Production and Application of Stable Isotope-Labeled Internal Standards for RNA Modification Analysis

Post-transcriptional RNA modifications have been found to be present in a wide variety of organisms and in different types of RNA. Nucleoside modifications are interesting due to their already known roles in translation fidelity, enzyme recognition, disease progression, and RNA stability. In addition, the abundance of modified nucleosides fluctuates based on growth phase, external stress, or possibly other factors not yet explored. With modifications ever changing, a method to determine absolute quantities for multiple nucleoside modifications is required. Here, we report metabolic isotope labeling to produce isotopically labeled internal standards in bacteria and yeast. These can be used for the quantification of 26 different modified nucleosides. We explain in detail how these internal standards are produced and show their mass spectrometric characterization. We apply our internal standards and quantify the modification content of transfer RNA (tRNA) from bacteria and various eukaryotes. We can show that the origin of the internal standard has no impact on the quantification result. Furthermore, we use our internal standard for the quantification of modified nucleosides in mouse tissue messenger RNA (mRNA), where we find different modification profiles in liver and brain tissue.


Introduction
In the central dogma of molecular biology, messenger RNA (mRNA) is the molecule linking the genetic code, DNA, with the cellular work force, proteins. mRNA is translated into proteins with the help of transfer RNA (tRNA) and ribosomal RNA (rRNA). In addition to these key players, many other RNA species have been identified such as long non-coding RNAs (lncRNAs), micro RNAs (miRNAs) or even enzymatic RNAs like RNase P [1]. For over half a century, it is known that tRNAs In addition, we compare the modification profiles of tRNA from various eukaryotes, including fungi, amoebozoa, worms, mouse tissue and human cell culture. Finally, we demonstrate the use of a tRNA derived SILIS for the quantification of modified nucleosides in purified mRNA samples from mouse brain and liver tissue.
All solutions and buffers were made with water from a Millipore device (Milli-Q, Merck, Kenilworth, NJ, USA).
Supplementary Materials Table S1 shows all synthetic standards of modified nucleosides and their respective vendors.

Labeling of Escherichia coli
A single colony of E. coli BW25113 grown on a lysogeny broth (LB) agar plate was selected for inoculation of 5 mL M9 media liquid pre-culture.
10× M9 stock solution was made with Na 2 HPO 4 , KH 2 PO 4 , NaCl and 15 N-NH 4 Cl at the respective final concentrations (482, 220, 43, 187 mM). All solutions were sterile filtered and stored at room temperature until use.
The pre-culture was allowed to grow overnight at 37 • C with 250 rpm shaking and was then used to inoculate a 200 mL culture. The 200 mL culture was grown overnight and was harvested when it reached an OD 600 (optical density) of 2.2 in early stationary phase.

Labeling of Saccharomyces cerevisiae
A single colony of S. cerevisiae BY4741 grown on a yeast extract peptone dextrose (YPD) plate agar plate was selected for inoculated of 5 mL supplemented Silantes 13 C media for liquid culture. Silantes 13 C media was supplemented with 13 C 6 -glucose (final concentration 1%) and CD 3 methionine (final concentration 0.1 g/L). Liquid pre-culture was incubated overnight at 30 • C at 250 rpm. The pre-culture was used to inoculate two 100mL cultures. Cultures were grown overnight to late exponential/early stationary with an OD 600 of 3.5.

Isolation of Total RNA for Stable Isotope-Labeled Internal Standards (SILIS) Production
Bacteria or yeast cultures were centrifuged at 1200× g for 5 min to pellet the cells. The resulting cell pellet was resuspended in 1 mL TRI-Reagent ® (Sigma-Aldrich) per 5-10 mL of culture. For every 1 mL of the TRI-Reagent ® used, 200 µL of chloroform (≥99% purity, Roth, Karlsruhe, Germany) was added to the solution and the mixture was vortexed. In case of S. cerevisiae, cell lysis was aided by addition of acid-washed glass beads as previously described [21]. The biphasic solution rested at room temperature for 5 min and was centrifuged at room temperature for 10 min at 12,000× g. The resulting clear upper phase was precipitated with an equal volume of isopropanol (Roth) After incubation at −20 • C overnight, the precipitated total RNA was pelleted by centrifugation for 20 min at 12,000× g at 4 • C. The RNA pellet was washed two times with 100-200 µL of 70% EtOH and dissolved in 30 µL water.

Dictyostelium Discoideum
The cultivation of the Dictyostelium discoideum AX2 wild-type strain was carried out at 22 • C in constant light. Cells were grown axenically in HL5+ medium (Formedium, Norfolk, UK) in shaking culture. 2 × 10 7 cells were collected via centrifugation at room temperature and washed once with 1× Soerensen buffer (2 mM Na 2 HPO 4 , 15 mM KH 2 PO 4 , adjusted with H 3 PO 4 to pH 6.0). Cells were lysed with 1 mL Trizol ® reagent with 20 mM EDTA (ethylenediaminetetraacetic acid) and incubated for 5 min at room temperature. Isolation of total RNA was performed according to manufacturer's protocol and subsequent size exclusion chromatography (SEC) purification as previously described [22].

Caenorhabditis Elegans
Caenorhabditis elegans cultures were grown to the gravid adult stage and harvested and flash frozen as in [23]. Total RNA was isolated with TRI-Reagent ® as described in Section 2.4.

Cell Culture
HEK 293T cells were cultured in Dulbecco's modified Eagle medium (DMEM) (Sigma Aldrich) with 10% fetal bovine serum (FBS) (Sigma) at 37 • C with 10% CO 2 . Cell were seeded at 20% conflunce and allowed to grow to 80% confluence prior to harvesting. Cells were harvested directly in cell culture flasks using 1 mL TRI-Reagent ® and total RNA was isolated as previously reported [22].

Mouse Brain and Liver
Female C57BL/6J mice (six months old) were used. The mice were anesthetized with isoflurane and subsequently euthanized by cervical dislocation. Mouse organs were isolated and frozen in liquid nitrogen. The organs were kept at −80 • C until RNA extraction was performed. For RNA isolation, 50-100 mg of tissue were sliced on ice and homogenized in 1 mL TRI-Reagent ® using a Qiagen TissueRuptor II and disposable probes (Hilden, Germany). Subsequent isolation of total RNA was performed as previously described.

Purification of Ribosomal RNA and Transfer RNA by Size Exclusion Chromatography
For purification of tRNA from total RNA SEC [24] was used on an Agilent 1100 high-performance liquid chromatography (HPLC) system (Degasser, G1279A; Quat Pump, G1311A; ALS, G1313A; COLCOM, G1316A; VWD, G1314A; Analyt FC, G1364C) with an AdvanceBio column, 300 Å pore size, 2.7 µm particle size, 7.8 × 300 mm (Agilent, Waldbronn, Germany). RNA was eluted with an isocratic gradient with a flow rate of 1mL/min of 0.1 M ammonium acetate (≥98%). Eluting RNA was detected at 254 nm with a diode array detector. Under these conditions tRNA elutes at a retention time between 7 and 8 min. The 1 mL tRNA fraction was collected and evaporated (GeneVac, EZ-2 PLUS, Ipswich, UK) to a volume of~100 µL before precipitation by a standard ammonium acetate is the precipitation at −20 • C overnight. The tRNA was pelleted by centrifugation (12,000× g, 20 min, 4 • C) and washed with 70% ethanol and resuspended in 30 µL water.

Messenger RNA Purification
From isolated total RNA, a 100 µg aliquot was used for mRNA purification with a Dynabeads mRNA purification kit (catalog number 61006) purchased from Thermo Fisher Scientific (Waltham, MA, USA). Manufacturer instructions were followed exactly. Resulting RNA was further treated with Ribominus Transcriptome Kit (catalog number K155001, Thermo Fisher Scientific) to remove any residual rRNA. Again manufacturer instructions were followed without deviation. Resulting RNA was treated again with a Dynabeads mRNA purification kit. To verify the depletion of rRNA and tRNA, the mRNA samples were analyzed with the Bionalyzer RNA 6000 Nano assay on an Agilent 2100 Bioanalyzer System.

High-Resolution Mass Spectrometry
The ribonucleosides were separated using a Dionex Ultimate 3000 HPLC system on an RP-18 column (Synergi, 2.5 µm Fusion-RP C18 100 Å, 100 × 2 mm; Phenomenex ® , Torrance, CA, USA). Mobile phase A was 10 mM ammonium acetate and mobile phase B was 80% acetonitrile. The gradient began with 0% B and increased to 20% B over 10 min and to 80% by 12 min. After a 4 min hold at 80% B, the column returned to 100% A over 1 min. The column was re-equilibrated with 100% A for 8 min. The flow rate was 0.2 mL/min with a column temperature of 30 • C. For untargeted analysis, the slower flow rate resulted in better peak separation and thus mass spectra generation for individual compounds.
High-resolution mass spectra of precursor and product ions were recorded by a Thermo Finnigan LTQ Orbitrap XL operated in positive ionization mode with a capillary voltage of 20 V and temperature of 275 • C. Sheath gas flow was set to 5, and auxiliary gas was set to 35.

Dilution of RNA Digests for 10× SILIS Generation
Serial dilutions of each SILIS type were prepared for analysis to determine the proper dilution A dilution with peaks at least 10-fold above the lower limit of quantification (LLOQ) for the lowest abundant modified nucleosides was considered ideal.

Liquid Chromatography-Mass Spectrometry (LC-MS/MS) Analysis
For quantification a 1290 Infinity II (Agilent Technologies, Waldbronn, Germany) equipped with a diode array detector (DAD) combined with a G6470A Triple Quad system (Agilent Technologies) and electro-spray ionization mass spectrometry (ESI-MS, Agilent Jetstream; Agilent Technologies) was used. Operating parameters were as follows: positive ion mode, skimmer voltage 15 V, Cell Accelerator Voltage 5 V, N 2 gas temperature 230 • C and N 2 gas flow 6 L/min, sheath gas (N 2 ) temperature 400 • C with a flow of 12 L/min, Capillary Voltage of 2500 V, Nozzle Voltage of 0 V and the Nebulizer at 40 psi. The instrument was operated in dynamic multiple reaction monitoring (MRM) mode with the method listed in Table S2 and the individual mass transitions for the nucleosides are given in Supplementary  Materials Table S3. The MRM approach allowed shorter runs as separation of individual compounds was not crucial for analysis. The mobile phases were: A as 5 mM NH 4 OAc (≥99%, HiPerSolv CHROMANORM ® , VWR) aqueous buffer, brought to pH = 5.6 with glacial acetic acid (≥99%, HiPerSolv CHROMANORM ® , VWR) and B as pure acetonitrile (Roth, liquid chromatography-mass spectrometry (LC-MS) grade, purity ≥ 99.95). A Synergi Fusion-RP column (Phenomenex ® , Torrance, CA, USA; Synergi ® 2.5 µm Fusion-RP 100Å, 150 × 2.0 mm) at 35 • C and a flow rate of 0.35 mL/min was used. The gradient began with 100% A for one minute, increased to 10% B by 5 min, and to 40% B by 7 min. The column was flushed with 40% B for 1 min and returned to starting conditions to 100% A by 8.5 min followed by re-equilibration at 100% A for 2.5 additional minutes.

Generation and Characterization of SILIS
Our laboratory analyzes samples from bacteria and various eukaryotes on a regular basis. While many modified nucleosides overlap in both domains of life, some are unique to one of them. For example, 2-methyladenosine (m 2 A) is only found in bacteria, while 1-methyladenosine (m 1 A) and 3-methylcytidine (m 3 C) are common in eukaryotes but not in bacteria. For absolute quantification, isotope dilution mass spectrometry is the method of choice and thus stable isotope labeled internal standards, SILIS, are needed. In 2014 the production of 11 modified nucleosides by metabolic labeling was shown. Important modified nucleosides e.g., the hypermodified uridines and adenosines (e.g., mcm 5 s 2 U or i 6 A) were not reported. With the goal to expand the number of metabolically produced stable isotope labeled nucleosides, we decided to prepare SILIS in the bacterium E. coli and in addition, in the eukaryote S. cerevisiae. At this point, it was necessary to decide which labeling strategy should be used. For standard quantification of unlabeled samples, a simple labeling scheme (e.g., in E. coli only carbon-13) is possible and most economic. We do not recommend nitrogen-15 labeling alone in E. coli for SILIS production. The resulting heavy uridine nucleosides would be only 2 Dalton heavier compared to the unlabeled nucleosides, which is not sufficient due to the natural abundance of 13 C in the natural nucleosides. Our laboratory is specialized in the analysis of isotopically labeled nucleosides from e.g., nucleic acid isotope labeling coupled mass spectrometry (NAIL-MS) pulse-chase experiments [21,26]. These samples already contain isotopically labeled nucleosides and are thus up to 8 Dalton heavier than unlabeled nucleosides. In our context, the SILIS must be even heavier than the samples. Therefore, we decided to use a simultaneous nitrogen-15 and carbon-13 labeling in E. coli and a combined carbon-13 and CD 3 -methionine labeling in S. cerevisiae. Deuterium labeling of a single methyl-group, as introduced by CD 3 -methionine, does not lead to detectable changes in the physicochemical properties of the methylated nucleosides and thus the labeling strategy is suitable for SILIS production [13].
With the goal to produce enough SILIS to last several months or even years, E. coli was cultured in 200 mL of carbon-13 and nitrogen-15 containing M9 medium. S. cerevisiae was grown in two separate 100 mL carbon-13 full medium flasks (Silantes, Munich, Germany) supplemented with 13 C 6 -glucose and CD 3 methionine (final concentration 0.1 g/L). Both organisms were grown to late exponential phase and harvested by centrifugation. The total RNA was isolated using TRI ® reagent. In our previous work [14], the total RNA was used for generation of the SILIS. Thus, an unnecessary excess of canonical nucleosides was introduced into the sample, which complicated the quantification of canonical nucleosides. Therefore, we decided to fractionate the total RNA into the long RNAs and the heavily modified tRNA fraction by size exclusion chromatography [24] to reduce the amount of canonical nucleosides in the later tRNA SILIS. An aliquot of the purified RNA was enzymatically digested to the nucleoside building block. The digest was immediately injected on a Thermo Orbitrap high resolution mass spectrometer for characterization of the produced nucleoside isotopomers. The principle of SILIS generation is shown in Figure 1. The mass spectra of the heavy isotope labeled RNA digests showed a high labeling efficiency for both organisms. We used synthetic standards for retention time comparison and could thus assign all extracted mass spectra to their corresponding modified nucleoside. In Figure 2 (left), the chemical structure, mass spectra and labeled positions are shown for the canonical nucleoside adenosine. In yeast, a 13 C10-labeled adenosine is formed and thus the mass is increased by an m/z of 10. In E. coli, we see a 13 C10-15 N5-labeled adenosine and a mass increase of +15. In both RNA digests, the abundance of unlabeled adenosine is negligible. In Figure 2 (right), the results for 6-methyladenosine (m 6 A) are shown. In yeast, we see the formation of the main product, a 13 C10-CD3-labeled m 6 A (m/z 295.17). We also observe some m/z 293.17, which corresponds to a 13 C11-labeled m 6 A. The CD3-label is introduced by supplementing CD3-methionine to the media. Since we used a 13 C full media, some 13 CH3-methionine is also available to the cells and, thus, we also find 13 CH3-methylated nucleosides in the yeast RNA. For E. coli, we see a distinct labeling of 13 C11-15 N5-labeled m 6 A (+16). Supplementary Materials Figure S1 shows the mass spectra of various other nucleosides from the produced labeled RNA digests. Figure 2. The mass spectra of adenosine (left) and 6-methyladenosine (m 6 A, (right)) are shown for the synthetic standard, the E. coli stable isotope-labeled internal standard (E. c. SILIS) and S. cerevisiae SILIS (S. c. SILIS). Atoms that were stable isotope labeled are indicated in red for carbon-13, blue for deuterium and green for nitrogen-15. The mass spectra of the heavy isotope labeled RNA digests showed a high labeling efficiency for both organisms. We used synthetic standards for retention time comparison and could thus assign all extracted mass spectra to their corresponding modified nucleoside. In Figure 2 (left), the chemical structure, mass spectra and labeled positions are shown for the canonical nucleoside adenosine. In yeast, a 13 C 10 -labeled adenosine is formed and thus the mass is increased by an m/z of 10. In E. coli, we see a 13 C 10 -15 N 5 -labeled adenosine and a mass increase of +15. In both RNA digests, the abundance of unlabeled adenosine is negligible. In Figure 2 (right), the results for 6-methyladenosine (m 6 A) are shown. In yeast, we see the formation of the main product, a 13 C 10 -CD 3 -labeled m 6 A (m/z 295.17). We also observe some m/z 293.17, which corresponds to a 13 C 11 -labeled m 6 A. The CD 3 -label is introduced by supplementing CD 3 -methionine to the media. Since we used a 13 C full media, some 13 CH 3 -methionine is also available to the cells and, thus, we also find 13 CH 3 -methylated nucleosides in the yeast RNA. For E. coli, we see a distinct labeling of 13 C 11 -15 N 5 -labeled m 6 A (+16). Supplementary Materials Figure S1 shows the mass spectra of various other nucleosides from the produced labeled RNA digests. The mass spectra of the heavy isotope labeled RNA digests showed a high labeling efficiency for both organisms. We used synthetic standards for retention time comparison and could thus assign all extracted mass spectra to their corresponding modified nucleoside. In Figure 2 (left), the chemical structure, mass spectra and labeled positions are shown for the canonical nucleoside adenosine. In yeast, a 13 C10-labeled adenosine is formed and thus the mass is increased by an m/z of 10. In E. coli, we see a 13 C10-15 N5-labeled adenosine and a mass increase of +15. In both RNA digests, the abundance of unlabeled adenosine is negligible. In Figure 2 (right), the results for 6-methyladenosine (m 6 A) are shown. In yeast, we see the formation of the main product, a 13 C10-CD3-labeled m 6 A (m/z 295.17). We also observe some m/z 293.17, which corresponds to a 13 C11-labeled m 6 A. The CD3-label is introduced by supplementing CD3-methionine to the media. Since we used a 13 C full media, some 13 CH3-methionine is also available to the cells and, thus, we also find 13 CH3-methylated nucleosides in the yeast RNA. For E. coli, we see a distinct labeling of 13 C11-15 N5-labeled m 6 A (+16). Supplementary Materials Figure S1 shows the mass spectra of various other nucleosides from the produced labeled RNA digests. Figure 2. The mass spectra of adenosine (left) and 6-methyladenosine (m 6 A, (right)) are shown for the synthetic standard, the E. coli stable isotope-labeled internal standard (E. c. SILIS) and S. cerevisiae SILIS (S. c. SILIS). Atoms that were stable isotope labeled are indicated in red for carbon-13, blue for deuterium and green for nitrogen-15. The mass spectra of adenosine (left) and 6-methyladenosine (m 6 A, (right)) are shown for the synthetic standard, the E. coli stable isotope-labeled internal standard (E. c. SILIS) and S. cerevisiae SILIS (S. c. SILIS). Atoms that were stable isotope labeled are indicated in red for carbon-13, blue for deuterium and green for nitrogen-15.
After mass spectrometric characterization, we serially diluted the isotopically labeled RNA digests and analyzed them on a sensitive triple quadrupole instrument. For highest sensitivity, we used the multiple reaction monitoring function of our Agilent software. (For instruments from other suppliers this function is also referred to as single reaction monitoring, SRM) The key parameters for the E. coli and S. cerevisiae heavy isotope labeled nucleosides are given in Supplementary Materials Tables S2a and S2b. We analyzed the dilutions of the digests with their respective parameters and noted the peak intensity of all modified nucleosides. For SILIS preparation, we decided to use a dilution which produces peaks at least 10-fold above the LLOQ of the lowest abundant modified nucleoside. The LLOQ is a unique parameter for each modified nucleoside in the used analytical setup and is determined by analysis of serial dilutions of synthetic standards. From this calibration curve ( Figure S2), the LLOQ (Table S4) is found at the concentration/injected amount where the peak height of the analyte is 10-fold higher than the surrounding noise (S/N > 10).
After determination of the final SILIS concentration, we multiplied the concentration by 10 as we have positive experience with using a 10× SILIS. As an example: For yeast we used 10 µg labeled tRNA and digested the RNA in 30 µL digestion buffer. This digest is diluted 20-fold for production of 600 µL of a 10× yeast SILIS, which is sufficient for analysis of 200-300 samples. In addition, we add a UV-detectable amount of theophylline to our 10× SILIS. Theophyllin absorbs at 260 nm and elutes after adenosine. The theophylline peak area is used to control the correct mixing of SILIS with sample (theophylline acts as an external standard).

Guideline for SILIS Generation
By high-resolution MS characterization, we found a unique signature of modified nucleosides in each of the four produced SILISs (yeast tRNA and largeRNA SILIS and bacteria tRNA and large RNA SILIS). The results are summarized in the Venn diagram of Figure 3 and Table 1. With this information, it is now possible to choose the right organism and RNA type for production of the modified nucleoside of interest. 9 modified nucleosides, such as pseudouridine (Ψ), inosine (I), 7-methylguanosine (m 7 G) and 6-methyladenosine (m 6 A), are found in both RNA types of both organisms (center of Venn diagram, Figure 3) and thus the scientist can decide which organism to use for SILIS generation of these nucleosides. After mass spectrometric characterization, we serially diluted the isotopically labeled RNA digests and analyzed them on a sensitive triple quadrupole instrument. For highest sensitivity, we used the multiple reaction monitoring function of our Agilent software. (For instruments from other suppliers this function is also referred to as single reaction monitoring, SRM) The key parameters for the E. coli and S. cerevisiae heavy isotope labeled nucleosides are given in Supplementary Materials Tables S2a and S2b. We analyzed the dilutions of the digests with their respective parameters and noted the peak intensity of all modified nucleosides. For SILIS preparation, we decided to use a dilution which produces peaks at least 10-fold above the LLOQ of the lowest abundant modified nucleoside. The LLOQ is a unique parameter for each modified nucleoside in the used analytical setup and is determined by analysis of serial dilutions of synthetic standards. From this calibration curve ( Figure S2), the LLOQ (Table S4) is found at the concentration/injected amount where the peak height of the analyte is 10-fold higher than the surrounding noise (S/N > 10).
After determination of the final SILIS concentration, we multiplied the concentration by 10 as we have positive experience with using a 10× SILIS. As an example: For yeast we used 10 µg labeled tRNA and digested the RNA in 30 µL digestion buffer. This digest is diluted 20-fold for production of 600 µL of a 10× yeast SILIS, which is sufficient for analysis of 200-300 samples. In addition, we add a UV-detectable amount of theophylline to our 10× SILIS. Theophyllin absorbs at 260 nm and elutes after adenosine. The theophylline peak area is used to control the correct mixing of SILIS with sample (theophylline acts as an external standard).

Guideline for SILIS Generation
By high-resolution MS characterization, we found a unique signature of modified nucleosides in each of the four produced SILISs (yeast tRNA and largeRNA SILIS and bacteria tRNA and large RNA SILIS). The results are summarized in the Venn diagram of Figure 3 and Table 1. With this information, it is now possible to choose the right organism and RNA type for production of the modified nucleoside of interest. 9 modified nucleosides, such as pseudouridine (Ψ), inosine (I), 7-methylguanosine (m 7 G) and 6-methyladenosine (m 6 A), are found in both RNA types of both organisms (center of Venn diagram, Figure 3) and thus the scientist can decide which organism to use for SILIS generation of these nucleosides.   Table 1.
Queuosine (Q) 0 0 0 1 Very low abundance in this RNA. If available, use yeast RNA instead. 2 Due to matrix effects, this modification cannot be reliably quantified in our chromatographic system.
The modified nucleosides, 4-thiouridine (s 4 U), 2-thiocytidine (s 2 C) and 2-methyladenosine (m 2 A) are unique for E. coli tRNA and thus E. coli has to be used for SILIS production. While 2-methylguanosine (m 2 G) and 5-methylcytidine (m 5 C) are present in the large RNA of E. coli, they are more commonly found in the tRNA of yeast. Here, the production is possible in E. coli, but production in yeast would lead to higher yields of SILIS. 5-methyluridine (m 5 U, sometimes also ribothymidine rT), dihydrouridine (D), 6-threonyladenosine (t 6 A) and 6-isopentenyladenosine (i 6 A) are unique tRNA modifications and thus only found in tRNA from both organisms. As a eukaryotic organism, S. cerevisiae has several unique RNA modifications. For example 3-methylcytidine (m 3 C), 1-methyladenosine (m 1 A), 2,2,-dimethylguanosine (m 22 G) and 4-acetylcytidine (ac 4 C) are uniquely found in the yeast SILIS and thus must be produced in yeast. Eukaryotes also carry hypermodified uridine derivatives at position 34 of their tRNAs. These wobble uridines are also found in the yeast tRNA SILIS. At this stage we already want to stress that wobble uridines are prone to decomposition due to the conditions of enzymatic digestion. Therefore, the samples and also the yeast tRNA SILIS need to be prepared fresh for absolute quantification of these modified nucleosides. Another hypermodified nucleoside is queuosine (Q). We could clearly find Q in our yeast tRNA SILIS. Q is one of the most complex modified nucleosides, and its synthesis is highly complicated [27]. Therefore, Q is not available as a stable isotope labeled standard or even as an unlabeled standard, which highlights the strength of metabolic labeling for SILIS production. Table 1 gives an overview of all modified nucleosides, we identified in our SILIS in alphabetical order. This list was generated from all the high-resolution mass spectra we acquired. In principle, it is also possible to predict the mass transition of an unlisted modified nucleoside and search for it in the respective SILIS. The arising signals should be referenced with the unlabeled synthetic standard for verification. With this method, even lower abundant modified nucleosides are identified in the SILIS and absolute quantification of samples becomes possible.

Absolute Quantification with SILIS
With our 10× SILISs in hand, we decided to use them for quantification of samples of various origins. We used the Venn diagram ( Figure 3) to decide which SILIS should be used for which sample type/organism. In general, we digest samples in 20-30 µL and after filtration we mix 18 µL of sample with 2 µL of the respective 10× SILIS and inject 10 µL of the mix onto the LC-MS/MS. For calibration we prepare serial dilutions of the synthetic standards and add 10% SILIS to each solution before injecting 10 µL. A detailed description is given in the Materials and Methods section. After analysis of the calibration solutions, we normalize the signal of the synthetic standard to the signal of the respective internal standard and receive the calibration curve. With the calibration curve, we can then determine the quantities of modified nucleosides in the sample. The quantities of modified nucleosides are normalized against the quantity of one or all canonical nucleoside(s), e.g., guanosine (G) is commonly used [28]. With SILIS quantification from metabolic labeling, we introduce an additional challenge to the quantification of canonical nucleosides. The SILIS contains both modified and canonical nucleosides. Especially total RNA and rRNA SILIS contain a huge excess of canonical nucleosides and these overlap with the sample canonical signal during UV detection. Simple UV detection does not allow the discrimination of the canonicals from SILIS and from the sample. Thus, the amount of sample canonicals is difficult to quantify by UV detection.
To overcome this problem, we use the mass spectrometer to detect and distinguish the canonical nucleosides. With MS detection, we can clearly distinguish SILIS canonicals from sample canonicals due to their different mass transitions. To avoid saturation of the mass spectrometer by the highly abundant canonical nucleosides, we use non-optimal ionization and collision parameters (e.g., elevated fragmentor voltage and high collision energy). Thus, we can clearly quantify the abundance of sample canonicals and use them for normalization of the less abundant modified nucleosides. The quantity of each modified nucleoside per G is then plotted in %. A detailed description of the calculations and normalization steps is given in the Supplementary Materials and Figure S3 In our first experiment, we wanted to demonstrate that the origin of the SILIS used has no impact on the quantification result. For this purpose, we cultured HEK 293T cells and purified the total tRNA [22]. After digestion we split the sample in two aliquots and analyzed one using the E. coli tRNA SILIS and the other using the yeast tRNA SILIS. As shown in Figure 4a, we see comparable quantities of modified nucleosides independently of the chosen SILIS. Only Cm shows a 10% difference and i 6 A around 32% difference in dependence of the chosen SILIS. The low abundance of i 6 A in the sample (slightly above LLOQ) is causative for the high difference. The difference for Cm is currently under investigation. Thus we recommend caution upon absolute quantification of Cm. For all other modified nucleosides found in both SILISs, like dihydrouridine (D), 1-methylguanosine (m 1 G), 7-methylguanosine (m 7 G) or 5-methyluridine (m 5 U), we see identical quantification results. For those modified nucleosides that are absent in the E. coli tRNA SILIS, no normalization against the stable isotope labeled modified nucleoside was possible and the quantification is less robust (Figure 4b).
Here, we used external calibration for quantification of m 1 A, m 22 G, mcm 5 s 2 U and ac 4 C. While m 22 G and mcm 5 s 2 U seem to be robust for correct external calibration, the results for ac 4 C indicate that external calibration is not sufficient and that internal standards are necessary for correct quantification for these modified nucleosides. For four modified nucleosides, we used a chemically similar modified nucleoside as the internal standard in the E. coli SILIS. For m 2 G we used the SILIS signal of m 1 G, for Um quantification the SILIS signal for Cm and for m 5 C and m 3 C we used the SILIS signal of m 5 U. In most of these cases, the quantification result is significantly different from the yeast SILIS quantification. Using a chemically similar compound is sometimes sufficient for reliable quantification.
However, such an internal standard is unable to normalize the matrix effects and pH effects and thus the quantification results are perturbed as in the case for m 3 C. This experiment highlights the importance of choosing the right SILIS for the analyzed sample type. Our data demonstrates that the origin of the SILIS is mostly without consequence as long as it contains the modified nucleoside of interest. All data can be found in Supplementary Materials Table S5a. signal of m 1 G, for Um quantification the SILIS signal for Cm and for m 5 C and m 3 C we used the SILIS signal of m 5 U. In most of these cases, the quantification result is significantly different from the yeast SILIS quantification. Using a chemically similar compound is sometimes sufficient for reliable quantification. However, such an internal standard is unable to normalize the matrix effects and pH effects and thus the quantification results are perturbed as in the case for m 3 C. This experiment highlights the importance of choosing the right SILIS for the analyzed sample type. Our data demonstrates that the origin of the SILIS is mostly without consequence as long as it contains the modified nucleoside of interest. All data can be found in Supplementary Materials Table S5a.

Modified Nucleosides in Pseudomonas Aeruginosa Total tRNA
Bacteria contain a vast variety of modified nucleosides and tRNA modifications are especially getting more attention from microbiologists and infectiologists. Only recently, the tRNA modification status of the pathogen P. aeruginosa (PA) was determined [29]. Unfortunately, no absolute quantification of modified nucleosides was possible in this study. Therefore, we decided to grow P. aeruginosa PA14 in minimal M9 medium and compare the quantity of modified nucleosides with tRNA from LB and M9 cultured E. coli (in stationary growth phase only). In general, we found the same modified nucleosides in E. coli and PA tRNA (Figure 5a). For the modified nucleosides m 1 G, m 2 A, m 2 G, s 2 C, m 5 U, m 6 A, m 7 G, Cm and I, we found comparable quantities in both bacterial strains. However, we observed a lower abundance of s 4 U (enzyme: thiI), Gm (TrmH) and Um (TrmJ) in P. aeruginosa. Gm, in bacterial tRNA, is connected to inhibition of TLR7, which results in a suppression of the hosts immune system [30]. Our data suggests, that PA tRNA might be more immune-stimulatory compared to E. coli tRNA due to the lower abundance of Gm. TrmJ and thus Um on the other hand was shown to be important for oxidative stress survival in PA [29]. At this point, we cannot predict the impact of these low abundant tRNA modifications in PA, but we are confident that future studies will address these findings. Overall, bacteria show a very low modification density in their tRNAs. The absolute quantities are given in Supplementary Materials Table S5b.

Modified Nucleosides in Total tRNA from Various Eukaryotes
In a next step, we applied our yeast tRNA SILIS to assess the modification profile of tRNAs from increasingly complex eukaryotes. We analyzed S. cerevisiae, D. discoideum, C. elegans, the human cell culture strain HEK 293T and tissue samples from adult mice (here liver and brain). The results are shown in Figure 5b,c and are listed in Supplementary Materials Table S5b. In comparison to the modification profile of E. coli and PA in Figure 5a, we see a higher variety and abundance of

Modified Nucleosides in Pseudomonas Aeruginosa Total tRNA
Bacteria contain a vast variety of modified nucleosides and tRNA modifications are especially getting more attention from microbiologists and infectiologists. Only recently, the tRNA modification status of the pathogen P. aeruginosa (PA) was determined [29]. Unfortunately, no absolute quantification of modified nucleosides was possible in this study. Therefore, we decided to grow P. aeruginosa PA14 in minimal M9 medium and compare the quantity of modified nucleosides with tRNA from LB and M9 cultured E. coli (in stationary growth phase only). In general, we found the same modified nucleosides in E. coli and PA tRNA (Figure 5a). For the modified nucleosides m 1 G, m 2 A, m 2 G, s 2 C, m 5 U, m 6 A, m 7 G, Cm and I, we found comparable quantities in both bacterial strains. However, we observed a lower abundance of s 4 U (enzyme: thiI), Gm (TrmH) and Um (TrmJ) in P. aeruginosa. Gm, in bacterial tRNA, is connected to inhibition of TLR7, which results in a suppression of the hosts immune system [30]. Our data suggests, that PA tRNA might be more immune-stimulatory compared to E. coli tRNA due to the lower abundance of Gm. TrmJ and thus Um on the other hand was shown to be important for oxidative stress survival in PA [29]. At this point, we cannot predict the impact of these low abundant tRNA modifications in PA, but we are confident that future studies will address these findings. Overall, bacteria show a very low modification density in their tRNAs. The absolute quantities are given in Supplementary Materials Table S5b.

Modified Nucleosides in Total tRNA from Various Eukaryotes
In a next step, we applied our yeast tRNA SILIS to assess the modification profile of tRNAs from increasingly complex eukaryotes. We analyzed S. cerevisiae, D. discoideum, C. elegans, the human cell culture strain HEK 293T and tissue samples from adult mice (here liver and brain). The results are shown in Figure 5b,c and are listed in Supplementary Materials Table S5b. In comparison to the modification profile of E. coli and PA in Figure 5a, we see a higher variety and abundance of modified nucleosides in the eukaryotic samples. This is in accordance with the literature [31]. For the fungus S. cerevisiae and the amoebozoa D. discoideum, we only observe minor differences in the total abundance of modified nucleosides. One of the most interesting organisms in our analysis is C. elegans. C. elegans has a similar chemical variety of modified nucleosides as the other eukaryotes, but the abundance is lower compared to the other eukaryotes. The only exception are the ribose methylated nucleosides Um, Gm and Cm, which are of comparable abundance or even increased abundance in the worms (Am). From our data, many hypotheses are possible. For example, the tRNA maturation and modification might be less efficient in the worm and thus more tRNAs remain as unmodified transcripts. It is also possible, that worms contain large quantities of an unknown transcript of 60-100 nucleotide lengths, which is heavily modified with ribose methylation. We are currently investigating why worms display such a bizarre modification profile. Our analysis of the human cell culture samples and the mouse tissue samples revealed an overall high similarity between the sample types. From our data, we observe the previously reported lower abundance of m 2 G, m 1 G and m 22 G in mouse brain compared to mouse liver samples. In addition, we can confirm the comparable abundance of m 1 A and t 6 A in the tRNAs from these tissues [13]. Furthermore, we see a reduced abundance of Gm and Um in the mouse brain tissue compared to liver.
Samples from eukaryotic organisms, especially cell culture samples, can be contaminated with bacteria. Sometimes it is difficult to detect such contaminations. Here, tRNA modification analysis is a useful tool to determine the contamination status of a sample. Bacteria contain several unique tRNA modifications such as s 4 U, s 2 C or m 2 A. Any of these modified nucleosides can be used as an indicator for bacterial contamination in a eukaryotic sample. However, m 2 A is the most simple of all because of its (a) high abundance in bacterial tRNAs, (b) high detection efficiency and (c) similar retention time to m 6 A. m 6 A is a common eukaryotic modification, which is part of most RNA modification analyses. In case of bacterial contamination, a second peak right before m 6 A appears in the chromatogram of a sample. This second peak is m 2 A and clearly indicates the presence of bacteria. We have had several cases in the past, where we analyzed tRNA from inconspicuous cell cultures and we observed a more or less pronounced peak of m 2 A (see Supplementary Materials Figure S4). Only later, testing for mycobacteria revealed the actual contamination of the cell culture and the experiment was discarded. Therefore, we recommend careful analysis of m 6 A in eukaryotic samples in order to assess the potential presence of bacterial contaminations in form of m 2 A. modified nucleosides in the eukaryotic samples. This is in accordance with the literature [31]. For the fungus S. cerevisiae and the amoebozoa D. discoideum, we only observe minor differences in the total abundance of modified nucleosides. One of the most interesting organisms in our analysis is C. elegans. C. elegans has a similar chemical variety of modified nucleosides as the other eukaryotes, but the abundance is lower compared to the other eukaryotes. The only exception are the ribose methylated nucleosides Um, Gm and Cm, which are of comparable abundance or even increased abundance in the worms (Am). From our data, many hypotheses are possible. For example, the tRNA maturation and modification might be less efficient in the worm and thus more tRNAs remain as unmodified transcripts. It is also possible, that worms contain large quantities of an unknown transcript of 60-100 nucleotide lengths, which is heavily modified with ribose methylation. We are currently investigating why worms display such a bizarre modification profile. Our analysis of the human cell culture samples and the mouse tissue samples revealed an overall high similarity between the sample types. From our data, we observe the previously reported lower abundance of m 2 G, m 1 G and m 22 G in mouse brain compared to mouse liver samples. In addition, we can confirm the comparable abundance of m 1 A and t 6 A in the tRNAs from these tissues [13]. Furthermore, we see a reduced abundance of Gm and Um in the mouse brain tissue compared to liver.
Samples from eukaryotic organisms, especially cell culture samples, can be contaminated with bacteria. Sometimes it is difficult to detect such contaminations. Here, tRNA modification analysis is a useful tool to determine the contamination status of a sample. Bacteria contain several unique tRNA modifications such as s 4 U, s 2 C or m 2 A. Any of these modified nucleosides can be used as an indicator for bacterial contamination in a eukaryotic sample. However, m 2 A is the most simple of all because of its (a) high abundance in bacterial tRNAs, (b) high detection efficiency and (c) similar retention time to m 6 A. m 6 A is a common eukaryotic modification, which is part of most RNA modification analyses. In case of bacterial contamination, a second peak right before m 6 A appears in the chromatogram of a sample. This second peak is m 2 A and clearly indicates the presence of bacteria. We have had several cases in the past, where we analyzed tRNA from inconspicuous cell cultures and we observed a more or less pronounced peak of m 2 A (see Supplementary Materials Figure S4). Only later, testing for mycobacteria revealed the actual contamination of the cell culture and the experiment was discarded. Therefore, we recommend careful analysis of m 6 A in eukaryotic samples in order to assess the potential presence of bacterial contaminations in form of m 2 A.

The Abundance of Modified Nucleosides in Messenger RNA Is Different among Tissues
Messenger RNA has become one of the most intensively studied RNA types since the discovery of its dynamic modification status. It was found that m 6 A is incorporated into mRNA by RNA modifying enzymes (RNA writers), where it regulates the binding of RNA reader proteins. In addition, m 6 A and its ribose-methylated form m 6 Am can be removed enzymatically from the mRNA by RNA erasers [32][33][34]. The dynamics of the m 6 A modification in mRNA prompted the start of the field of epitranscriptomics. In addition to m 6 A, other modified nucleosides have been found in mRNA. Some of these are of very low abundance, such as m 5 C, m 3 C and m 1 A. From a mass spectrometrist's perspective, one of the main challenges of mRNA modification profiling is the availability of a truly pure sample. The Lyko and Helm lab [35] recently demonstrated the impact of the currently used purification protocol on the quantification result and presented evidence for the constant contamination with rRNA. We share their conclusion, that true purification of mRNA is currently not possible and that modified nucleosides found in low abundances are most likely from

The Abundance of Modified Nucleosides in Messenger RNA Is Different among Tissues
Messenger RNA has become one of the most intensively studied RNA types since the discovery of its dynamic modification status. It was found that m 6 A is incorporated into mRNA by RNA modifying enzymes (RNA writers), where it regulates the binding of RNA reader proteins. In addition, m 6 A and its ribose-methylated form m 6 Am can be removed enzymatically from the mRNA by RNA erasers [32][33][34]. The dynamics of the m 6 A modification in mRNA prompted the start of the field of epitranscriptomics. In addition to m 6 A, other modified nucleosides have been found in mRNA. Some of these are of very low abundance, such as m 5 C, m 3 C and m 1 A. From a mass spectrometrist's perspective, one of the main challenges of mRNA modification profiling is the availability of a truly pure sample. The Lyko and Helm lab [35] recently demonstrated the impact of the currently used purification protocol on the quantification result and presented evidence for the constant contamination with rRNA. We share their conclusion, that true purification of mRNA is currently not possible and that modified nucleosides found in low abundances are most likely from contaminating RNAs and not from mRNA. The purpose of our next analysis was to demonstrate the difficulties and challenges in mRNA modification analysis. In accordance with the protocol suggested by Lyko and Helm, we have purified total mRNA from mouse liver and mouse brain using first a Poly-A enrichment step, followed by rRNA removal and a second Poly-A enrichment. The quality of the eluted RNA was checked by chip gel electrophoresis. As shown in Figure 6a, we see a size distribution of approximately 500 to 7000 nucleotides, which is to be expected for mRNA. We see no distinct peaks for tRNA or rRNA in the purified RNA. However, the amount of isolated mRNA is very low, and we cannot completely exclude contamination by remnant tRNA or rRNA hidden underneath the mRNA population. We digested the purified mRNA sample and quantified the abundance of modified nucleosides using the yeast tRNA SILIS. Figure 6b shows the results. mRNA is polyadenylated and the length of the Poly-A tail is highly variable and might fluctuate from organ to organ. Thus, we decided against the normalization against A and normalized against G. The most abundant modified nucleoside in both samples is m 6 A. Interestingly, the abundance of m 6 A is significantly higher in mRNA from brain (0.32% m 6 A per G) compared to liver (0.19% m 6 A per G, p-value: 0.00005). m 66 A (also referred to as m 6 2 A) and m 6 Am were not quantifiable due to their absence in the used dilution of yeast SILIS. The cap modification m 7 G is found in comparable amounts in both tissues. Other modified nucleosides are far less abundant in our isolated mRNA samples and potentially from tRNA or rRNA contamination. Mass spectrometry is a highly sensitive tool, and thus even sub-stoichiometric quantities can be assessed. However, in the case of mRNA preparations, we cannot exclude which origin small quantities of modified nucleosides such as Gm, Cm, Am and Um have. It is quite possible that these signals arise from low amounts of contaminating rRNA. Thus, we recommend caution upon biological data interpretation as many of the quantified nucleosides might be false positive results. Orthogonal techniques, such as sequencing (for review see [36]) must be employed to verify such findings. The only modified nucleoside we want to highlight in addition to m 6 A is m 3 C. This modification was recently described as an mRNA modification in mouse liver and a human cell line, incorporated by METTL8 [37]. The authors of this study found very low abundances of m 3 C (~30-fold less m 3 C than m 6 A) in their cell culture samples. We find a rather high amount of m 3 C in our mouse liver mRNA (5-fold less m 3 C than m 6 A), while in the mouse brain mRNA the abundance is drastically lower (60-fold less m 3 C than m 6 A). The biological significance and correctness needs validation by tools such as the recently presented AlkAnilin-seq [38]. A sequence-specific localization of m 3 C in large RNAs is crucial to understand the origin of the modification. We and others showed that position 3 of cytidine is prone to direct methylation and thus sub-stoichiometric signals of m 3 C might hint towards a chemical methylation and not an enzymatic one [26]. contaminating RNAs and not from mRNA. The purpose of our next analysis was to demonstrate the difficulties and challenges in mRNA modification analysis. In accordance with the protocol suggested by Lyko and Helm, we have purified total mRNA from mouse liver and mouse brain using first a Poly-A enrichment step, followed by rRNA removal and a second Poly-A enrichment. The quality of the eluted RNA was checked by chip gel electrophoresis. As shown in Figure 6a, we see a size distribution of approximately 500 to 7000 nucleotides, which is to be expected for mRNA. We see no distinct peaks for tRNA or rRNA in the purified RNA. However, the amount of isolated mRNA is very low, and we cannot completely exclude contamination by remnant tRNA or rRNA hidden underneath the mRNA population. We digested the purified mRNA sample and quantified the abundance of modified nucleosides using the yeast tRNA SILIS. Figure 6b shows the results. mRNA is polyadenylated and the length of the Poly-A tail is highly variable and might fluctuate from organ to organ. Thus, we decided against the normalization against A and normalized against G. The most abundant modified nucleoside in both samples is m 6 A. Interestingly, the abundance of m 6 A is significantly higher in mRNA from brain (0.32% m 6 A per G) compared to liver (0.19% m 6 A per G, p-value: 0.00005). m 66 A (also referred to as m 6 2A) and m 6 Am were not quantifiable due to their absence in the used dilution of yeast SILIS. The cap modification m 7 G is found in comparable amounts in both tissues. Other modified nucleosides are far less abundant in our isolated mRNA samples and potentially from tRNA or rRNA contamination. Mass spectrometry is a highly sensitive tool, and thus even sub-stoichiometric quantities can be assessed. However, in the case of mRNA preparations, we cannot exclude which origin small quantities of modified nucleosides such as Gm, Cm, Am and Um have. It is quite possible that these signals arise from low amounts of contaminating rRNA. Thus, we recommend caution upon biological data interpretation as many of the quantified nucleosides might be false positive results. Orthogonal techniques, such as sequencing (for review see [36]) must be employed to verify such findings. The only modified nucleoside we want to highlight in addition to m 6 A is m 3 C. This modification was recently described as an mRNA modification in mouse liver and a human cell line, incorporated by METTL8 [37]. The authors of this study found very low abundances of m 3 C (~30-fold less m 3 C than m 6 A) in their cell culture samples. We find a rather high amount of m 3 C in our mouse liver mRNA (5-fold less m 3 C than m 6 A), while in the mouse brain mRNA the abundance is drastically lower (60-fold less m 3 C than m 6 A). The biological significance and correctness needs validation by tools such as the recently presented AlkAnilin-seq [38]. A sequence-specific localization of m 3 C in large RNAs is crucial to understand the origin of the modification. We and others showed that position 3 of cytidine is prone to direct methylation and thus sub-stoichiometric signals of m 3 C might hint towards a chemical methylation and not an enzymatic one [26]. Modified nucleosides found and quantified in mouse mRNA from brain and liver. Legend: Arrow: Most interesting differences between liver and brain tissue mRNA. *signal detectable but not Figure 6. Absolute quantification of mRNA purified from mouse liver and mouse brain. (a) The chip electropherogram of mouse brain mRNA shows the expected size distribution from 500-7000 nucleotides. The location of tRNA and ribosomal RNA (18S and 28S) are indicated in grey. (b) Modified nucleosides found and quantified in mouse mRNA from brain and liver. Legend: Arrow: Most interesting differences between liver and brain tissue mRNA. *signal detectable but not quantifiable. The average of n = 3 biol. replicates are plotted. The error bars reflect the standard deviation.

Discussion
Internal standards are crucial for the correct and robust quantification of modified nucleosides. We have presented the preparation of such standards, also referred to as SILIS, in two microorganisms and characterized the heavy labeled modified nucleosides. In summary, 26 modified nucleosides are easily accessible by our approach, including hypermodified nucleosides. In addition, we present a method which allows the quantification of canonical nucleosides for normalization by mass spectrometry. The key here is the use of non-optimal MS parameters to avoid saturation of the mass spectrometer. By this approach, the canonical nucleosides from the sample can be quantified even in the presence of the canonical nucleosides introduced through the SILIS. From the provided overview, it becomes clear that yeast tRNA is the ideal source for production of SILIS. However, the growth medium for yeast is more expensive than for bacteria (1L costs~2000 €/yeast and~400 €/bacteria). In bacteria, 16 eukaryotic modified nucleosides can be produced, however, most only in low abundance (e.g., m 5 C and m 2 G). Nevertheless, bacterial SILIS production might be an economic choice for the analysis of eukaryotic samples. We could show that the origin of the SILIS is of no consequence to the result of quantification as long as the modified nucleoside of interest is actually present in the used SILIS. Overall, we can now perform absolute quantification of up to 26 modified nucleosides, which is 15 more than we previously reported. Among these modified nucleosides, crucial analytes such as the hypermodified uridines and queuosine are found.
We have applied our produced SILISs to a variety of sample types and showed its vast usability for a multitude of experiments. While we focused on the analysis of non-isotopically labeled samples from various organisms in the presented work, we have also utilized the produced SILISs successfully in our NAIL-MS studies in bacteria [26] and yeast [21].
We have studied the effect of nutrition on the abundance of modified nucleosides in the tRNA of E. coli by growing the bacteria in either rich LB broth or minimal M9 salt solution. Interestingly, we found only minimal to no effects of the medium on the abundance of the studied nucleosides (analysis in stationary phase). Furthermore, we analyzed the modification profile of P. aeruginosa tRNA. While the overall modification profile is similar to the profile of E. coli, three modified nucleosides were significantly different. The first is s 4 U, a modified nucleoside commonly located at position 8 of bacterial tRNAs. This modification is known to act as a sensor for UV irradiation [39], which leads to delayed growth during UV light exposure [40]. At this point, it is difficult to judge how the reduced abundance of s 4 U in P. aeruginosa affects its UV sensitivity. From the literature it is known, that P. aeruginosa has a higher UV-A sensitivity compared to E. coli [41], but it remains unclear how this finding is connected to the abundance of s 4 U. In addition to s 4 U, Gm is 4-fold lower in P. aeruginosa compared to E. coli. It would be interesting to compare the TLR7 activation of total tRNA from both organisms [30].
We have used the yeast tRNA SILIS to study the abundance of modified nucleosides in total tRNA from various eukaryotic organisms. We find the same chemical diversity of modified nucleosides in all analyzed tRNAs, however, C. elegans has a very low abundance of most modified nucleosides. Only the ribose-methylated nucleosides are of similar abundance compared to the other organisms and Am is significantly higher. Our analysis used tRNA prepared by size exclusion chromatography. It is possible, that worms have a unique set of ribose-methylated RNAs which are of comparable length to tRNA. It is possible that these RNAs dilute the actual tRNA modification profile and thus the low modification density can be explained. This unique modification profile of C. elegans and other worms should be subject to future studies. For the mammalian cells and tissue samples, we find comparable modification densities in the tRNAs. The comparison of HEK and mouse tissue tRNA showed a slightly lower abundance of structure stabilizing tRNA modifications such as 1-methyladenosine, 7-methylguanosine and 5-methyluridine in the tissue-derived tRNAs. The nutritional environment of cultured cells might be the explanation for these minor differences. Our results for mouse brain and mouse liver are comparable to previous reports [13]. In addition, we find a 2-and 1.5-fold reduced abundance of Gm and Um, respectively, in the mouse brain tRNA compared to the mouse liver tRNA. Gm is located at position 34 (wobble) of tRNA Phe GAA and Um in tRNA Glu UUG and thus both modifications are directly linked to translation. In fact, defects in tRNA anticodon 2 -O-methylation are implicated in nonsyndromic X-linked intellectual disability due to mutations in FTSJ1 [42]. Therefore, we assume that the lower abundance of these modified nucleosides in brain tRNA is of physiological importance.
The analysis of eukaryotic hypermodified uridine derivatives, the wobble uridines, has always proven to be difficult. In our hands, the alkaline reaction conditions of the enzymatic digestion reaction (dictated by the mandatory use of alkaline phosphatase) results in a slow chemical decomposition of the modified nucleosides mcm 5 U, mcm 5 s 2 U and ncm 5 s 2 U. Storage of digested tRNA at −20 • C does not stop this decomposition. From our experience, the addition of 1/3 Vol of our LC-MS mobile buffer (pH 5.3) after the digestion slows down the decomposition process. Nevertheless, analysis should be performed on the same day. A SILIS for wobble uridine quantification cannot be stored either and should be produced on the same day of sample analysis. Therefore, we recommend keeping aliquots of 5-10 µg total tRNA (from SILIS production) and digest once needed. Higher quantities of s 2 U and ncm 5 s 2 U SILIS can be produced in knockout S. c. strains [43].
The bottleneck in wobble uridine analysis is the low abundance and detection efficiency of these modified nucleosides in tRNA. For mRNA modification analysis, the key bottleneck is the production of truly pure samples. We have used a stringent purification protocol, which uses a series of Poly-A enrichment, Ribominus treatment and again Poly-A enrichment steps to produce mRNA samples of best achievable purity. Such a procedure is necessary to ensure removal of especially ribosomal RNA as recently presented [35]. Although the procedure is highly expensive (~200 € per sample), the purity of the sample is not guaranteed. As we show in our chip electropherogram, the yield of total mRNA is very low and small amounts of tRNA and rRNA contaminations may be hidden within the mRNA signal. Our results reflect our circumspection. We detect a variety of modified nucleosides in our purified mRNA samples, but all in very low abundance. A validation to confirm their nature as true mRNA modifications is currently only possible for m 6 A, m 7 G, m 1 A, m 5 C and m 3 C by modern sequencing techniques. Due to the nature of LC-MS/MS analysis and the necessarily complete digestion, the biological meaning is difficult to interpret. Nevertheless, LC-MS/MS analysis, and especially absolute quantification using metabolically produced SILIS, remains a key tool to study the abundance and potential dynamics of modifications in mRNA once their presence has been verified using orthogonal techniques.
Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4425/10/1/26/s1, Figure S1: High-resolution mass spectra of nucleosides, Figure S2: Calibration curves, Figure S3: Calculations for absolute quantification of modified nucleosides, Figure S4: Chromatogram of methylated adenosines to determine bacterial contamination in eukaryotic samples, Table S1: Synthetic modified nucleosides and their suppliers, Table S2a: MRM method for detection of nucleosides using the E. coli SILIS, Table S2b: MRM method for detection of nucleosides using the yeast SILIS, Table S3a: Mass transition of nucleosides in the E. coli SILIS, Table S3b: Mass transition of nucleosides in the yeast SILIS, Table S4: Lower limits of quantification for modified nucleosides in general and for mRNA,