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Mass Spectrometry (LC-MS)

LC-MS Based tRNA Modification Analysis — Full-Spectrum Quantification of 55 Nucleoside Modifications

Arraystar LC-MS tRNA Modification Analysis Service analyzes 55 nucleoside modifications and characterizes the global modification profile of tRNAs as tRNA biochemical properties vital to tRNA biogenesis, structure, functioning, and implication in diseases.

Sample-to-data service · 55 nucleoside modifications in one assay · state-of-the-art UHPLC-MS/MS

Overview

Quantitative, full-spectrum tRNA modification profiling from total RNA

Arraystar LC-MS tRNA Modification Analysis Service offers the sample-to-data solution for simultaneous profiling of 55 nucleoside modifications important to tRNA, using total RNA as the starting material. The service includes tRNA isolation from the total RNA, complete hydrolysis, and dephosphorylation to prepare single nucleosides.

The ultra-high-performance LC-MS system delivers a new level of sensitivity, precision, accuracy, dynamic range, and robustness of the quantification results. The full service runs from sample QC, tRNA isolation, nucleoside analyte preparation, LC-MS/MS data acquisition, and analysis to report.

What is LC-MS based tRNA modification analysis?

LC-MS based tRNA modification analysis quantifies modified nucleosides in tRNA by mass spectrometry. tRNAs are isolated from total RNA, completely hydrolyzed and dephosphorylated into single nucleosides, and analyzed by ultra-high-performance LC-MS/MS to simultaneously profile 55 nucleoside modifications and characterize the global modification profile of tRNAs.

Service at a Glance

Standard service options — custom projects available on request

Service NameCatalog NoSizePrice
LC-MS tRNA Modification Analysis Service - EukaryoticAS-LC-t-S1 sample
Total RNA Extraction ServiceAS-RE-S1 sample

Benefits

Key advantages of Arraystar LC-MS tRNA Modification Analysis

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Quantitative Analysis

Quantitative analysis of complex tRNA modifications from total RNA samples.

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Full Sample-to-Data Service

From sample QC, tRNA isolation, nucleoside analyte preparation, LC-MS/MS data acquisition, analysis to report.

High Performance

Highly optimized experimental procedures, an ultra-high-performance LC-MS system, and expertise in operation.

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Best Analytical Coverage

Simultaneous profiling of 55 nucleoside modifications in tRNAs.

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Comprehensive Data

Raw and normalized peak data, Total Ion Current chromatograms of nucleosides, and differential modification among samples.

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Integrated tRNA Solutions

Works together with tRNA sequencing and base-resolution modification services across Arraystar's tRNA research portfolio.

Background

Why tRNA modifications matter

tRNAs are the fundamental component of mRNA decoding and protein translation. tRNAs undergo by far the greatest number of and the most chemically diverse post-transcriptional modifications. These modifications are critical for all core aspects of tRNA function, such as folding, stability, and decoding [1].

Typically, modifications in the main body of tRNA are crucial for tRNA structure folding, stability, rigidity, and flexibility, whereas modifications in the anticodon loop affect decoding by open loop structure, codon-anticodon pairing, wobbling, and preventing translational frameshifts. Additionally, modified nucleosides serve as identity determinants for aminoacyl-tRNA synthetase (AARS) for extra amino acid recognition accuracy [2]. In general, hypomodified tRNAs are targeted for degradation. Studying tRNA modification is perhaps just as important as tRNA expression profiling.

Defects in tRNA modifications and modification enzymes are linked with human diseases such as cancers, diabetes, neurological syndromes, cardiac conditions, and mitochondrial-linked disorders (Fig. 1) [3]. Analysis of tRNA modification profiles is key to establish the link with the disease, tRNA modification enzymes, and tRNA molecular functioning.

Figure 1. tRNA modifications in human diseases.
Figure 1. tRNA modifications in human diseases.

Background References

  1. Kirchner S, Ignatova Z. Emerging roles of tRNA in adaptive translation, signalling dynamics and disease. Nature Reviews Genetics, 2015. PMID: 25534324
  2. El Yacoubi B, et al. Biosynthesis and function of posttranscriptional modifications of transfer RNAs. Annual Review of Genetics, 2012. PMID: 22905870
  3. Torres AG, et al. Role of tRNA modifications in human diseases. Trends in Molecular Medicine, 2014. PMID: 24581449

LC-MS tRNA Modification Analysis Workflow

From total RNA to quantitative modification profiles

1

Sample QC

RNA quality and quantity assessment before the project proceeds.

2

tRNA Isolation

tRNA isolation from total RNA samples.

3

Nucleoside Preparation

Complete hydrolysis and dephosphorylation of tRNA to prepare single nucleosides.

4

LC-MS/MS Data Acquisition

Ultra-high-performance LC-MS/MS acquisition delivering high sensitivity, precision, accuracy, dynamic range, and robustness.

5

Data Analysis & Report

Raw and normalized peak data, Total Ion Current chromatograms of nucleosides, and differential modification of nucleosides among samples.

Figure 2. Workflow of Arraystar LC-MS tRNA Modification Analysis.
Figure 2. Workflow of Arraystar LC-MS tRNA Modification Analysis.

Bioinformatics & Deliverables

Detected modifications and data deliverables

Arraystar LC-MS tRNA Modification Analysis Service analyzes 55 nucleoside modifications and characterizes the global modification profile of tRNAs. Check if your modifications of interest are in the standard detection list below; if yes, click Request Quote and our technical sales team will follow up shortly.

Standard Deliverables

Table 1. Nucleoside modifications profiled by Arraystar LC-MS tRNA Modification Analysis

NumberNucleosideSymbol
13′-O-methyladenosine3′-OMeA
23′-O-methyluridine3′-OMeU
32′-O-methylcytidineCm
45-methyl-2-thiouridinem5s2U
53-methylcytidinem3C
65-methoxyuridinemo5U
75-methylcytidinem5C
8pseudouridineΨ
9N6-isopentenyladenosinei6A
102′-O-methylinosineIm
115,2′-O-dimethylcytidinem5Cm
123-methyluridinem3U
131-methyladenosinem1A
141-methylpseudouridinem1
152-thiocytidines2C
165-hydroxymethylcytidinehm5C
17N2, N2, 7-trimethylguanosinem2,2,7G
185,2′-O-dimethyluridinem5Um
19N4-acetyl-2′-O-methylcytidineac4Cm
20N6-threonylcarbamoyladenosinet6A
21N6-methyladenosinem6A
222-methylthio-N6-threonylcarbamoyladenosinems2t6A
233′-O-methylcytidine3′-OmeC
245-carboxymethyluridinecm5U
252′-O-methyladenosineAm
265-methoxycarbonylmethyl-2-thiouridinemcm5s2U
27N2, N2-dimethylguanosinem22G
285-Methoxycarbonylmethyluridinemcm5U
295′-O-methylthymidine5′-OMeT
302-methylthio-N6-isopentenyladenosinems2i6A
312′-O-methyluridineUm
32Peroxywybutosineo2w
33inosineI
345-taurinomethyl-2-thiouridinetm5s2U
352′-O-methylguanosineGm
365-oxyacetic acid uridinecmo5U
371-methylguanosinem1G
385-carbamoylmethyuridinencm5U
397-methylguanosinem7G
40QueuosineQ
41N2-methylguanosinm2G
425-taurinomethyluridinetm5U
433′-O-methylinosine3′-OMeI
445-formyl-2′-O-methylcytidinef5Cm
452-thiouridines2U
46dihydrouridineD
474-thiouridines4U
485-formylcytidinef5c
495-methyluridinem5U
50wybutosineW
51N4-acetylcytidineac4C
525-methoxycarbonylmethyl-2′-o-methyluridinemcm5Um
53N6, O2′-methyladenosinem6Am
545-methylaminomethyl-2-thiouridinemnm5s2U
555-hydroxyuridineho5U
Figure 3. Total Ion Current chromatogram of nucleosides.
Figure 3. Total Ion Current chromatogram of nucleosides.
Figure 4. Differential modification of nucleosides among samples.
Figure 4. Differential modification of nucleosides among samples.

Research Applications

tRNA modification profiling across biology and disease

Disease Research

Defects in tRNA modifications and modification enzymes are linked with cancers, diabetes, neurological syndromes, cardiac conditions, and mitochondrial-linked disorders (Trends Mol Med, 2014).

Translation & Codon Usage

tRNA modifications are critical for folding, stability, and decoding; anticodon-loop modifications affect codon-anticodon pairing, wobbling, and prevention of translational frameshifts.

Aminoacyl-tRNA Synthetase Biology

Modified nucleosides serve as identity determinants for aminoacyl-tRNA synthetase (AARS) for extra amino acid recognition accuracy.

tRNA Quality Control

Hypomodified tRNAs are targeted for degradation, linking modification status to tRNA stability.

Epitranscriptomics

Global modification profiling complements base-resolution sequencing for quantitative, position-resolved tRNA modification studies.

Sample Requirements

Official Arraystar sample submission requirements for LC-MS tRNA modification analysis

RNA Amount & Quality

  • Total RNA input: 10*–15 µg for tRNA/mRNA modification LC-MS (official recommended minimum for the entire experiment in a single attempt, including sample QC). Supply twice the recommended minimum to avoid project delays.
  • Purification: TRIzol / RNA precipitation or an RNA isolation kit. Because tRNA is < 200 nt, use a kit specified to retain small RNAs (e.g. Qiagen miRNeasy).
  • Concentration: > 20 ng/µL by Nanodrop; OD260/280 ~2.0 (acceptable 1.7–2.1); OD260/230 > 1.8.
  • Integrity: sharp 18S/28S rRNA bands by gel, or RIN > 7.0 by Bioanalyzer (serum/plasma/exosome/FFPE RNA exempt).
  • DNase treatment: optional for gDNA removal; required if the sample is also used for qPCR.

Shipping Instructions

  • Ship RNA in nuclease-free water (> 20 ng/µL), freeze-dried, or in ethanol; store at −80 °C or in liquid nitrogen.
  • Use nuclease-free certified, screw-cap 1.5 mL microtubes; seal caps with Parafilm; place tubes in a plastic bag.
  • Use 10 kg dry ice as refrigerant; include a signed Project Form and the sample list.
  • Ship to: Arraystar Inc., 9430 Key West Avenue #128, Rockville, MD 20850, USA. Contact us before shipping.

FAQ

Common questions about LC-MS based tRNA modification analysis

How many nucleoside modifications does the service detect?
The service analyzes 55 nucleoside modifications and characterizes the global modification profile of tRNAs. Check if your modifications of interest are in the standard detection list (Table 1) before ordering; if yes, click Request Quote and our technical sales team will follow up shortly.
What is the starting material?
Total RNA is used as the starting material. The service includes tRNA isolation from the total RNA, complete hydrolysis and dephosphorylation to prepare single nucleosides, and LC-MS/MS data acquisition on an ultra-high-performance LC-MS system. The full service runs from sample QC through data analysis to the final report.
What is the minimum amount of RNA required?
We recommend 10–15 µg of total RNA per sample — the official Arraystar recommended minimum for the entire experiment in a single attempt, including sample QC. Supplying twice the recommended minimum helps avoid project delays. RNA integrity must be preserved, because degraded RNA cannot be rescued by downstream steps.
What data will I receive?
Projects deliver raw and normalized peak data, Total Ion Current chromatograms of nucleosides, and differential modification of nucleosides among samples, together with the report of quantified modification levels for the 55 nucleoside modifications. All data are delivered with documentation for straightforward interpretation.
Can I add total RNA extraction to my project?
Yes. A Total RNA Extraction Service (catalog AS-RE-S, 1 sample) is available as an add-on, so you can ship raw samples and Arraystar will perform RNA extraction before the LC-MS tRNA modification analysis. This option is useful when RNA isolation capacity or small RNA retention is a concern in your lab.
How does LC-MS complement sequencing-based modification analysis?
LC-MS simultaneously quantifies 55 nucleoside modifications from total RNA and characterizes the global modification profile, while sequencing methods such as m7G TRAC-Seq, m3C HAC-Seq, tRNA Modification Seq, and tRNA Charging Seq map modifications at base resolution. Combining both routes provides quantitative, position-resolved tRNA modification studies.

Selected Publications

Featured Client Publications in tRNA Modification Research

  1. Arnskötter F, et al. Loss of Elp1 in cerebellar granule cell progenitors models ataxia phenotype of Familial Dysautonomia. Neurobiology of Disease, 2024. PMID: 38996985
  2. Li G, et al. Unconventional secretion of Magnaporthe oryzae effectors in rice cells is regulated by tRNA modification and codon usage control. Nature Microbiology, 2023. PMID: 37563288
  3. Chen D, et al. Elp3-mediated codon-dependent translation promotes mTORC2 activation and regulates macrophage polarization. The EMBO Journal, 2022. PMID: 35920020
  4. Usha A, et al. Phytophthora capsici infection causes dynamic alterations in tRNA modifications and their associated gene candidates in black pepper. Computational and Structural Biotechnology Journal, 2022. PMID: 36420148
  5. Rosu A, et al. Loss of tRNA-modifying enzyme Elp3 activates a p53-dependent antitumor checkpoint in hematopoiesis. Journal of Experimental Medicine, 2021. PMID: 33507234
  6. Rojas-Benítez D, et al. Elongator Subunit 3 (Elp3) Is Required for Zebrafish Trunk Development. International Journal of Molecular Sciences, 2020. PMID: 32023806
  7. Ahmad AA, et al. Papillary Renal Cell Carcinomas Rewire Glutathione Metabolism and Are Deficient in Both Anabolic Glucose Synthesis and Oxidative Phosphorylation. Cancers, 2019. PMID: 31484429
  8. Goffena J, et al. Elongator and codon bias regulate protein levels in mammalian peripheral neurons. Nature Communications, 2018. PMID: 29497044

Ready to Quantify the Full Spectrum of tRNA Modifications?

Arraystar LC-MS tRNA Modification Analysis profiles 55 nucleoside modifications from total RNA — get a quote and a project timeline tailored to your study.