Discovery of Cysteine Carboxyalkylations by Real-Time Isotopic Signature Targeted Profiling

Data-dependent acquisition (DDA)
is widely applied in
shotgun proteomics.
However, restricted by the scanning speed of mass spectrometry (MS)
instruments, it remains challenging for DDA to directly detect peptides
with low abundance. Herein, we developed a real-time targeted MS data
acquisition method, “isoSTAR”, which identifies target
peptides by their unique isotopic signatures during the stage of full-MS
scanning and subjects them to targeted MS/MS scans immediately. The
method showed dramatic improvement in sensitivity in identifying target
peptides with low abundance compared to traditional MS acquisition
methods. Using this method, we discovered a series of carboxyalkylations
on cysteines during fatty acid metabolism and verified their modification
structures using synthetic peptide standards. We envision that isoSTAR
will become a powerful and versatile tool to enhance shotgun proteomics
applications in profiling protein-centric modifications.