In-Capillary Chemoselective Tagging-Facilitated Direct nESI-MS Profiling of Carboxyl- and Carbonyl-Containing Metabolites in Urine

Carboxyl- and carbonyl-containing metabolites (CCMs) are essential for energy metabolism and signaling in living cells and can serve as diagnostic biomarkers for various diseases. However, their low ESI ionization efficiencies and structural diversities could bring difficulties for their direct detection by mass spectrometry. In this work, a chemoselective tagging and acid-trigged release (CTAR) strategy-based nanoelectrospray ionization-mass spectrometry (nESI-MS) platform was developed by immobilizing a cis-diol-containing amine probe (i.e., 3-((2-aminoethyl)amino)propane-1,2-diol, denoted as AEAP-diol) on a coated ESI capillary with boronate esters as acid cleavage site, which can directly and selectively “fish out” targeted CCMs from biological matrices. Following the removal of unwanted species, the AEAP-diol tagged CCMs derivatives can be online released and directly detected by nESI-MS within 1 min. MS/MS fragmentation profiles of these CCMs derivatives exhibited a neutral loss of 91 Da and diagnostic fragments at m/z 118 for carboxyl- and carbonyl-containing metabolites, respectively, thereby enabling unambiguous structural elucidation of unknown CCMs. With high specificity, superior salt tolerance, and outstanding robustness, our CTAR-nESI-MS platform enabled direct and sensitive analysis of CCMs from a single drop of biofluid (2.5 μL of urine), achieving a low detection limit of 0.1 ppb for butyric acid. Using this method, 26 carboxyl- and carbonyl-containing metabolites were identified and quantified in urine samples from esophageal cancer patients and healthy volunteers, revealing significant differences in several reported CCM biomarkers. These findings collectively underscore the potential of CTAR-nESI-MS as a sensitive, cost-effective, and versatile tool for comprehensive metabolite profiling and high-throughput screening applications.