A successful mass spectrometry-based phosphoproteomics
analysis
relies on effective sample preparation strategies. Suspension trapping
(S-Trap) is a novel, rapid, and universal method of sample preparation
that is increasingly applied in bottom-up proteomics studies. However,
the performance of the S-Trap protocol for phosphoproteomics studies
is unclear. In the existing S-Trap protocol, the addition of phosphoric
acid (PA) and methanol buffer creates a fine protein suspension to
capture proteins on a filter and is a critical step for subsequent
protein digestion. Herein, we demonstrate that this addition of PA
is detrimental to downstream phosphopeptide enrichment, rendering
the standard S-Trap protocol suboptimal for phosphoproteomics. In
this study, the performance of the S-Trap digestion for proteomics
and phosphoproteomics is systematically evaluated in large-scale and
small-scale samples. The results of this comparative analysis show
that an optimized S-Trap approach, where trifluoroacetic acid is substituted
for PA, is a simple and effective method to prepare samples for phosphoproteomics.
Our optimized S-Trap protocol is applied to extracellular vesicles
to demonstrate superior sample preparation workflow for low-abundance,
membrane-rich samples.