Integrated Solid-Phase Enzymatic Digestion and Porous Graphitic Carbon Chromatography for Rapid LC–MS Analysis of RNA Modifications

A research study recently introduced a streamlined, chromatography-compatible workflow for the rapid analysis of RNA modifications by integrating solid-phase enzymatic digestion with downstream liquid chromatography–mass spectrometry (LC–MS). RNases—including nuclease P1, phosphodiesterase I, and shrimp alkaline phosphatase—were immobilized on agarose beads via a bioorthogonal click chemistry approach, enabling efficient on-bead digestion of RNA into ribonucleosides within 30 minutes.

The system incorporates a microspin device packed with porous graphitic carbon (PGC), facilitating direct chromatographic separation and MS detection without the need for extensive sample preparation. The use of PGC provides enhanced retention and separation of polar ribonucleosides, improving analytical performance for modification profiling.

LCGC International spoke to Yixuan (Axe) Xie, an assistant professor at Fudan University (Shanghai, China) and one of the authors of a paper resulting from this study,¹ about the group’s findings.

What are the chromatographic retention mechanisms of porous graphitic carbon (PGC), and why is it particularly suitable for separating structurally similar ribonucleosides and isomeric RNA modifications?
Porous graphitic carbon (PGC) retains compounds through three main mechanisms. First, it shows dispersive interactions with analytes, so more hydrophobic molecules are usually retained longer. Second, because the graphite surface is flat, retention also depends on the size and shape of the molecule. Compounds that can make better contact with the flat surface are retained more strongly. Third, polar compounds can interact with the graphite surface through the polar retention effect on graphite (PREG). These properties make PGC very useful for separating ribonucleosides and isomeric RNA modifications. Even small structural differences, such as the position of a methyl group, can change how a molecule interacts with the carbon surface. As a result, PGC can separate closely related compounds that are difficult to resolve by other chromatography methods.

RNA modifications such as N1-methyladenosine (m1A) and N6-methyladenosine (m6A) are positional isomers. How would you optimize liquid chromatography (LC) conditions to resolve such isomers chromatographically before MS detection?
Fortunately, extensive LC optimization may not be necessary for m1A and m6A on a PGC column. Although they are positional isomers, the different position of the methyl group changes their molecular shape and their interaction with the flat graphite surface. Since PGC shows strong shape selectivity and is very sensitive to small structural differences, m1A and m6A can often be well resolved chromatographically and elute at different acetonitrile (CAN) percentages.

How does chemical derivatization (for example, permethylation) alter ribonucleoside polarity and chromatographic retention behavior, and what adjustments in mobile phase composition might be necessary?
Chemical derivatization generally converts polar functional groups into less polar ones. For example, in permethylation, hydroxyl and amino groups can be methylated, which reduces hydrogen-bonding capacity and increases the hydrophobicity of ribonucleosides. As a result, the derivatized compounds are more amenable to reversed-phase LC, for example on C18 columns, and usually show stronger retention than the underivatized forms. Because of this change in polarity, the LC conditions often need to be adjusted accordingly. First, a certain proportion of organic solvent is usually required to reconstitute the derivatized analytes before injection. Second, since the compounds become more hydrophobic, the separation is typically performed with a relatively higher organic content in the mobile phase, including a higher initial percentage of mobile phase B if needed. Overall, the ACN composition should be adjusted to ensure good solubility, appropriate retention, and efficient elution before MS detection.

When coupling LC to mass spectrometry (MS) for RNA modification analysis, what are the chromatographic trade-offs between nano-LC and microflow LC in terms of sensitivity, robustness, peak capacity, and reproducibility?
When coupling LC to MS for RNA modification analysis, nano-LC generally offers higher sensitivity because the lower flow rate improves electrospray ionization efficiency and is therefore better for detecting low-abundance modifications, and it can also provide high separation efficiency and peak capacity; however, it is usually less robust and more susceptible to clogging, unstable spray, and retention time variation, which can reduce reproducibility. In contrast, microflow LC usually has lower sensitivity than nano-LC, but it is more robust, easier to operate, and more reproducible, with more stable chromatography and ionization, making it more suitable for routine or quantitative workflows. Therefore, the main trade-off is that nano-LC favors maximum sensitivity and separation performance, whereas microflow LC favors robustness and reproducibility.

The study integrates immobilized RNase digestion with PGC-based cleanup in a single tube. From a chromatographic standpoint, how does minimizing sample transfers improve analyte recovery and chromatographic reproducibility?
Minimizing sample transfers reduces sample loss caused by adsorption to tube walls and pipette tips, incomplete transfer, evaporation, or unintended dilution, which is especially important for low-abundance RNA digestion products. It also decreases sample-to-sample variability introduced during handling, including differences in sample composition, injection solvent, volume, and residual salts or contaminants. As a result, analytes are introduced into the LC-MS system more consistently, leading to better recovery and more reproducible retention times, peak shapes, and signal intensities.

How does PGC-based solid-phase extraction reduce matrix effects prior to MS detection, and what chromatographic parameters would you monitor to ensure consistent cleanup performance?
PGC-based solid-phase extraction is used to reduce matrix effects by retaining the target digestion products while salts, buffers, enzymes, and other highly polar matrix components are removed during the washing step, thereby lowering chemical background and reducing ion suppression before MS detection. To ensure consistent cleanup performance, chromatographic parameters such as retention time, peak shape, peak width, signal intensity or peak area, and background noise should be monitored.

Compare the retention mechanisms of PGC with hydrophilic interaction liquid chromatography (HILIC) for highly polar ribonucleosides. Under what circumstances might HILIC outperform PGC?
PGC and HILIC show different selectivity for highly polar ribonucleosides. Notably t6A and related RNA modifications are retained too strongly on PGC, which can result in broad or poorly eluting peaks and make reliable identification and quantification difficult. By contrast, HILIC often provides better elution and separation for these modifications. In addition, because peak broadening is more pronounced on PGC for some analytes, HILIC may sometimes give higher MS response, especially for certain low-abundance ribonucleosides. Therefore, HILIC may outperform PGC when t6A-related modifications are analyzed or when improved peak shape and more practical quantitative performance are required.

Immobilized enzymes (NP1, PDE I, SAP) were functionalized via inverse electron-demand Diels–Alder chemistry. How does immobilization improve chromatographic compatibility compared to solution-phase digestion?
Immobilization improves chromatographic compatibility by keeping the digestion enzymes on a solid support, so they can be removed by filtration before LC-MS analysis. This also makes the digestion more controllable, since the reaction can be terminated at any time by removing the immobilized nucleases, and it may enhance digestion efficiency because of the higher local enzyme concentration on the support. Compared with solution-phase digestion, fewer soluble proteins and enzyme-derived contaminants are introduced into the sample, leading to lower matrix complexity, cleaner chromatography, and better MS compatibility.

In isotope-dilution LC–MS workflows (which use labeled internal standards), what chromatographic factors are critical to ensure accurate quantification of low-abundance RNA modifications?
In isotope-dilution LC–MS workflows, accurate quantification of low-abundance RNA modifications depends heavily on chromatographic performance. The analyte and the labeled internal standard should co-elute as closely as possible, so that they are affected similarly by matrix effects and ionization variability. In addition, retention times need to be stable and reproducible to ensure reliable peak assignment. For low-abundance modifications, sufficient separation from isomeric species, isobaric interferences, and highly abundant neighboring nucleosides is also critical, because even minor co-elution can cause substantial quantitative bias. Good peak shape is equally important, since broad or tailing peaks reduce signal-to-noise ratio and make peak integration less reliable. Therefore, even when isotope-labeled internal standards are used, clean separation, reproducible retention, and good peak shape remain essential for accurate quantification.

During TGF-β–induced epithelial–mesenchymal transition (EMT) studies, RNA modification levels change dynamically. How would you validate chromatographic robustness (retention time stability, resolution, carryover) for longitudinal biological experiments?
For longitudinal experiments, chromatographic robustness should be validated through a system suitability and QC strategy applied across the entire analytical sequence. Retention time stability should be assessed by repeated injections of standard mixtures and pooled QC samples, with predefined acceptance windows established for each nucleoside and RNA modification. Resolution should be monitored for critical peak pairs, especially isomeric modifications and low-abundance analytes eluting near highly abundant canonical nucleosides, to ensure that sufficient separation is maintained over time. Carryover should be evaluated by inserting solvent blanks after high-concentration standards or high-signal samples, and residual signals should remain below a predefined threshold. In addition, sample order should be randomized, QC samples should be injected periodically throughout the run, and internal standards should be used to distinguish chromatographic drift from true biological variation. Overall, chromatographic robustness should be demonstrated by stable retention times, consistent resolution and peak shape, and negligible carryover throughout the full longitudinal experiment.

References

1. Li, Z.; Yu, L.; Liu, X. et al. Development of an Immobilized System for RNA Modification Analysis. Mol Omics 2026, 22 (1), aaiaf005. DOI: 10.1093/momics/aaiaf005