NAIL-MS (short for
nucleic acid
Nucleic acids are biopolymers, macromolecules, essential to all known forms of life. They are composed of nucleotides, which are the monomers made of three components: a 5-carbon sugar, a phosphate group and a nitrogenous base. The two main cl ...
isotope
Isotopes are two or more types of atoms that have the same atomic number (number of protons in their nuclei) and position in the periodic table (and hence belong to the same chemical element), and that differ in nucleon numbers (mass numbers) ...
labeling coupled
mass spectrometry
Mass spectrometry (MS) is an analytical technique that is used to measure the mass-to-charge ratio of ions. The results are presented as a ''mass spectrum'', a plot of intensity as a function of the mass-to-charge ratio. Mass spectrometry is use ...
) 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
Studies that are ''in vivo'' (Latin for "within the living"; often not italicized in English) are those in which the effects of various biological entities are tested on whole, living organisms or cells, usually animals, including humans, and ...
''. For example, the dynamic behaviour of nucleic acids in living cells, especially of
RNA modification
RNA editing (also RNA modification) 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 ...
s, can be followed in more detail.
Theory
![Labeling of cells](https://upload.wikimedia.org/wikipedia/commons/1/13/Labeling_of_cells.png)
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
Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and deoxyribonucleic acid ( DNA) are nucleic acids. Along with lipids, proteins, and carbohydra ...
, analysis is done by mass spectrometry. Mass spectrometry is an analytical technique that measures the
mass-to-charge ratio
The mass-to-charge ratio (''m''/''Q'') is a physical quantity relating the ''mass'' (quantity of matter) and the ''electric charge'' of a given particle, expressed in units of kilograms per coulomb (kg/C). It is most widely used in the electrody ...
of
ion
An ion () is an atom or molecule with a net electrical charge.
The charge of an electron is considered to be negative by convention and this charge is equal and opposite to the charge of a proton, which is considered to be positive by conven ...
s. Pairs of chemically identical nucleosides of different stable-isotope composition can be differentiated in a mass spectrometer due to their mass difference. Unlabeled
nucleoside
Nucleosides are glycosylamines that can be thought of as nucleotides without a phosphate group. A nucleoside consists simply of a nucleobase (also termed a nitrogenous base) and a five-carbon sugar (ribose or 2'-deoxyribose) whereas a nucleotide ...
s can therefore be distinguished from their stable isotope labeled
isotopologue
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.
An exampl ...
s. 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.
General procedure
![General workflow](https://upload.wikimedia.org/wikipedia/commons/6/68/General_workflow.jpg)
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
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.
Detection by mass spectrometry
A mass ...
atoms (
13C) instead of the normal
carbon-12
Carbon-12 (12C) is the most abundant of the two stable isotopes of carbon (carbon-13 being the other), amounting to 98.93% of element carbon on Earth; its abundance is due to the triple-alpha process by which it is created in stars. Carbon-12 i ...
(
12C). Cells growing in this medium, will, depending on model organism, incorporate the heavy glucose into all of their RNA molecules. Thereafter, all
nucleotide
Nucleotides are organic molecules consisting of a nucleoside and a phosphate. They serve as monomeric units of the nucleic acid polymers – deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), both of which are essential biomolecules wi ...
s 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
Size-exclusion chromatography (SEC), also known as molecular sieve chromatography, is a chromatographic method in which molecules in solution are separated by their size, and in some cases molecular weight. It is usually applied to large molecules ...
(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. 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
''Escherichia coli'' (),Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. also known as ''E. coli'' (), is a Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus ''Esche ...
'' (
bacteria
Bacteria (; singular: bacterium) are ubiquitous, mostly free-living organisms often consisting of one biological cell. They constitute a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria were among ...
) 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.
In ''
S.cerevisiae
''Saccharomyces cerevisiae'' () (brewer's yeast or baker's yeast) is a species of yeast (single-celled fungus microorganisms). The species has been instrumental in winemaking, baking, and brewing since ancient times. It is believed to have been ...
'' (
yeast
Yeasts are eukaryotic, single-celled microorganisms classified as members of the fungus kingdom. The first yeast originated hundreds of millions of years ago, and at least 1,500 species are currently recognized. They are estimated to constitut ...
) 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 acid
Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha am ...
s and
glucose
Glucose is a simple sugar with the molecular formula . Glucose is overall the most abundant monosaccharide, a subcategory of carbohydrates. Glucose is mainly made by plants and most algae during photosynthesis from water and carbon dioxide, using ...
which can be added as stable isotope labeled variants in order to achieve
13C-carbon,
15N-nitrogen and
2H-hydrogen labeling of RNA.
While labeling in model organisms like ''E.coli'' and ''S.cerevisiae'' is fairly simple, stable isotope labeling in
cell culture
Cell culture or tissue culture is the process by which cells are grown under controlled conditions, generally outside of their natural environment. The term "tissue culture" was coined by American pathologist Montrose Thomas Burrows. This te ...
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
Glutamine (symbol Gln or Q) is an α-amino acid that is used in the biosynthesis of proteins. Its side chain is similar to that of glutamic acid, except the carboxylic acid group is replaced by an amide. It is classified as a charge-neutral, ...
and/or
aspartate
Aspartic acid (symbol Asp or D; the ionic form is known as aspartate), is an α-amino acid that is used in the biosynthesis of proteins. Like all other amino acids, it contains an amino group and a carboxylic acid. Its α-amino group is in the pro ...
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. 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). 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 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 quantitativ ...
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.
Discovery of new RNA modifications
For the discovery of uncharacterized modifications cells are grown in unlabeled or
13C‑labeled or
15N‑labeled or
2H‑labeled or
34S‑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.
[{{Cite journal, last1=Hagelskamp, first1=Felix, last2=Borland, first2=Kayla, last3=Ramos, first3=Jillian, last4=Hendrick, first4=Alan G., last5=Fu, first5=Dragony, last6=Kellner, first6=Stefanie, date=21 February 2020, title=Broadly applicable oligonucleotide mass spectrometry for the analysis of RNA writers and erasers in vitro, journal=Nucleic Acids Research, volume=48, issue=7, pages=e41, doi=10.1093/nar/gkaa091, issn=1362-4962, pmid=32083657, pmc=7144906]
References
External links
* https://www.cup.lmu.de/oc/kellner/research/
* https://iimcb.genesilico.pl/modomics/
Biochemistry detection methods
Biotechnology
Epigenetics
Genetics techniques
RNA
Isotopes
Mass spectrometry