Development of a comprehensive and ultrasensitive isotope calibration method for soil amino acid profiles using Orbitrap mass spectrometry

Bound amino acids constitute a significant portion of soil organic nitrogen, which represents an essential source of nitrogen for plant and microbial nutrition. The analysis of the content and isotope enrichment of bound amino acids still represents a significant challenge due to the degradation of certain amino acids following the conventional acid hydrolysis method, due to the low isotope enrichment levels reached under near-native soil conditions and due to the lack of isotopically labelled standards for some key amino acids. In this study, we used both a ¹³C-labeled and unlabeled 16 algal amino acid mixture to establish standard calibration curves for various amino acids, using the 6-Aminoquinolyl-N-hydroxysccinimidyl carbamate (AQC) derivatization method and the ultra-high-performance liquid chromatography with high-resolution Orbitrap mass spectrometry (UPLC-Orbitrap MS) platform. Molecular ions of AQC-derivatives for all amino acids were identified at the expected m/z values of the respective isotopologues, and the isotope calibration curves exhibited excellent linearity for those amino acids where we had isotope standards at hand (polynomial R² > 0.9896). However, the polynomial fitting terms differed between single amino acids. Subsequently, we developed equations to relate the calibrated regression terms to physicochemical properties of the respective amino acids. First, we conducted a linear regression using the Orbitrap-derived ¹³C atom % of unlabeled standards against the carbon atom number of the specific amino acid-AQC derivative molecules, demonstrating great linearity (R² = 0.9728). This linear regression curve allowed us to predict the natural ¹³C abundance of amino acids unavailable as isotopically labelled standards (e.g. hydroxyproline, meso-diaminopimelic acid). Consequently, based on further regressions, we could ultimately develop isotope calibration curves for those amino acids unavailable as ¹³C labelled standards based on the integrated isotope calibration functions. This general predictive model can be applied to comprehensively and highly sensitively (¹³C enrichment ~0.01 at %) quantify isotope enrichments of the whole soil amino acids profile, providing valuable insights for a better understanding of the overall fate of different amino acids in soils.