Post-Deconvolution MS/MS Spectra Extraction with Data-Independent Acquisition for Comprehensive Profiling of Urinary Glucuronide-Conjugated Metabolome

Conjugation reactions
are of critical significance in human metabolism.
Identification of these conjugated metabolites is still challenging.
Here, we propose a strategy, post-deconvolution MS/MS spectra extraction
with data-independent acquisition (PDMS2E-DIA), to comprehensively
profile the glucuronide-conjugated metabolome. PDMS2E-DIA enables
the identification of conjugated and unconjugated metabolite pairs
through neutral loss filtering combined with a significant change
in abundance after the deconjugation reaction. Purified DIA MS/MS
spectra were constructed by extracting MS/MS fragments shared between
spectra derived from conjugated and unconjugated metabolites. The
feasibility of this approach was first demonstrated by the identification
of two glucuronide-conjugated metabolite standards spiked in urine
samples. For human urine samples, 479 features were structurally annotated
as potential glucuronide-conjugated metabolites, resulting in the
identification of 211 metabolites. Fragment peaks derived from interferents
were found to be removed by PDMS2E-DIA, which increased about 6 times
the number of identified urine metabolites compared with those calculated
from raw DIA deconvoluted MS/MS spectra. This approach was found to
have great potential for identifying glucuronide-conjugated metabolites,
as well as other kinds of chemical conjugations.