Comprehensive Online Reversed-Phase x Chiral Two-Dimensional Liquid Chromatography-Mass Spectrometry with Data-Independent Sequential Window Acquisition of All Theoretical Fragment-Ion Spectra-Acquisition for Untargeted Enantioselective Amino Acid

Karongo, Ryan; Horak, Jeannie und Lämmerhofer, Michael (2022): Comprehensive Online Reversed-Phase x Chiral Two-Dimensional Liquid Chromatography-Mass Spectrometry with Data-Independent Sequential Window Acquisition of All Theoretical Fragment-Ion Spectra-Acquisition for Untargeted Enantioselective Amino Acid. In: Analytical Chemistry, Bd. 94, Nr. 49: S. 17063-17072

Abstract

This work presents an advanced analytical platform for untargeted enantioselective amino acid analysis (eAAA) by comprehensive achiral x chiral 2D-LC hyphenated to ESI-QTOF-MS/MS utilizing data-independent SWATH (sequential window acquisition of all theoretical fragment-ion spectra) technology. The methodology involves N-terminal pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC;AccQ) as retention, selectivity, and MS tag, supporting retention and UV detection in RPLC (1D), chiral recognition, and thus enantioselectivity by the core-shell tandem column composed of a quinine carbamate weak anion exchanger (QNAX) and a zwitterionic chiral ion-exchanger (ZWIX(+)) (2D) as well as the ionization efficiency during positive electrospray ionization due to a high proton affinity of the AQC label. Furthermore, the urea-type MS tag gives rise to the generation of AQC-tag characteristic signature fragments in MS2. The latter allows the chemoselective mass spectrometric filtering of targeted and untargeted N-derivatized amino acids or related labeled species. The chiral core-shell tandem column provides a complete enantioselective amino acid profile of all proteinogenic amino acids within 1 min, with full baseline separation of all enantiomers, but without resolution of isomeric Ile/allo-Ile (aIle)/Leu, which can be resolved by RPLC. The entire LC x LC separation occurs within a total run time of 60 min (1D), with the chiral 2D operated in gradient elution mode and a cycle time of 60 s. A strategy to mine the 2D-LC-SWATH data is presented and demonstrated for the qualitative eAAA of two peptide hydrolysate samples of therapeutic peptides containing common and uncommon as well as primary and secondary amino acids. Absolute configuration assignment of amino acids using template matching for all proteinogenic amino acids was made feasible due to method robustness and the inclusion of an isotopically labeled L-[U-13C15N]-AA standard. The quantification performance of this LC x LC-MS/MS assay was also evaluated. Accuracies were acceptable for the majority of AAs enabling AA composition determination in peptide hydrolysates simultaneously with configuration assignment, as exemplified by oxytocin. This methodology represents a step toward truly untargeted 2D enantioselective amino acid analysis and metabolomics.

• Dokumententyp: Zeitschriftenartikel
• Fakultät: Medizin
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• ISSN: 0003-2700
• Sprache: Englisch