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The Pytheas Software Suite

Tools for Identifying RNA Modifications from MS2 Fragmentation Spectra

The Pytheas Software Suite

At Cassia, LLC, we continue to not only empower scientists with new cutting‑edge reagents, but also software tools to explore one of the most dynamic and influential areas of RNA research.  The Pytheas2 software uses state‑of‑the‑art LC‑MS/MS data to accurately detect and identify nucleoside modifications in mRNA, rRNA, tRNA, and custom RNA samples.  Our system is adaptive and can match every MS2 spectrum against the fragments your RNA of interest can produce, scoring each match, and tests them against decoy sequences, so every result comes with a false discovery rate.  It is the engine behind Cassia’s RNA Mapping Service, offering comprehensive qualitative and quantitative insights into RNA modification landscapes. Pytheas2 represents a comprehensive redesign of Pytheas1, an open-source software package originally published in 2022 (D’Ascenzo et al., Nature Communications).  Rebuilt from the ground up, this latest iteration introduces a modern interface, expanded functionality, and performance enhancements that increase processing speeds by approximately 200-fold.

Three Ways to Use Pytheas

Pytheas1 Pytheas2-Lite Pytheas2 (Through Cassia)
What is it? Original Open-Source Pytheas
Refreshed Desktop GUI in 2026
 Pytheas2 Rebuilt from Pytheas1
With New Web Browser GUI
 Full Pytheas2 Engine
Tech Support and/or Service
Who it is for? Anyone Academic Labs Companies and Academic Projects
Needing Full Features & Services
What it Runs? Digest & Match
On Your Computer
 Digest & Match
Through Your Browser
 Digest & Match; Quantitation via Isotope
Labels; Tolerance Tuning from Your Data;
Discovery of Unexpected Modifications
Cost? Free (MIT License) Free for Academic Research Collaboration or Fee-For-Service
How to Start? Coming Soon on GitHub Request an Account
Sign In Here
 Tell Us About Your Project

What Pytheas2 Does

  • Digest: Builds every fragment your nuclease can make from your sequences, with known modifications, partial modification, end chemistries and heavy-isotope labels.
  • Match: Scores every candidate for every spectrum and keeps close calls visible, such as a modification that could sit on either of two neighbouring nucleotides.
  • False Discovery Rate: Every target sequence has a decoy of the same composition and mass, and each run reports how many identifications pass at each FDR.
  • Results You Can Check: Sortable tables of every candidate, annotated spectra, sequence maps of each RNA, and exports in the standard idXML and mzTab-NA formats.
  • Modification Library: each residue is cross-referenced to the MODOMICS database.

What Data Inputs You Need

  • RNA Sequence: Your RNA sequence in FASTA file format.
  • MS2 Spectra: Your MS2 spectra should be in .mgf file format. Pytheas2 contains an MS Data Prep guide showing how to convert instrument files using ProteoWizard MSConvert.
  • Modifications: Either the specific modification you expect, or use the built-in library.

Getting Started

  • Academic Labs: Request a free Pytheas2-Lite account. We routinely approve requests within two (2) business days and email your sign-in details. Please use your institutional email address.
  • ASMS Workshop Participants: Use the workshop account given out in the session.
  • Companies & Special Projects Needing the Full Pytheas2 Engine: Tell us about your project. We’ll reply to discuss a collaboration or fee-for-service analysis. Please use your company or institutional email address.
  • Have an Account?: Sign In Here
  • Questions: rna_ms@cassiallc.com

How to Cite Pytheas

  • D’Ascenzo L, Popova AM, Abernathy S, Sheng K, Limbach PA, Williamson JR. (2022) Pytheas: A Software Package for the Automated Analysis of RNA Sequences and Modifications via Tandem Mass Spectrometry. Nature Communications 13, 2424-2436. (https://doi.org/10.1038/s41467-022-30057-5)

Abstract: Ribosomal RNA modifications in prokaryotes have been sporadically studied, but there is a lack of a comprehensive picture of modification sites across bacterial phylogeny. Bacillus subtilis is a preeminent model organism for gram-positive bacteria, with a well-annotated and editable genome, convenient for fundamental studies and industrial use. Yet remarkably, there has been no complete characterization of its rRNA modification inventory. By expanding modern MS tools for the discovery of RNA modifications, we found a total of 25 modification sites in 16S and 23S rRNA of B. subtilis, including the chemical identity of the modified nucleosides and their precise sequence location. Furthermore, by perturbing large subunit biogenesis using depletion of an essential factor rbgA and measuring the completion of 23S modifications in the accumulated intermediate, we provide a first look at the order of modification steps during the late stages of assembly in B. subtilis. While our work expands the knowledge of bacterial rRNA modification patterns, adding B. subtilis to the list of fully annotated species after Escherichia coli and Thermus thermophilus, in a broader context, it provides the experimental framework for discovery and functional profiling of rRNA modifications to ultimately elucidate their role in ribosome biogenesis and translation.

[https://doi.org/10.1093/nar/gkae626]

Abstract: PubChem serves as a comprehensive repository, housing over 100 million unique chemical structures representing the breadth of our chemical knowledge across numerous fields including metabolism, pharmaceuticals, toxicology, cosmetics, agriculture, and many more. Rapid identification of these small molecules increasingly relies on electrospray ionization (ESI) paired with tandem mass spectrometry (MS/MS), particularly by comparison to genuine standard MS/MS data sets. Despite its widespread application, achieving consistency in MS/MS data across various analytical platforms remains an unaddressed concern. This study evaluated MS/MS data derived from one hundred molecular standards utilizing instruments from five manufacturers, inclusive of quadrupole time-of-flight (QTOF) and quadrupole orbitrap “exactive” (QE) mass spectrometers by Agilent (QTOF), Bruker (QTOF), SCIEX (QTOF), Waters (QTOF), and Thermo QE. We assessed fragment ion variations at multiple collisional energies (0, 10, 20, and 40 eV) using the cosine scoring algorithm for comparisons and the number of fragments observed. A parallel visual analysis of the MS/MS spectra across instruments was conducted, consistent with a standard procedure that is used to circumvent the still prevalent issue of mischaracterizations as shown for dimethyl sphingosine and C20 sphingosine. Our analysis revealed a notable consistency in MS/MS data and identifications, with fragment ions’ m/z values exhibiting the highest concordance between instrument platforms at 20 eV, the other collisional energies (0, 10, and 40 eV) were significantly lower. While moving toward a standardized ESI MS/MS protocol is required for dependable molecular characterization, our results also underscore the continued importance of corroborating MS/MS data against standards to ensure accurate identifications. Our findings suggest that ESI MS/MS manufacturers, akin to the established norms for gas chromatography mass spectrometry instruments, should standardize the collision energy at 20 eV across different instrument platforms.

[https://doi.org/10.1021/acs.analchem.3c05576]

Abstract: Mass spectrometry is an important method for analysis of modified nucleosides ubiquitously present in cellular RNAs, in particular for ribosomal and transfer RNAs that play crucial roles in mRNA translation and decoding. Furthermore, modifications have effect on the lifetimes of nucleic acids in plasma and cells and are consequently incorporated into RNA therapeutics. To provide an analytical tool for sequence characterization of modified RNAs, we developed Pytheas, an open-source software package for automated analysis of tandem MS data for RNA. The main features of Pytheas are flexible handling of isotope labeling and RNA modifications, with false discovery rate statistical validation based on sequence decoys. We demonstrate bottom-up mass spectrometry characterization of diverse RNA sequences, with broad applications in the biology of stable RNAs, and quality control of RNA therapeutics and mRNA vaccines.

[https://doi.org/10.1038/s41467-022-30057-5]

Abstract: Post-transcriptional RNA modifications that are introduced during the multistep ribosome biogenesis process are essential for protein synthesis. The current lack of a comprehensive method for a fast quantitative analysis of rRNA modifications significantly limits our understanding of how individual modification steps are coordinated during biogenesis inside the cell. Here, an LC-MS approach has been developed and successfully applied for quantitative monitoring of 29 out of 36 modified residues in the 16S and 23S rRNA from Escherichia coli. An isotope labeling strategy is described for efficient identification of ribose and base methylations, and a novel metabolic labeling approach is presented to allow identification of MS-silent pseudouridine modifications. The method was used to measure relative abundances of modified residues in incomplete ribosomal subunits compared to a mature (15)N-labeled rRNA standard, and a number of modifications in both 16S and 23S rRNA were present in substoichiometric amounts in the preribosomal particles. The RNA modification levels correlate well with previously obtained profiles for the ribosomal proteins, suggesting that RNA is modified in a schedule comparable to the association of the ribosomal proteins. Importantly, this study establishes an efficient workflow for a global monitoring of ribosomal modifications that will contribute to a better understanding of mechanisms of RNA modifications and their impact on intracellular processes in the future.

[https://doi.org/10.1021/ja412084b]

Abstract: The ribosome is an essential and highly complex biological system in all living cells. A large body of literature on the assembly of the ribosome in vitro is available, but a clear picture of this process inside the cell has yet to emerge. Here, we directly characterized in vivo ribosome assembly intermediates and associated assembly factors from wild-type Escherichia coli cells using a general quantitative mass spectrometry (qMS) approach. The presence of distinct populations of ribosome assembly intermediates was verified using an in vivo stable isotope pulse-labeling approach, and their exact ribosomal protein contents were characterized against an isotopically labeled standard. The model-free clustering analysis of the resultant protein levels for the different ribosomal particles produced four 30S assembly groups that correlate very well with previous in vitro assembly studies of the small ribosomal subunit and six 50S assembly groups that clearly define an in vivo assembly landscape for the larger ribosomal subunit. In addition, de novo proteomics identified a total of 21 known and potentially new ribosome assembly factors co-localized with various ribosomal particles. These results represent new in vivo assembly maps of the E. coli 30S and 50S subunits, and the general qMS approach should prove to be a solid platform for future studies of ribosome biogenesis across a host of model organisms.

[https://doi.org/10.1016/j.jmb.2012.11.040]

Abstract: The ribosome is an essential organelle responsible for cellular protein synthesis. Until recently, the study of ribosome assembly has been largely limited to in vitro assays, with few attempts to reconcile these results with the more complex in vivo ribosome biogenesis process. Here, we characterize the ribosome synthesis and assembly pathway for each E. coli ribosomal protein (r-protein) in vivo using a stable isotope pulse-labeling timecourse. Isotope incorporation into assembled ribosomes was measured by quantitative mass spectrometry (qMS) and fit using steady-state flux models. Most r-proteins exhibit precursor pools ranging in size from 0% to 7% of completed ribosomes, and that the sizes of these individual r-protein pools correlate well with the order of r-protein binding in vitro. Additionally, we observe anomalously large precursor pools for specific r-proteins with known extra-ribosomal functions and we have detected three r-proteins with significant turnover during steady-state growth. Taken together, this highly precise, time-dependent proteomic qMS approach should prove useful in future studies of ribosome biogenesis and could be easily extended to explore other complex biological processes in a cellular context.

[https://doi.org/10.1039/c2mb25310k]

Abstract: Complete characterization of a biomolecule’s chemical structure is crucial in the full understanding of the relations between their structure and function. The dominating components in ribosomes are ribosomal RNAs (rRNAs), and the entire rRNA – but a single modified nucleoside at position 2501 in 23S rRNA – has previously been characterized in the bacterium Escherichia coli. Despite a first report nearly 20 years ago, the chemical nature of the modification at position 2501 has remained elusive, and attempts to isolate it have so far been unsuccessful. We unambiguously identify this last unknown modification as 5-hydroxycytidine – a novel modification in RNA. Identification of 5-hydroxycytidine was completed by liquid chromatography under nonoxidizing conditions using a graphitized carbon stationary phase in combination with ion trap tandem mass spectrometry and by comparing the fragmentation behavior of the natural nucleoside with that of a chemically synthesized ditto. Furthermore, we show that 5-hydroxycytidine is also present in the equivalent position of 23S rRNA from the bacterium Deinococcus radiodurans. Given the unstable nature of 5-hydroxycytidine, this modification might be found in other RNAs when applying the proper analytical conditions as described here.

[https://doi.org/10.1016/j.jmb.2011.06.036]

Abstract: LC/MS analysis of ribonucleosides is traditionally performed by reverse phase chromatography on silica based C18 type stationary phases using MS compatible buffers and methanol or acetonitrile gradients. Due to the hydrophilic and polar nature of nucleosides, down-scaling C18 analytical methods to a two-column nano-flow setup is inherently difficult. We present a nano-chip LC/MS ion-trap strategy for routine characterization of RNA nucleosides in the fmol range. Nucleosides were analyzed in positive ion mode by reverse phase chromatography using a 75 μ × 150 mm, 5 μ particle porous graphitic carbon (PGC) chip with an integrated 9 mm, 160 nL trapping column. Nucleosides were separated using a formic acid/acetonitrile gradient. The method was able to separate isobaric nucleosides as well as nucleosides with isotopic overlap to allow unambiguous MSn identification on a low resolution ion-trap. Synthesis of 5-hydroxycytidine (oh5C) was achieved from 5-hydroxyuracil in a novel three-step enzymatic process. When operated in its native state using formic acid/acetonitrile, PGC oxidized oh5C to its corresponding glycols and formic acid conjugates. Reduction of the PGC stationary phase was achieved by flushing the chip with 2.5 mM oxalic acid and adding 1 mM oxalic acid to the online solvents. Analyzed under reduced chromatographic conditions oh5C was readily identified by its MH+ m/z 260 and MSn fragmentation pattern. This investigation is, to our knowledge, the first instance where oxalic acid has been used as an online reducing agent for LC/MS. The method was subsequently used for complete characterization of nucleosides found in tRNAs using both PGC and C18 chips.

[https://doi.org/10.1007/s13361-011-0126-8]

Abstract: Stable isotope mass spectrometry has become a widespread tool in quantitative biology. Pulse-chase monitored by quantitative mass spectrometry (PC/QMS) is a recently developed stable isotope approach that provides a powerful means of studying the in vitro self-assembly kinetics of macromolecular complexes. This method has been applied to the Escherichia coli 30S ribosomal subunit, but could be applied to any stable self-assembling complex that can be reconstituted from its component parts and purified from a mixture of components and complex. The binding rates of 18 out of the 20 ribosomal proteins have been measured at several temperatures using PC/QMS. Here, PC/QMS experiments on 30S ribosomal subunit assembly are described, and the potential application of the method to other complexes is discussed. A variation on the PC/QMS experiment is introduced that enables measurement of kinetic cooperativity between proteins. In addition, several related approaches to stable isotope labeling and quantitative mass spectrometry data analysis are compared and contrasted.

[https://doi.org/10.1016/j.ymeth.2009.06.002]

Abstract: Quantitative proteomic mass spectrometry involves comparison of the amplitudes of peaks resulting from different isotope labeling patterns, including fractional atomic labeling and fractional residue labeling. We have developed a general and flexible analytical treatment of the complex isotope distributions that arise in these experiments, using Fourier transform convolution to calculate labeled isotope distributions and least-squares for quantitative comparison with experimental peaks. The degree of fractional atomic and fractional residue labeling can be determined from experimental peaks at the same time as the integrated intensity of all of the isotopomers in the isotope distribution. The approach is illustrated using data with fractional (15)N-labeling and fractional (13)C-isoleucine labeling. The least-squares Fourier transform convolution approach can be applied to many types of quantitative proteomic data, including data from stable isotope labeling by amino acids in cell culture and pulse labeling experiments.

[https://doi.org/10.1021/ac800080v]

Abstract: An important aspect of proteomic mass spectrometry involves quantifying and interpreting the isotope distributions arising from mixtures of macromolecules with different isotope labeling patterns. These patterns can be quite complex, in particular with in vivo metabolic labeling experiments producing fractional atomic labeling or fractional residue labeling of peptides or other macromolecules. In general, it can be difficult to distinguish the contributions of species with different labeling patterns to an experimental spectrum and difficult to calculate a theoretical isotope distribution to fit such data. There is a need for interactive and user-friendly software that can calculate and fit the entire isotope distribution of a complex mixture while comparing these calculations with experimental data and extracting the contributions from the differently labeled species. Envelope has been developed to be user-friendly while still being as flexible and powerful as possible. Envelope can simultaneously calculate the isotope distributions for any number of different labeling patterns for a given peptide or oligonucleotide, while automatically summing these into a single overall isotope distribution. Envelope can handle fractional or complete atom or residue-based labeling, and the contribution from each different user-defined labeling pattern is clearly illustrated in the interactive display and is individually adjustable. At present, Envelope supports labeling with 2H, 13C, and 15N, and supports adjustments for baseline correction, an instrument accuracy offset in the m/z domain, and peak width. Furthermore, Envelope can display experimental data superimposed on calculated isotope distributions, and calculate a least-squares goodness of fit between the two. All of this information is displayed on the screen in a single graphical user interface. Envelope supports high-quality output of experimental and calculated distributions in PNG or PDF format. Beyond simply comparing calculated distributions to experimental data, Envelope is useful for planning or designing metabolic labeling experiments, by visualizing hypothetical isotope distributions in order to evaluate the feasibility of a labeling strategy. Envelope is also useful as a teaching tool, with its real-time display capabilities providing a straightforward way to illustrate the key variable factors that contribute to an observed isotope distribution. Envelope is a powerful tool for the interactive calculation and visualization of complex isotope distributions for comparison to experimental data.

[https://doi.org/10.1186/1471-2105-9-446]

Abstract: The apparent mass resolution of oligonucleotides in time-of-flight (TOF) mass spectrometers has been examined. In a reflectron TOF instrument, where the isotopic profile can be completely resolved, the apparent resolution matches the instrument’s resolving power. In a linear TOF instrument, unresolved isotopic profiles limit the apparent resolution to much lower values than the actual instrument resolution. By using 12C/14N-enriched oligonucleotides, the apparent resolution can be improved significantly. The isotope enrichment method also enhances the signal-to-noise ratio.

[https://doi.org/10.1021/ac010804c]

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