An UPLC-ESI-MS/MS Assay Using 6-Aminoquinolyl-N-Hydroxysuccinimidyl Carbamate Derivatization for Targeted Amino Acid Analysis: Application to Screening of Arabidopsis thaliana Mutants

Abstract

In spite of the large arsenal of methodologies developed for amino acid assessment in complex matrices, their implementation in metabolomics studies involving wide-ranging mutant screening is hampered by their lack of high-throughput, sensitivity, reproducibility, and/or wide dynamic range. In response to the challenge of developing amino acid analysis methods that satisfy the criteria required for metabolomic studies, improved reverse-phase high-performance liquid chromatography-mass spectrometry (RPHPLC-MS) methods have been recently reported for large-scale screening of metabolic phenotypes. However, these methods focus on the direct analysis of underivatized amino acids and, therefore, problems associated with insufficient retention and resolution are observed due to the hydrophilic nature of amino acids. It is well known that derivatization methods render amino acids more amenable for reverse phase chromatographic analysis by introducing highly-hydrophobic tags in their carboxylic acid or amino functional group. Therefore, an analytical platform that combines the 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) pre-column derivatization method with ultra performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) is presented in this article. For numerous reasons typical amino acid derivatization methods would be inadequate for large scale metabolic projects. However, AQC derivatization is a simple, rapid and reproducible way of obtaining stable amino acid adducts amenable for UPLC-ESI-MS/MS and the applicability of the method for high-throughput metabolomic analysis in Arabidopsis thaliana is demonstrated in this study. Overall, the major advantages offered by this amino acid analysis method include high-throughput, enhanced sensitivity and selectivity; characteristics that showcase its utility for the rapid screening of the preselected plant metabolites without compromising the quality of the metabolic data. The presented method enabled thirty-eight metabolites (proteinogenic amino acids and related compounds) to be analyzed within 10 min with detection limits down to 1.02 × 10−11 M (i.e., atomole level on column), which represents an improved sensitivity of 1 to 5 orders of magnitude compared to existing methods. Our UPLC-ESI-MS/MS method is one of the seven analytical platforms used by the Arabidopsis Metabolomics Consortium. The amino acid dataset obtained by analysis of Arabidopsis T-DNA mutant stocks with our platform is captured and open to the public in the web portal PlantMetabolomics.org. The analytical platform herein described could find important applications in other studies where the rapid, high-throughput and sensitive assessment of low abundance amino acids in complex biosamples is necessary.

Application of 6‐aminoquinolyl‐N‐hydroxysuccinimidyl carbamate derivatization for enhanced peptide mapping analysis of non‐biological complex drug glatiramer acetate using liquid chromatography/electrospray ionization collision‐induced dissociation high‐resolution mass spectrometry

“…However, when the same amounts of peptide digests were used, the AQC derivatization produced better results. As expected, signal enhancement upon derivatization was observed in the ESI positive‐ion mode because of quinoline ring basicity introduced by the AQC label . Peptide modification of the digest sample produced an increase in the number of identified peptide peaks.…”

Glutamine rapidly induces the expression of key transcription factor genes involved in nitrogen and stress responses in rice roots

“…Roots and shoots were harvested separately for extraction of free amino acids. Amino acids were extracted with 50 % ( v / v ) methanol: H 2 O solution spiked with internal standard 50 μM Norvaline as described [ 41 ]. Free amino acids were derivatized with the AccQ•Tag Ultra derivatization kit (Waters, Milford, MA, USA); for derivatization, 10 μL of rice extract was mixed with 70 μL of AccQ•Tag Ultra borate buffer and 20 μL of AccQ•Tag Ultra reagent, followed by incubation for 15 min at 55 °C.…”

Simple Detection Methods for Antinutritive Factor β-ODAP Present in Lathyrus sativus L. by High Pressure Liquid Chromatography and Thin Layer Chromatography

“…FMOC method requires removal of excess reaction solution with hexane-ethyl acetate which may interfere with the separation of the amino acid derivatives. OPA, PITC derivatization may lead to rapid RP-LC column deterioration [ 17 ]. Also, in some cases excess reagent needs to be evaporated making it time consuming process [ 18 ].…”