NAIL-MS

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NAIL-MS (short for nucleic acid isotope labeling coupled mass spectrometry) is a technique based on mass spectrometry used for the investigation of nucleic acids and its modifications. It enables a variety of experiment designs to study the underlying mechanism of RNA biology in vivo . For example, the dynamic behaviour of nucleic acids in living cells, especially of RNA modifications, can be followed in more detail. [1]

Contents

Theory

Labeling of cytidine. Left: unlabeled cytidine, right: ribose-labeled cytidine (red dots = C). Labeling of cells.png
Labeling of cytidine. Left: unlabeled cytidine, right: ribose-labeled cytidine (red dots = C).

NAIL-MS is used to study RNA modification mechanisms. Therefore, cells in culture are first fed with stable isotope labeled nutrients and the cells incorporate these into their biomolecules. After purification of the nucleic acids, most often RNA, analysis is done by mass spectrometry. Mass spectrometry is an analytical technique that measures the mass-to-charge ratio of ions. Pairs of chemically identical nucleosides of different stable-isotope composition can be differentiated in a mass spectrometer due to their mass difference. Unlabeled nucleosides can therefore be distinguished from their stable isotope labeled isotopologues. For most NAIL-MS approaches it is crucial that the labeled nucleosides are more than 2 Da heavier than the unlabeled ones. This is because 1.1% of naturally occurring carbon atoms are 13C isotopes. In the case of nucleosides this leads to a mass increase of 1 Da in ~10% of the nucleosides. This signal would disturb the final evaluation of the measurement.

NAIL-MS can be used to investigate RNA modification dynamics by changing the labeled nutrients of the corresponding growth medium during the experiment. Furthermore, cell populations can be compared directly with each other without effects of purification bias. Furthermore, it can be used for the production of biosynthetic isotopologues of most nucleosides which are needed for quantification by mass spectrometry and even for the discovery of yet unknown RNA modifications. [2] [3] [4]

General procedure

General workflow of NAIL-MS assays. Cells are cultivated in the appropriately labeled medium before harvesting and RNA isolation. Further RNA purification is followed by digestion to nucleosides and subsequent triple quadrupole mass spectrometry. General workflow.jpg
General workflow of NAIL-MS assays. Cells are cultivated in the appropriately labeled medium before harvesting and RNA isolation. Further RNA purification is followed by digestion to nucleosides and subsequent triple quadrupole mass spectrometry.

In general, cells are cultivated in unlabeled or stable (non-radioactive) isotope labeled media. For example, the medium can contain glucose labeled with six carbon-13 atoms (13C) instead of the normal carbon-12 (12C). Cells growing in this medium, will, depending on model organism, incorporate the heavy glucose into all of their RNA molecules. Thereafter, all nucleotides are 5 Da heavier than their unlabeled isotopologues due to a complete carbon labeling of the ribose. After cultivation and appropriate labeling of the cells, they are generally harvested using phenol/chloroform/guanidinium isothiocyanate. Other extraction methods are possible and sometimes needed (e.g. for yeast). RNA is then isolated by Phenol-Chloroform extraction and iso-Propanol precipitation. Further purification of specific RNA species (e.g. rRNA, tRNA) is usually done by size-exclusion chromatography (SEC) but other approaches are available as well. For most applications the final product needs to be enzymatically digested to nucleosides before analysis by LC-MS. Therefore, digestion enzymes such as benzonase, NP1 and CIP are used. [5] [6] Typically, a triple quadrupole in MRM mode is used for the measurements.

Labeling of cells

How the labeling of RNA molecules is achieved depends on the model organism. For E.coli (bacteria) the minimum medium M9 can be used and supplemented with the stable isotope labeled variants of the needed salts. This enables labeling with 13C-carbon, 15N-nitrogen, 34S-sulfur and 2H-hydrogen. [7] In S.cerevisiae (yeast) there are currently two possibilities: First, the use of commercially available complete growth medium, which enables labeling with 13C-carbon and/or 15N-nitrogen and second the use of minimal YNB medium which has to be supplemented with several amino acids and glucose which can be added as stable isotope labeled variants in order to achieve 13C-carbon, 15N-nitrogen and 2H-hydrogen labeling of RNA. [8]

While labeling in model organisms like E.coli and S.cerevisiae is fairly simple, stable isotope labeling in cell culture is much more challenging as the composition of the growth media is much more complex. Neither the supplementation of stable isotope labeled glucose nor the supplementation of stable isotope labeled variants of simple precursors of nucleoside biosynthesis such as glutamine and/or aspartate is sufficient for a defined mass increase higher than 2 Da. Instead, most cells kept in cell culture can be fed with stable isotope labeled methionine for labeling of methyl groups and with stable isotope labeled variants of adenin and uridine for labeling of the nucleoside's base body. [9] Special care must be taken when supplementing the medium with FBS (fetal bovine serum), as it also contains small metabolites used for the biosynthesis of nucleosides. The use of dialyzed FBS is therefore advisable when defined labeling of all nucleosides is desired.

Applications

With NAIL-MS different experiment designs are possible.

Production of SILIS

NAIL-MS can be used to produce stable isotope labeled internal standards (ISTD). Therefore, cells are grown in medium which results in complete labeling of all nucleosides. The purified mix of nucleosides can then be used as ISTD which is needed for accurate absolute quantification of nucleosides by mass spectrometry. This mixture of labeled nucleosides is also referred to as SILIS (stable isotope labeled internal standard). [10] The advantage of this approach is, that all modifications present in an organism can thereby be biosynthesized as labeled compounds. The production of SILIS was already done before the term NAIL-MS emerged.

Comparative Experiments

A comparative NAIL-MS experiment is quite similar to a SILAC experiment but for RNA instead of proteins. First, two populations of the respective cells are cultivated. One of the cell populations is fed with growth medium containing unlabeled nutrients, whereas the second population is fed with growth medium containing stable isotope labeled nutrients. The cells then incorporate the respective isotopologues into their RNA molecules. One of the cell populations serves as a control group whereas the other is subject to the associated research (e.g. KO strain, stress). Upon harvesting of the two cell populations they are mixed and co-processed together to exclude purification-bias. Due to the distinct masses of incorporated nutrients into the nucleosides a differentiation of the two cell populations is possible by mass spectrometry.

Pulse-Chase Experiments

Upon initiation of a pulse-chase experiment the medium is switched from medium(1) to medium(2). The two media must only differ in their isotope content. Thereby it is possible to distinguish between RNA molecules already existent before experiment initiation (= RNA molecules grown in medium(1)) and RNA molecules that are newly transcribed after experiment initiation (= RNA molecules grown in medium(2)). This allows the detailed study of modification dynamics in vivo. The supplementation of labeled methionine in either medium(1) or medium(2) allows the tracing of methylation processes. Other isotopically labeled metabolites potentially allow for further modification analysis.

Altogether NAIL-MS enables the investigation of RNA modification dynamics by mass spectrometry. With this technique, enzymatic demethylation has been observed for several RNA damages inside living bacteria. [4] [7]

Discovery of new RNA modifications

For the discovery of uncharacterized modifications cells are grown in unlabeled or13C‑labeled or15N‑labeled or2H‑labeled or34S‑labeled medium. Unknown signals occurring during mass spectrometry are then inspected in all differentially labeled cultures. If retention times of unknown compounds with appropriately divergent m/z values overlap, a sum formula of the compound can be postulated by calculating the mass differences of the overlapping signal in the differentially labeled cultures. With this method several new RNA modifications could be discovered. This experimental design also was the initial idea that started the concept of NAIL-MS.

Oligonucleotide NAIL-MS

NAIL-MS can also be applied to oligonucleotide analysis by mass spectrometry. This is useful when the sequence information is to be retained. [11]

Related Research Articles

Oligonucleotides are short DNA or RNA molecules, oligomers, that have a wide range of applications in genetic testing, research, and forensics. Commonly made in the laboratory by solid-phase chemical synthesis, these small fragments of nucleic acids can be manufactured as single-stranded molecules with any user-specified sequence, and so are vital for artificial gene synthesis, polymerase chain reaction (PCR), DNA sequencing, molecular cloning and as molecular probes. In nature, oligonucleotides are usually found as small RNA molecules that function in the regulation of gene expression, or are degradation intermediates derived from the breakdown of larger nucleic acid molecules.

<span class="mw-page-title-main">Fluorescent tag</span>

In molecular biology and biotechnology, a fluorescent tag, also known as a fluorescent label or fluorescent probe, is a molecule that is attached chemically to aid in the detection of a biomolecule such as a protein, antibody, or amino acid. Generally, fluorescent tagging, or labeling, uses a reactive derivative of a fluorescent molecule known as a fluorophore. The fluorophore selectively binds to a specific region or functional group on the target molecule and can be attached chemically or biologically. Various labeling techniques such as enzymatic labeling, protein labeling, and genetic labeling are widely utilized. Ethidium bromide, fluorescein and green fluorescent protein are common tags. The most commonly labelled molecules are antibodies, proteins, amino acids and peptides which are then used as specific probes for detection of a particular target.

Carbon-13 (13C) is a natural, stable isotope of carbon with a nucleus containing six protons and seven neutrons. As one of the environmental isotopes, it makes up about 1.1% of all natural carbon on Earth.

Isotopic labeling is a technique used to track the passage of an isotope through chemical reaction, metabolic pathway, or a biological cell. The reactant is 'labeled' by replacing one or more specific atoms with their isotopes. The reactant is then allowed to undergo the reaction. The position of the isotopes in the products is measured to determine the sequence the isotopic atom followed in the reaction or the cell's metabolic pathway. The nuclides used in isotopic labeling may be stable nuclides or radionuclides. In the latter case, the labeling is called radiolabeling.

In chemistry, isotopologues are molecules that differ only in their isotopic composition. They have the same chemical formula and bonding arrangement of atoms, but at least one atom has a different number of neutrons than the parent.

Isotopomers or isotopic isomers are isomers which differ by isotopic substitution, and which have the same number of atoms of each isotope but in a different arrangement. For example, CH3OD and CH2DOH are two isotopomers of monodeuterated methanol.

<span class="mw-page-title-main">RNA editing</span> Molecular process

RNA editing is a molecular process through which some cells can make discrete changes to specific nucleotide sequences within an RNA molecule after it has been generated by RNA polymerase. It occurs in all living organisms and is one of the most evolutionarily conserved properties of RNAs. RNA editing may include the insertion, deletion, and base substitution of nucleotides within the RNA molecule. RNA editing is relatively rare, with common forms of RNA processing not usually considered as editing. It can affect the activity, localization as well as stability of RNAs, and has been linked with human diseases.

An isotopic signature is a ratio of non-radiogenic 'stable isotopes', stable radiogenic isotopes, or unstable radioactive isotopes of particular elements in an investigated material. The ratios of isotopes in a sample material are measured by isotope-ratio mass spectrometry against an isotopic reference material. This process is called isotope analysis.

<span class="mw-page-title-main">Doebner–Miller reaction</span>

The Doebner–Miller reaction is the organic reaction of an aniline with α,β-unsaturated carbonyl compounds to form quinolines.

<span class="mw-page-title-main">Stable isotope labeling by amino acids in cell culture</span>

Stable isotope labeling by/with amino acids in cell culture (SILAC) is a technique based on mass spectrometry that detects differences in protein abundance among samples using non-radioactive isotopic labeling. It is a popular method for quantitative proteomics.

A tandem mass tag (TMT) is a chemical label that facilitates sample multiplexing in mass spectrometry (MS)-based quantification and identification of biological macromolecules such as proteins, peptides and nucleic acids. TMT belongs to a family of reagents referred to as isobaric mass tags which are a set of molecules with the same mass, but yield reporter ions of differing mass after fragmentation. The relative ratio of the measured reporter ions represents the relative abundance of the tagged molecule, although ion suppression has a detrimental effect on accuracy. Despite these complications, TMT-based proteomics has been shown to afford higher precision than Label-free quantification. In addition to aiding in protein quantification, TMT tags can also increase the detection sensitivity of certain highly hydrophilic analytes, such as phosphopeptides, in RPLC-MS analyses.

<span class="mw-page-title-main">Protein mass spectrometry</span> Application of mass spectrometry

Protein mass spectrometry refers to the application of mass spectrometry to the study of proteins. Mass spectrometry is an important method for the accurate mass determination and characterization of proteins, and a variety of methods and instrumentations have been developed for its many uses. Its applications include the identification of proteins and their post-translational modifications, the elucidation of protein complexes, their subunits and functional interactions, as well as the global measurement of proteins in proteomics. It can also be used to localize proteins to the various organelles, and determine the interactions between different proteins as well as with membrane lipids.

<span class="mw-page-title-main">Quantitative proteomics</span> Analytical chemistry technique

Quantitative proteomics is an analytical chemistry technique for determining the amount of proteins in a sample. The methods for protein identification are identical to those used in general proteomics, but include quantification as an additional dimension. Rather than just providing lists of proteins identified in a certain sample, quantitative proteomics yields information about the physiological differences between two biological samples. For example, this approach can be used to compare samples from healthy and diseased patients. Quantitative proteomics is mainly performed by two-dimensional gel electrophoresis (2-DE), preparative native PAGE, or mass spectrometry (MS). However, a recent developed method of quantitative dot blot (QDB) analysis is able to measure both the absolute and relative quantity of an individual proteins in the sample in high throughput format, thus open a new direction for proteomic research. In contrast to 2-DE, which requires MS for the downstream protein identification, MS technology can identify and quantify the changes.

<span class="mw-page-title-main">Selected reaction monitoring</span> Tandem mass spectrometry method

Selected reaction monitoring (SRM), also called multiple reaction monitoring (MRM), is a method used in tandem mass spectrometry in which an ion of a particular mass is selected in the first stage of a tandem mass spectrometer and an ion product of a fragmentation reaction of the precursor ions is selected in the second mass spectrometer stage for detection.

Nucleic acid NMR is the use of nuclear magnetic resonance spectroscopy to obtain information about the structure and dynamics of nucleic acid molecules, such as DNA or RNA. It is useful for molecules of up to 100 nucleotides, and as of 2003, nearly half of all known RNA structures had been determined by NMR spectroscopy.

Stable-isotope probing (SIP) is a technique in microbial ecology for tracing uptake of nutrients in biogeochemical cycling by microorganisms. A substrate is enriched with a heavier stable isotope that is consumed by the organisms to be studied. Biomarkers with the heavier isotopes incorporated into them can be separated from biomarkers containing the more naturally abundant lighter isotope by isopycnic centrifugation. For example, 13CO2 can be used to find out which organisms are actively photosynthesizing or consuming new photosynthate. As the biomarker, DNA with 13C is then separated from DNA with 12C by centrifugation. Sequencing the DNA identifies which organisms were consuming existing carbohydrates and which were using carbohydrates more recently produced from photosynthesis. SIP with 18O-labeled water can be used to find out which organisms are actively growing, because oxygen from water is incorporated into DNA (and RNA) during synthesis.

<span class="mw-page-title-main">Nanoscale secondary ion mass spectrometry</span>

NanoSIMS is an analytical instrument manufactured by CAMECA which operates on the principle of secondary ion mass spectrometry. The NanoSIMS is used to acquire nanoscale resolution measurements of the elemental and isotopic composition of a sample. The NanoSIMS is able to create nanoscale maps of elemental or isotopic distribution, parallel acquisition of up to seven masses, isotopic identification, high mass resolution, subparts-per-million sensitivity with spatial resolution down to 50 nm.

<span class="mw-page-title-main">Position-specific isotope analysis</span>

Position-specific isotope analysis, also called site-specific isotope analysis, is a branch of isotope analysis aimed at determining the isotopic composition of a particular atom position in a molecule. Isotopes are elemental variants with different numbers of neutrons in their nuclei, thereby having different atomic masses. Isotopes are found in varying natural abundances depending on the element; their abundances in specific compounds can vary from random distributions due to environmental conditions that act on the mass variations differently. These differences in abundances are called "fractionations," which are characterized via stable isotope analysis.

Methane clumped isotopes are methane molecules that contain two or more rare isotopes. Methane (CH4) contains two elements, carbon and hydrogen, each of which has two stable isotopes. For carbon, 98.9% are in the form of carbon-12 (12C) and 1.1% are carbon-13 (13C); while for hydrogen, 99.99% are in the form of protium (1H) and 0.01% are deuterium (2H or D). Carbon-13 (13C) and deuterium (2H or D) are rare isotopes in methane molecules. The abundance of the clumped isotopes provides information independent from the traditional carbon or hydrogen isotope composition of methane molecules.

<span class="mw-page-title-main">Translatomics</span>

Translatomics is the study of all open reading frames (ORFs) that are being actively translated in a cell or organism. This collection of ORFs is called the translatome. Characterizing a cell's translatome can give insight into the array of biological pathways that are active in the cell. According to the central dogma of molecular biology, the DNA in a cell is transcribed to produce RNA, which is then translated to produce a protein. Thousands of proteins are encoded in an organism's genome, and the proteins present in a cell cooperatively carry out many functions to support the life of the cell. Under various conditions, such as during stress or specific timepoints in development, the cell may require different biological pathways to be active, and therefore require a different collection of proteins. Depending on intrinsic and environmental conditions, the collection of proteins being made at one time varies. Translatomic techniques can be used to take a "snapshot" of this collection of actively translating ORFs, which can give information about which biological pathways the cell is activating under the present conditions.

References

  1. ↑ Reichle, Valentin F.; Kaiser, Steffen; Heiss, Matthias; Hagelskamp, Felix; Borland, Kayla; Kellner, Stefanie (1 March 2019). "Surpassing limits of static RNA modification analysis with dynamic NAIL-MS". Methods. 156: 91–101. doi: 10.1016/j.ymeth.2018.10.025 . ISSN   1095-9130. PMID   30395967.
  2. ↑ Kellner, Stefanie; Neumann, Jennifer; Rosenkranz, David; Lebedeva, Svetlana; Ketting, René F.; Zischler, Hans; Schneider, Dirk; Helm, Mark (4 April 2014). "Profiling of RNA modifications by multiplexed stable isotope labelling". Chemical Communications. 50 (26): 3516–3518. doi:10.1039/c3cc49114e. ISSN   1364-548X. PMID   24567952.
  3. ↑ Dal Magro, Christina; Keller, Patrick; Kotter, Annika; Werner, Stephan; Duarte, Victor; Marchand, Virginie; Ignarski, Michael; Freiwald, Anja; Müller, Roman-Ulrich; Dieterich, Christoph; Motorin, Yuri (25 June 2018). "A Vastly Increased Chemical Variety of RNA Modifications Containing a Thioacetal Structure". Angewandte Chemie International Edition in English. 57 (26): 7893–7897. doi:10.1002/anie.201713188. ISSN   1521-3773. PMID   29624844.
  4. 1 2 Reichle, Valentin F.; Petrov, Dimitar P.; Weber, Verena; Jung, Kirsten; Kellner, Stefanie (6 December 2019). "NAIL-MS reveals the repair of 2-methylthiocytidine by AlkB in E. coli". Nature Communications. 10 (1): 5600. Bibcode:2019NatCo..10.5600R. doi:10.1038/s41467-019-13565-9. ISSN   2041-1723. PMC   6898146 . PMID   31811240.
  5. ↑ Quinlivan, Eoin P.; Gregory, Jesse F. (15 February 2008). "DNA digestion to deoxyribonucleoside: a simplified one-step procedure". Analytical Biochemistry. 373 (2): 383–385. doi:10.1016/j.ab.2007.09.031. ISSN   0003-2697. PMC   2239294 . PMID   18028864.
  6. ↑ Crain, P. F. (1990). "Preparation and enzymatic hydrolysis of DNA and RNA for mass spectrometry". Mass Spectrometry. Methods in Enzymology. Vol. 193. pp. 782–790. doi:10.1016/0076-6879(90)93450-y. ISBN   978-0-12-182094-7. ISSN   0076-6879. PMID   1706062.
  7. 1 2 Reichle, Valentin F.; Weber, Verena; Kellner, Stefanie (18 December 2018). "NAIL-MS in E. coli Determines the Source and Fate of Methylation in tRNA". ChemBioChem. 19 (24): 2575–2583. doi:10.1002/cbic.201800525. ISSN   1439-7633. PMC   6582434 . PMID   30328661.
  8. ↑ Heiss, Matthias; Reichle, Valentin F.; Kellner, Stefanie (2 September 2017). "Observing the fate of tRNA and its modifications by nucleic acid isotope labeling mass spectrometry: NAIL-MS". RNA Biology. 14 (9): 1260–1268. doi:10.1080/15476286.2017.1325063. ISSN   1555-8584. PMC   5699550 . PMID   28488916.
  9. ↑ Heiss, Matthias; Hagelskamp, Felix; Marchand, Virginie; Motorin, Yuri; Kellner, Stefanie (15 January 2021). "Cell culture NAIL-MS allows insight into human tRNA and rRNA modification dynamics in vivo". Nature Communications. 12 (1): 389. Bibcode:2021NatCo..12..389H. doi:10.1038/s41467-020-20576-4. ISSN   2041-1723. PMC   7810713 . PMID   33452242.
  10. ↑ Kellner, Stefanie; Ochel, Antonia; Thüring, Kathrin; Spenkuch, Felix; Neumann, Jennifer; Sharma, Sunny; Entian, Karl-Dieter; Schneider, Dirk; Helm, Mark (16 August 2014). "Absolute and relative quantification of RNA modifications via biosynthetic isotopomers". Nucleic Acids Research. 42 (18): e142. doi:10.1093/nar/gku733. ISSN   1362-4962. PMC   4191383 . PMID   25129236.
  11. ↑ Hagelskamp, Felix; Borland, Kayla; Ramos, Jillian; Hendrick, Alan G.; Fu, Dragony; Kellner, Stefanie (21 February 2020). "Broadly applicable oligonucleotide mass spectrometry for the analysis of RNA writers and erasers in vitro". Nucleic Acids Research. 48 (7): e41. doi:10.1093/nar/gkaa091. ISSN   1362-4962. PMC   7144906 . PMID   32083657.
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