Protocol for RNA modification analysis by UHPLC-QqQ MS

Summary

Over 170 RNA modifications have been reported across domains of life. Here, we present a protocol for identifying and quantifying modified nucleosides in RNA using ultra-high-performance liquid chromatography coupled with triple-quadrupole mass spectrometry (UHPLC-QqQ MS). We describe steps for preparing nucleoside standards, determining nucleoside retention time and mass transition, and building calibration curves for quantification. We next detail procedures for the digestion of RNA and sample analysis. This protocol was applied to RNA modification analysis in archaeal, bacterial, and eukaryotic cells.

For complete details on the use and execution of this protocol, please refer to Tsai et al.¹

Graphical abstract

Highlights

• •Steps for preparing standard solutions to identify RNA modifications by LC-MS/MS
• •Instructions for building calibration curves for MS-based nucleoside quantification
• •Guidance on digesting RNA to nucleosides and performing sample analysis

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Over 170 RNA modifications have been reported across domains of life. Here, we present a protocol for identifying and quantifying modified nucleosides in RNA using ultra-high-performance liquid chromatography coupled with triple-quadrupole mass spectrometry (UHPLC-QqQ MS). We describe steps for preparing nucleoside standards, determining nucleoside retention time and mass transition, and building calibration curves for quantification. We next detail procedures for the digestion of RNA and sample analysis. This protocol was applied to RNA modification analysis in archaeal, bacterial, and eukaryotic cells.

Before you begin

Extremophilic archaea thrive in some of the most extreme environments on earth while maintaining RNA integrity and functions in part due to diverse RNA modifications introduced by dedicated enzymes.^1,2,3 In thermophilic archaea, RNA modifications stabilize RNA structures and response to environmental cues.^{1,2,4,5,6,7,8} Therefore, the identification and quantification of modified nucleosides in distinct RNA species can help researchers understand the functions of RNA modifications and discover new RNA-modifying enzymes. However, identifying unknown RNA modifications or simultaneously detecting multiple modifications has proven challenging with conventional RNA sequencing. LC-MS/MS offers the potential for unbiased RNA modification detection within a single experiment.

This protocol describes the targeted detection and quantification of modified nucleosides in archaeal RNA samples using UHPLC-QqQ MS. We have also used this protocol for analysis of RNA isolated from Escherichia coli and Saccharomyces cerevisiae (S288C strain), and for a universal human reference RNA (Agilent #740000). The protocol comprises four main parts: (i) determining retention times and mass transitions for a panel of standard modified and unmodified nucleosides, (ii) building calibration curves, (iii) digesting RNA to nucleosides, and (iv) performing data analysis. We recently applied this protocol to profile RNA modifications from five archaeal species, resulting in the discovery of a new m⁷G methyltransferase.

Innovation

This protocol provides a step-by-step instruction for researchers to implement UHPLC-QqQ MS methods to identify and quantify a wide range of RNA modifications. Our approach provides researchers with a tool to globally profile RNA modifications complementing epitranscriptomic sequencing methods.

Preparation of the nucleoside standard solutions for mass transition determination

Timing: 1 h

Note: Timing may vary depending on the number of nucleosides in the panel.

• 1.Prepare a 20 mM stock of each standard nucleoside using LC-MS grade water in a 1.5 mL microcentrifuge tube. Vortex to ensure complete dissolution.
• 2.In an HPLC vial, dilute 200 μL of each 20 mM nucleoside standard stock solution to 100 μM with LC-MS grade water. The resulting solutions will be used for mass transition determination.

Note: 76 nucleoside standards were obtained from commercial sources.

Preparation of the nucleoside standard mix for retention time determination

Timing: 1 h

• 3.Calculate the monoisotopic mass for each standard nucleoside.
• 4.Combine 0.5–1 μL of each 20 mM stock into 1 mL of LC-MS grade water in a 1.5 mL HPLC vial to make a nucleoside standard mix.

Note: We recommend no more than 8 nucleosides per standard mix.

CRITICAL: Nucleosides with monoisotopic masses differing by $\le$2 Da should ideally be placed in separate mixes.

Preparation of the nucleoside standard solutions for building calibration curves

Timing: 1–2 h

• 5.Take 0.5–1 μL of each 20 mM stock and separately resuspend into 1 mL of LC-MS grade water in a 1.5 mL microcentrifuge tube.
• 6.Transfer 700–800 μL of each nucleoside standard solution into a quartz cuvette.
• 7.
  Measure absorbance of the diluted nucleoside standards using a UV spectrometer.

  • a.
    Calculate molar concentration of nucleosides using Beer’s law:

    $$c=A/\epsilon ·L$$
  • b.
    Transfer the measured nucleoside standard solutions from the quartz cuvettes back to microcentrifuge tubes. Record molar concentrations. This will be used to determine the highest concentration point of the calibration curve.

    Note: Molar concentration ($c$); Absorbance at $\lambda$ max ($A$); Extinction coefficient at $\lambda$ max ($\epsilon$); Light path length in cm ($L$).

    Note: To clean the quartz cuvettes for next use, incubate inner chamber with 98% sulfuric acid for 2 h to remove residual nucleosides. Wash the cuvettes thoroughly with deionized water.

• 8.Combine 100–200 μL of each measured nucleoside standard solution in same volume to make starting nucleoside standard mix in a 1.5 mL HPLC vial. If two nucleoside standards were combined, divide the recorded molar concentration from step 7 by two. The resulting number serves as the highest concentration point of the calibration curve.

Note: Include at least one internal standard in the nucleoside standard mix for relative quantification. Common internal standards are the four canonical nucleosides (A, U, G, C).

CRITICAL: Use calibrated electronic pipette to ensure precise volume ratio of each nucleoside standard.

• 9.Conduct a serial dilution using the starting nucleoside standard mix prepared from the last step in LC-MS grade water in ½ volume ratio to 16 concentration points. For example, if the combined volume of the starting nucleoside standard mix is 800 μL, prepare 400 μL of LC-MS grade water in fifteen 1.5 mL-HPLC vials. Transfer 400 μL of the starting nucleoside standard mix to one vial, vortex vigorously, then transfer 400 μL of the diluted nucleoside mix to the next vial. Repeat the transfer steps to obtain all 16 concentration points.

CRITICAL: Use a calibrated electronic pipette throughout the serial dilution process.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
N4-acetylcytidine	Biosynth	NA05753
N4-Acetyl-2′-O-methylcytidine	Biosynth	NA08375
N6-acetyladenosine	Biosynth	NA159478
3-(3-amino-3-carboxypropyl) uridine	Biosynth	NA170442
2′-O-methyladenosine	Biosynth	NM05694
5-carboxycytidine	Biosearch Technologies	PY7598
2′-O-methylcytidine	Biosynth	NM06302
5-carboxymethyluridine	Biosynth	NC159474
5-carboxymethylaminomethyluridine	Biosynth	NC159491
Dihydrouridine	Toronto Research Chemicals	D449668
5-formylcytidine	Biosearch Technologies	PY7599
N6-formyladenosine	Biosynth	NF146409
2′-O-methylguanosine	Biosynth	NM02941
5-hydroxymethylcytidine	Biosearch Technologies	PY7596
N6-hydroxymethyladenosine	Biosynth	NH178111
5-hydroxyuridine	Biosynth	NH32540
N6-isopentenyladenosine	Biosynth	ND08001
2′-O-methylinosine	Biosearch Technologies	PR3770-B500
4-demethylwyosine	Biosynth	ND159486
Isowyosine	Toronto Research Chemicals	I918635
N6-(cis-hydroxyisopentenyl) adenosine	Biosynth	XZ181120
1-methyladenosine	Biosynth	PR3032
1,2′-O-dimethyladenosine	Biosynth	ND159412
1-methylguanosine	Biosynth	NM08574
1-methylinosine	Biosynth	FM163160
1-methylpseudouridine	Biosearch Technologies	PYA11052
N2,N2,7-trimethylguanosine	Biosynth	NT08918
N2,N2-dimethylguanosine	Biosynth	ND05647
N2,7-dimethylguanosine	Biosynth	ND45563
2,8-dimethyladenosine	Biosynth	FD144777
2-methyladenosine	Biosynth	NM46832
N2-methylguanosine	Biosynth	NM35522
N2,2′-O-dimethylguanosine	Biosynth	ND159480
3-methylcytidine	ChemGenes	RP2317
3-methylpseudouridine	Biosynth	NM159403
3-methyluridine	Biosynth	NM06185
3,2′-O-dimethyluridine	Biosynth	NM163076
N4,N4-dimethylcytidine	Biosynth	ND159405
N4,N4,2′-O-trimethylcytidine	Biosynth	FT144591
N4-methylcytidine	Matrix Scientific	100567
N4,2′-O-dimethylcytidine	Biosynth	NM144592
5-methylcytidine	Biosynth	NM03720
5,2′-O-dimethylcytidine	Biosynth	NM06424
5-methyldihydrouridine	Biosynth	ND144868
5-methyl-2-thiouridine	Biosynth	NM08039
5-methyluridine	Biosynth	NM04922
5,2′-O-dimethyluridine	Biosearch Technologies	PY7650-B100
N6,N6-dimethyladenosine	Biosynth	ND04708
N6,N6,2′-O-trimethyladenosine	Biosynth	NT59826
N6-methyladenosine	Biosearch Technologies	PR3732
N6,2′-O-dimethyladenosine	Biosearch Technologies	PR3733
O6-methylguanine	Biosynth	NM02922
N6-methyl-N6-threonylcarbamoyladenosine	Toronto Research Chemicals	M696255
7-methylguanosine	Santa Cruz Biotechnology	sc-221113
8-methyladenosine	Biosearch Technologies	PR3007
5-methoxycarbonylmethyl-2-thiouridine	Biosynth	NM159492
5-methoxycarbonylmethyluridine	Biosynth	NM45525
5-methoxycarbonylmethyl-2′-O-methyluridine	Biosynth	FM144574
Uridine 5-oxyacetic acid methyl ester	Biosynth	NU159494
5-methylaminomethyl-2-thiouridine	Biosynth	NM159475
5-methoxyuridine	Biosynth	NM08028
2-methylthio-N6-isopentenyladenosine	Biosynth	NM30233
2-methylthio-N6-methyladenosine	Biosynth	NM159489
5-carbamoylmethyluridine	Biosynth	NC159473
Pseudouridine	Biosynth	NP11297
Adenosine	Biosearch Technologies	PR3005
Cytidine	Biosearch Technologies	PY7215
Guanosine	Biosearch Technologies	PR3703
Inosine	Biosynth	NI06297
Uridine	Biosearch Technologies	PY7780
2-thiocytidine	Biosynth	NT02911
2-thiouridine	Biosynth	NT02881
2-thio-2′-O-methyluridine	Biosynth	NM159407
4-thiouridine	Biosynth	NT06186
N6-threonylcarbamoyladenosine	Biosynth	NC06332
2′-O-methyluridine	Biosynth	NM04259
Sulfuric acid	MilliporeSigma	258105
Ammonium acetate	MilliporeSigma	73594-100G-F
2-propanol	MilliporeSigma	1.02781
Methanol	J.T. Baker	9830-03
LC-MS grade water	J.T. Baker	9831-03
Nucleoside digestion mix	New England Biolabs	M0649S
Qubit RNA high sensitivity (HS) assay kit	Thermo Fisher Scientific	Q32852
Software and algorithms
MassHunter Workstation for LC/TQ	Agilent Technologies	M5930AA
MassHunter Optimizer	Agilent Technologies	G3793AA
Other
Triple-quadrupole LC/MS	Agilent Technologies	6495C LC/TQ
Waters XSelect HSS T3 XP column (2.1 × 100 mm, 2.5 μm)	Waters	186006151
Evolution 220 UV-vis spectrophotometer	Thermo Fisher Scientific	912A0884
NanoDrop Onec microvolume UV-vis spectrophotometer	Thermo Fisher Scientific	ND-ONEC-W
Qubit 4 fluorometer	Thermo Fisher Scientific	Q33238
Eppendorf ThermoMixer F1.5	Eppendorf	EP5384000020

Materials and equipment

• •
  UHPLC gradient buffers.

  • ○
    Buffer A: 10 mM ammonium acetate (pH 4.5).
  • ○
    Buffer B: Methanol.
  • ○
    Store buffer A and B at 25°C. Maximum storage time: 48 months.

Needle wash solution

Reagent	Final concentration	Amount
Isopropanol	33%	350 mL
Methanol	33%	350 mL
LC-MS grade water	N/A	350 mL
Total	N/A	1050 mL

Store needle wash solution at 25°C. Maximum storage time: 48 months.

Step-by-step method details

Mass transition determination

Timing: 16 min per nucleoside standard

This step will determine the optimal mass transition in which to monitor detection of each nucleoside. Use the optimizer in the mass spectrometer software to automatically search for the collision energy that yields the most abundant product ion for each nucleoside.

• 1.Load the pre-made 100 μM nucleoside standards into the autosampler plates of the UHPLC-QqQ MS.

CRITICAL: Use solution containing a single standard, do not use standard mix.

• 2.Set the optimizer to positive ESI mode. Set the collision energy range between 2 to 60 V.

Note: Collision energy range varies across instruments, especially between different mass spectrometer manufacturers. The collision energy range provided here is specific to the UHPLC-QqQ MS (Agilent 6495C LC/TQ) used in this protocol.

• 3.Start program run. The instrument will scan for product ions of a given nucleoside precursor fragmented from the set collision energy range.
• 4.Record the collision energy that generates the highest product ion counts. This will be the optimal collision energy used for monitoring mass transition of individual nucleoside.
• 5.Record the mass transition at optimal collision energy.

Retention time determination

Timing: 15–30 min per nucleoside standard mix

Use the mass transitions obtained from the last step to search for the retention times of the nucleosides in Multiple Reaction Monitoring (MRM) mode.

• 6.Load the pre-made nucleoside standard mix into autosampler plate of UHPLC-QqQ MS.
• 7.
  Import MRM method parameters in the off-line method editor.

  • a.
    Set acquisition mode to MRM.
  • b.
    Adjust the gradient from 1% to 40% of buffer B in 26.5 min if conducting non-quantitative nucleoside identification; or 1% to 23% of buffer B in 7.5 min if conducting nucleoside quantification.
  • c.
    Import the mass transitions at optimal collision energy for nucleosides in the standard mix.
  • d.
    Save the MRM method.
• 8.Run the saved MRM method in the acquisition software.
• 9.Update the MRM method to Dynamic Multiple Reaction Monitoring (DMRM) mode using the acquired retention time for each nucleoside.

Note: In DMRM mode, the MS instrument only monitors the specific mass transitions at the specified retention time ranges instead of the entire chromatogram. This improves target sensitivity and peak shape.

• 10.Save the DMRM method.

Build calibration curves

Timing: 14 h

This step establishes a calibration curve for quantification of each nucleoside as part of the nucleoside standard mix.

• 11.Load the saved DMRM method with 7.5 min gradient in the acquisition software.
• 12.Load each of 16 concentration solutions of the nucleoside standard mix in incremental order into the autosampler plate of the UHPLC-QqQ MS.
• 13.Run at least six blanks prior to injecting the nucleoside standard mix solutions.
• 14.Run each nucleoside standard mix solution in triplicate from the lowest to the highest concentration.

CRITICAL: Injecting standards from lowest to highest concentrations minimizes issues with carryover from the previous injection.

RNA digestion to nucleosides

Timing: 12 h

This step uses the Nucleoside Digestion Mix (NEB, #M0649S) to achieve one-step complete digestion and dephosphorylation of the RNA samples.

• 15.Determine RNA concentration by Nanodrop or Qubit assay.
• 16.Set up the nucleoside digestion reaction as follows:

Nucleoside digestion setup

Reagent	Amount
RNA	up to 1 μg
Nucleoside Digestion Mix Reaction Buffer (10x)	2 μL
Nucleoside Digestion Mix	1 μL
Water	to 20 μL

• 17.Pipette up and down the reaction mixture at least 10 times.
• 18.Incubate the reaction at 37°C and shake at 300 rpm for 12 h in a thermomixer.
• 19.Transfer the reaction to an HPLC vial. The sample is ready for analysis without additional cleanup.

Pause point: Store the nucleoside digestions in −20°C freezer for no more than 6 months if not running the sample immediately.

Sample analysis

Timing: 15–30 min per injection

This step describes the parameter settings on UHPLC-QqQ MS while conducting both non-quantitative nucleoside identification and nucleoside quantification in digested RNA samples.

• 20.
  Non-quantitative nucleoside identification:

  • a.
    Load the 26.5 min gradient DMRM method in the acquisition software.
  • b.
    Load the digested RNA samples into the autosampler plate.
  • c.
    Inject greater than or equivalent to 100 ng of digested RNA.

Note: Increasing the injection volume up to the sample loop capacity (20 μL sample loop capacity in this protocol) can enhance the detection sensitivity for modified nucleosides present in relatively low abundance.

• 21.
  Nucleoside quantification:

  • a.
    Load the 7.5 min gradient DMRM method in the acquisition software.
  • b.
    Load the digested RNA samples into the autosampler plate.
  • c.
    Inject 1–5 ng of digested RNA for quantification. Ensure that the amount of each of the nucleosides to be quantified as well as the internal standard (e.g., one of the four canonical nucleosides) are within the range of the calibration curve.

Note: Inject the internal standard near its highest concentration point to maximize injection volume. Nucleoside abundance in RNA samples can be calculated by inputting the analyte mass response into the calibration curve equation. If the nucleoside abundance is outside of the upper or lower curve limit, one should adjust the sample injection volume to obtain an accurate quantification.

Expected outcomes

In this section, we describe how the combination of optimal mass transition and retention time of standards along with an accurate calibration curve can achieve maximal performance in nucleoside analysis.

Nucleoside precursor ions are prone to break at the N-glycosidic bond in the collision cell and yield nucleobase product ions (Figures 1A–1C). Therefore, the optimal m/z difference between nucleoside precursor and product ions is typically the mass of ribose. However, this difference varies depending on whether there is a modification at ribose 2′ position. For instance, the optimal precursor to product ion m/z difference is 132 for 5-methylcytidine (m⁵C), which does not have modifications at ribose 2′ position (Figure 1A); whereas such difference is 146 for 2′-O-methylcytidine (Cm) (Figure 1B). Nucleosides with hypermodified base could generate more than one primary product ions (Figure 1C). The precursor to product ion m/z difference is 132 for N6-isopentenyladenosine (i⁶A) at a lower collision energy, which is indicative of loss of ribose. While such m/z difference is 200 at a higher collision energy, suggesting simultaneous loss of ribose and the isopentenyl group (Figure 1C). We recommend monitoring all precursor to product ion transitions with mass responses of similar intensities. The most abundant product ions can be determined using the collision energy optimization program.

Figure 1.

Examples for optimal collision energy determination of nucleosides with simple base modification, ribose modification, and hyper base modification

(A) Predicted chemical structures of m⁵C precursor and product ions (258.1 -> 126.1) at optimal collision energy. The table displays the top four most abundant product ions m/z of m⁵C under three collision energies.

(B) Predicted chemical structures of Cm precursor and product ions (258.1 -> 112.1) at optimal collision energy. The table displays the top four most abundant product ions m/z of Cm under three collision energies.

(C) Predicted chemical structures of i⁶A precursor and product ions (336.1 -> 204.2; 336.1 -> 136.1) at optimal collision energies. The table displays the top four most abundant product ions m/z of i⁶A under four collision energies.

Using the non-quantitative nucleoside identification approach described here, we have established the mass transition and retention time of 76 standard nucleosides.¹ Each modified nucleoside can be distinguished by either its mass transition, retention time, or both (Figure 2A). Isomeric nucleosides can be effectively distinguished using this approach. For instance, N2,2-dimethylguanosine (m^2,2G) and N2,7-dimethylguanosine (m^2,7G) share the same 312.1 -> 180.1 mass transition. However, the two isomers have different retention times and can be distinguished thereby with high confidence (Figure 2B).

Figure 2.

Representative DMRM chromatograms of total RNA isolated from Thermococcus kodakarensis

(A) The graph shows the mass responses of the nucleosides detected in Thermococcus kodakarensis (T. kodakarensis) total RNA. Mass signals of the four canonical nucleosides are indicated.

(B) Representative DMRM chromatogram of m^2,2G and m^2,7G for the mass transition 312.1 -> 180.1 with the corresponding retention times.

For accurate nucleoside quantification, the linearity of the calibration curve is critical, and all concentration points should be within the linear range of mass response. A reliable calibration curve should contain $\ge$8 concentration points with a R² > 0.99 (Figure 3). To eliminate impacts of variation on the injection volume across samples, the quantification is performed by determining the relative abundance of the target nucleoside with the abundance of the internal standard, such as one of the four canonical nucleosides (e.g., m⁵U/U).

Figure 3.

Example of an optimal calibration curve for m⁵U

The graph shows a 5-methyluridine (m⁵U) calibration curve with 14 concentration points in the linear range and an R² > 0.99. Each concentration point contains the mass response values of m⁵U measured in three technical replicates from the corresponding nucleoside standard mix solutions.

Limitations

This protocol is designed for targeted modified nucleoside identification and quantification. To confirm the identity of modified nucleosides within RNA samples, standard nucleosides with pre-determined mass transitions and retention times are necessary. In addition, complete RNA digestion eliminates the sequence context of nucleosides in the transcriptome, so this approach is only suitable for global analysis of RNA modifications. Lastly, the availability of reliable extinction coefficients is crucial for preparing accurate nucleoside standard solutions for quantification experiments.

Troubleshooting

Problem 1

The software cannot automatically find the mass transition of a given standard nucleoside (related to Step 3).

Potential solution

• •Incorrect precursor ion mass input to the software could lead to wrong mass transition search. Re-calculate the monoisotopic mass of the precursor ion.
• •Confirm the presence of the nucleoside within the standard solution using a UV spectrometer. This troubleshooting step is to rule out the absence of the target nucleoside in the standard solution.
• •Increase the injection volume. The nucleoside standard solution could be too diluted. Measure the nucleoside concentration using a UV spectrometer to determine if the target nucleoside standard is within the range.
• •Manually search for product ions using the product ion mode, which scans for product ion masses of a given precursor at a specific collision energy. Select the product ion with a reasonable abundance and reliable mass loss (e.g., loss of a ribose). Manually find a collision energy that generates the highest mass response for the selected precursor-to-product ion mass transition using MRM mode. It is important to keep in mind that the sensitivity of MS detection is dependent on the nature of the nucleoside. A weak signal for certain nucleosides may be attributed to poor fragmentation.

Problem 2

Overlapping mass transition and retention time between isomeric nucleosides (related to Step 7).

Potential solution

• •We recommend switching to a different mobile phase, such as 0.1% formic acid. For example, N4-methylcytidine (m⁴C) and 5-methylcytidine (m⁵C) have the same primary mass transition (258.1 -> 126.1) and the same retention time in ammonium acetate-methanol mobile phase. However, the retention time of the two nucleosides can be effectively distinguishable in 0.1% formic acid gradient. For details, please see Fluke et al.³
• •Search for alternative product ions using the product ion mode. Unique product ions may be present for just one of the isomeric nucleosides and can be used for mass transition monitoring.

Problem 3

Calibration curve with low linearity (related to Step 14).

Potential solution

• •An accurate serial dilution of the nucleoside standard mix is critical to obtain a linear calibration curve. If in doubt, repeat the serial dilution and re-analyze the nucleoside standard mix.
• •Calibration curves not within the linear range of mass response may lead to low linearity. Consider discarding concentration points outside the linear range of mass response (Figure 4).
• •Carryover nucleosides could impact the calibration curve linearity, especially at the low concentration points. Run enough blanks to eliminate carryover prior to building the calibration curve.

Figure 4.

Example of a calibration curve with concentration points outside of the linear range

The two highest concentration data points in this calibration curve outside of the linear range (indicated by arrows). Since this calibration curve was designed to measure the concentration of relatively low abundant nucleosides, the two points outside of the linear range can be discarded. However, for highly abundant species, such as the four canonical nucleosides, we recommend redoing the calibration curve.

Problem 4

The retention time of the nucleoside in the RNA sample deviates from the retention time of the standard recorded in the DMRM method (related to Step 20 and 21).

Potential solution

• •The UHPLC column has reached its injection limit (i.e., column lifetime) and this can lead to retention time shifting and peak broadening. Change to a new column and determine the new retention time for each nucleoside.
• •Overloading the HPLC column can lead to retention time shifting. Reduce the injection amount to < 2 μg.
• •Run the standard of the nucleoside in question to confirm its retention time. If the standard runs at the expected retention time, the observed peak in the analyte may indicate the presence of a different nucleoside.

Problem 5

Inconsistent quantification of nucleosides for a given RNA sample (related to Step 21).

Potential solution

• •Incomplete RNA digestion can lead to inconsistent nucleoside quantification. RNA species containing complex secondary structures and/or highly modified backbones are particularly refractory to enzyme digestion. We recommend increasing the ratio of the digestion reagent to RNA substrate or increasing the incubation time.
• •Calibration curves lose accuracy over time. Moreover, calibration curves may be affected by MS instrument tuning and maintenance procedures. Re-run the nucleoside standard mix to build a new calibration curve periodically (every 4 weeks).
• •The nucleoside of interest may be chemically unstable. Shorten the RNA digestion reaction (for instance, to 1 h), protect from the light, and immediately analyze the RNA samples or preserve the sample after digestion at −20°C.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Ivan R. Corrêa, Jr. (correa@neb.com).

Technical contact

Questions on the technical specifics of performing the protocol should be directed to the technical contact, Yueh-Lin Tsai (atsai@neb.com).

Materials availability

This study did not generate new unique reagents.

Data and code availability

This study did not generate/analyze datasets.

Acknowledgments

We thank Donald Comb, Jim Ellard, Salvatore Russello, and Richard J. Roberts for the continuous support of research at New England Biolabs. This work was funded by New England Biolabs, Inc.

Author contributions

Y.-L.T. and I.R.C. conceptualized the study and wrote the manuscript. N.D. reviewed and edited the manuscript.

Declaration of interests

Y.-L.T., N.D., and I.R.C. are employees of New England Biolabs, Inc. New England Biolabs is a manufacturer and vendor of molecular biology reagents. The authors declare that this affiliation does not affect the authors’ impartiality, adherence to journal standards and policies, or the availability of data.