Broad-range RNA modification analysis of complex biological samples using rapid C18-UPLC-MS

Abstract

Post-transcriptional RNA modifications play an important role in cellular metabolism with homoeostatic disturbances manifesting as a wide repertoire of phenotypes, reduced stress tolerance and translational perturbation, developmental defects, and diseases, such as type II diabetes, leukaemia, and carcinomas. Hence, there has been an intense effort to develop various methods for investigating RNA modifications and their roles in various organisms, including sequencing-based approaches and, more frequently, liquid chromatography–mass spectrometry (LC-MS)-based methods. Although LC-MS offers numerous advantages, such as being highly sensitive and quantitative over a broad detection range, some stationary phase chemistries struggle to resolve positional isomers. Furthermore, the demand for detailed analyses of complex biological samples often necessitates long separation times, hampering sample-to-sample turnover and making multisample analyses time consuming. To overcome this limitation, we have developed an ultra-performance LC-MS (UPLC-MS) method that uses an octadecyl carbon chain (C18)-bonded silica matrix for the efficient separation of 50 modified ribonucleosides, including positional isomers, in a single 9-min sample-to-sample run. To validate the performance and versatility of our method, we analysed tRNA modification patterns of representative microorganisms from each domain of life, namely Archaea ( Methanosarcina acetivorans ), Bacteria ( Pseudomonas syringae ), and Eukarya ( Saccharomyces cerevisiae ). Additionally, our method is flexible and readily applicable for detection and relative quantification using stable isotope labelling and targeted approaches like multiple reaction monitoring (MRM). In conclusion, this method represents a fast and robust tool for broad-range exploration and quantification of ribonucleosides, facilitating future homoeostasis studies of RNA modification in complex biological samples.

Global analysis of aging-related protein structural changes uncovers enzyme polymerization-based control of longevity

“…For data normalization, cleaved monoribonucleosides were spiked with 15 N-labeled ribonucleosides, which served as an internal standard (25 ng 15 N-labelled ribonucleosides per 100 ng of sample). Samples were analyzed as described in 90 using a Waters Acquity® UPLC system attached to a Waters Synapt G2 HDMS mass spectrometer via an ESI ion source.…”

Bacteriophage Infection of the Marine Bacterium Shewanella glacialimarina Induces Dynamic Changes in tRNA Modifications

“…As the codon usage analysis revealed an interesting dichotomy for specific viral genes, we next decided to investigate how post-transcriptional tRNA modifications, which are known modulators of translation during stress, are altered during the infection cycle. To this end, we performed a quantitative C18-UPLC-MS [ 23 ] analysis and detected 18 modifications that were consistently present in all samples. Furthermore, none of the modifications disappeared, nor did any new modification appear in the infected or mock-infected cells throughout the infection cycle ( Figure 3 A).…”

“…Dephosphorylated monoribonucleosides were prepared as previously described [ 22 ]. Ribonucleoside standards and samples (500 ng) were separated and detected identically to the previously described C18-UPLC method [ 23 ]. MS data were analyzed in MZmine2 (version 2.52) and exported in csv format [ 24 ].…”

Cold-Active Shewanella glacialimarina TZS-4T nov. Features a Temperature-Dependent Fatty Acid Profile and Putative Sialic Acid Metabolism

“…RNA isolation was performed using acidic phenol:bromochloropropane (BCP; Chomczynski and Mackey, 1995;Gregorova et al, 2020). The cells were grown from the starter culture in 50 ml of rMB until it reached OD 600 of 0.8.…”