A Comprehensive and Ultrasensitive Isotope Calibration Method for Soil Amino Compounds Using Orbitrap Mass Spectrometry

Bound amino compounds (amino acid and amino sugar polymers) comprise a significant proportion (∼40%) of soil organic nitrogen and therefore represent an essential source of nitrogen for plant and microbial nutrition. The analysis of their content and isotope enrichment still represents a significant challenge due to the low isotope enrichment levels reached under near-native soil conditions and the lack of isotopically labeled standards for some key amino compounds. In this study, we used both a ¹³C-labeled and an unlabeled amino acid mixture to establish isotope calibration curves for 16 amino compounds, using the 6-aminoquinolyl-N-hydroxysccinimidyl carbamate (AQC) derivatization method and ultrahigh-performance liquid chromatography with high-resolution Orbitrap mass spectrometry (UPLC-Orbitrap MS). Molecular ions of AQC-derivatives for all standard amino compounds were identified at the expected m/z values of the respective isotopologues. The isotope calibration curves exhibited excellent linear fits across the whole ¹³C enrichment range and polynomial fits in the low ¹³C enrichment range (R² > 0.990). However, the polynomial fitting terms differed between individual amino acids. Subsequently, we developed equations to relate the calibrated regression terms to the physicochemical properties of the respective amino acids, here mainly the ratio of amino compound-C atoms to total C atoms in AQC-amino compound derivatives. Based on these regressions, we could ultimately predict isotope calibration curves for those amino compounds unavailable as ¹³C labeled standards, for example, muramic acid, hydroxyproline, and diaminopimelic acid. To test the model accuracy, we compared the outcomes of measured calibrations with predicted calibrations for amino acids where we had isotopically enriched standards. The results of linear regression between measured and predicted data were excellent, where R² was >0.97, and mean absolute (percentage) deviations, MAD and MAPD, were 0.334 and 15.8%. Finally, we applied both standard and predicted calibration curves to low ¹³C amended soil samples and unlabeled controls to test the applicability of our model. The limit of detection (LOD) as the minimum detectable atom % ¹³C incorporation of amino compounds ranged from 0.0003 to 0.14 atom % among different amino compounds. This general predictive model can be used to comprehensively quantify isotope enrichments across the entire soil amino compound profile, including amino sugars and proteinogenic and nonproteinogenic amino acids, providing valuable insights for a better understanding of the overall fate of different amino compounds in soils and other complex environmental systems.