1 research outputs found
Large-Scale Differentiation and Site Specific Discrimination of Hydroxyproline Isomers by Electron Transfer/Higher-Energy Collision Dissociation (EThcD) Mass Spectrometry
3- and 4-Hydroxyprolines
(HyP) are regioisomers that play different
roles in various species and organs. Despite their distinct functions
inside cells, they are generally considered indistinguishable using
mass spectrometry due to their identical masses. Here, we demonstrate,
for the first time, that characteristic w ions can be produced by
electron-transfer/higher energy collision dissociation (EThcD) dual
fragmentation technique to confidently discriminate 3-HyP/4-HyP isomers.
An integrated and high throughput strategy was developed which combined
online LC separation with EThcD for large-scale differentiation of
3-HyP/4-HyP in complex samples. An automated algorithm was developed
for charge state dependent characterization of 3-HyP/4-HyP isomers.
Using this combined discrimination approach, we identified 108 3-HyP
sites and 530 4-HyP sites from decellularized pancreas, allowing more
than 5-fold increase of both 3-HyP and 4-HyP identifications compared
to previous reports. This approach outperformed ETD and HCD in the
analysis of HyP-containing peptides with unique capacity to generate
w ions for HyP discrimination, improved fragmentation of precursor
ions, as well as unambiguous localization of modifications. A high
content of 3-HyP was observed in the C-terminal (GPP)<sub>n</sub> domain
of human CO1A1, which was previously only identified in vertebrate
fibrillar collagens from tendon. Unexpectedly, some unusual HyP sites
at Xaa position in Gly-HyP-Ala, Gly-HyP-Val, Gly-HyP-Gln, Gly-HyP-Ser,
and Gly-HyP-Arg were also confirmed to be 3-hydroxylated, whose functions
and enzymes are yet to be discovered. Overall, this novel discrimination
strategy can be readily implemented into de novo sequencing or other
proteomic search engines