Dansylhydrazine Isotope Labeling LC-MS for Comprehensive Carboxylic Acid Submetabolome Profiling

High-performance
chemical isotope labeling (CIL) LC-MS is an important
tool for profiling chemical-group-based submetabolomes using different
labeling chemistries for quantitative metabolomics. Metabolites containing
carboxylic acid groups are a class of molecules playing diverse and
significant roles in many metabolic pathways. We report a relatively
simple and convenient method for analyzing the carboxyl submetabolome
with high coverage. Dansylhydrazine (DnsHz) labeling of the carboxylic
acid group in metabolites is used to improve both separation and ionization
in LC-MS. Using differential 12C- and 13C-DnsHz
reagents for labeling individual samples and a reference sample (e.g.,
a pooled sample), accurate relative quantification of individual metabolites
among comparative samples can be achieved. DnsHz labeling of carboxylic
acids could be carried out at room temperature in water-containing
solution in 2 h. A labeled-carboxyl-standard library consisting of
193 endogenous human metabolites was constructed for metabolite identification,
which covers more than 55 pathways. To demonstrate the performance
of this method, analysis of triplicate human urine samples with duplicate
injections (n = 6) was carried out. A total of 2266
peak pairs or metabolites were commonly detected in all six runs;
among them, 81 peak pairs were positively identified and 517 and 1445
peak pairs were matched in the zero- and one-reaction MyCompoundID
metabolome libraries, respectively, on the basis of accurate mass
search. The possibility of using this method for comprehensive profiling
of the carboxyl submetabolome in real world samples was demonstrated
in metabolome comparison of 30 urine samples of female and male individuals
as well as in absolute quantification of two urinary acids.