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. 2024 Dec 11;52(22):e107.
doi: 10.1093/nar/gkae1134.

Simultaneous determination of cytosolic aminoacyl-tRNA synthetase activities by LC-MS/MS

Marisa I Mendes  1   2 , Nicole I Wolf  3 , Joëlle Rudinger-Thirion  4 , Dominic Lenz  5 , Magali Frugier  4 , Patrick Verloo  6 , Hanna Mandel  7 , Joshua Manor  8 , Rachel Kassel  9 , Willemijn E Corpeleijn  2   10   11 , Sanne van der Rijt  1   2 , Elsbeth M Schroor  1   2 , Silvy J M van Dooren  1   2 , Christian Staufner  5 , Gajja S Salomons  1   2   10 , Desirée E C Smith  1   2
Affiliations

Simultaneous determination of cytosolic aminoacyl-tRNA synthetase activities by LC-MS/MS

Marisa I Mendes et al. Nucleic Acids Res. .

Abstract

In recent years, pathogenic variants in ARS genes, encoding aminoacyl-tRNA synthetases (aaRSs), have been associated with human disease. Patients harbouring pathogenic variants in ARS genes have clinical signs partly unique to certain aaRSs defects, partly overlapping between the different aaRSs defects. Diagnosis relies mostly on genetics and remains challenging, often requiring functional validation of new ARS variants. In this study, we present the development and validation of a method to simultaneously determine aminoacylation activities of all cytosolic aaRSs (encoded by ARS1 genes) in one single cell lysate, improving diagnosis in suspected ARS1 disorders and facilitating functional characterization of ARS1 variants of unknown significance. As proof of concept, we show enzyme activities of five individuals with variants in different ARS1 genes, demonstrating the usability and convenience of the presented method.

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Figures

Graphical Abstract
Graphical Abstract
Figure 1.
Figure 1.
Schematic representation depicting each step of the simultaneous aminoacylation assay. Step 1: Aminoacylation reaction performed in polymerase chain reaction strips. Step 2: After incubation, the reaction mix is transferred to a 96-well filter plate filled with TCA in order to stop the reaction (denaturated aaRS) and rinsed with TCA to remove unbound amino acids. Step 3: Incubation with ammonia to release amino acids from the aminoacyl-tRNA molecules and addition of amino acid internal standard. Step 4: Elution of amino acids with nonafluoropentanoic acid. Step 5: A typical LC–MS/MS chromatogram showing separation of amino acids using a Symmetry Shield C18 analytical column (2.0 × 100 mm; 3.5 μm; Waters).
Figure 2.
Figure 2.
Aminoacylation activities (PheRS, LeuRS, TyrRS, MetRS and HisRS) of five individuals with variants in LARS1, FARSA, YARS1, MARS1 and FARSB. Plots represent average with standard deviation of three different measurements. Black bar corresponds to the aaRS for which the variants were identified.
See this image and copyright information in PMC

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