A Chemically Derivatized in Silico Mass Spectral Library for Fine-Structure Annotation of Phosphoinositides

Phosphoinositides (PIPx) are structurally complex lipids
with essential
roles in cellular signaling and disease. Their biological functions
critically depend on subtle molecular characteristics, including
headgroup identity, acyl-chain composition, and regioisomerism. However,
comprehensive structural annotation of PIPx species by mass spectrometry
remains challenging due to their intrinsically low abundance, extensive
isomerism, and limited availability of reference spectra. Herein,
we report a chemically derivatized in silico mass spectral library
that enables the fine-structure annotation of PIPx. A chemical derivatization
strategy using (4-(diazomethyl)­phenyl)-N,N-dimethyl­methanamine
(DMPDA) markedly improves the liquid chromatographic behavior and
ionization efficiency of PIPx species, resulting in up to a 10-fold
increase in detection sensitivity. More importantly, the resulting
DMPDA-PIPx derivatives exhibit reprogrammed fragmentation behavior
in tandem mass spectrometry, generating diagnostic ions that differentiate
phosphate positional isomers as well as acyl-chain composition and
sn-positional variants. General fragmentation rules were established
and applied to 1,736,028 simulated DMPDA-PIPx structures, yielding
an in-depth in silico mass spectral library that spans millions of
PIPx structures. Integration of chemical derivatization with in silico
library-based spectral matching enables automated annotation of PIPx
isomers that are indistinguishable using conventional MS/MS approaches.
Application of this workflow to aging mouse tissues reveals pronounced
organ-specific heterogeneity in PIPx profiles and distinct tissue-specific
remodeling of PIPx isomers during aging.