FAIMS) coupled to Orbitrap Tribrid
mass spectrometers for the analysis of TMT-labeled phosphopeptides.
We determined that using FAIMS-MS3 with three compensation voltages
(CV) in a single method (e.g., CV = −40/–60/–80
V) maximizes phosphopeptide coverage while minimizing inter-CV overlap.
Furthermore, consecutive analyses using MSA-CID (multistage activation
collision-induced dissociation) and HCD (higher-energy collisional
dissociation) fragmentation at the MS2 stage increases the depth of
phosphorylation analysis. The methodology and results outlined herein
provide a template for tailoring optimized FAIMS-based methods.