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Peptide Identification Using Tandem Mass Spectrometry01:33

Peptide Identification Using Tandem Mass Spectrometry

Tandem mass spectrometry, also known as MS/MS or MS2, is an analytical technique that employs two mass analyzers. Essentially it is a series of mass spectrometers that helps isolate a particular biomolecule and then helps study its chemical properties.
This technique helps gather information regarding the protein from which the peptide was obtained and to study the peptides’ amino acid sequence. Identifying peptides from a complex mixture is an important component of the growing field of...

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Strategies to Avoid Artifacts in Mass Spectrometry-Based Epitranscriptome Analyses.

Steffen Kaiser1,2, Shane R Byrne3, Gregor Ammann1

  • 1Ludwig-Maximilians Universität München, Butenandtstr. 5-13, 81137, München, Deutschland.

Angewandte Chemie (International Ed. in English)
|August 2, 2021
PubMed
Summary

This study disproves the existence of RNA phosphorothioate (PT) modifications in bacteria and eukaryotes, including humans. An MS artifact was identified as the cause of previous misidentifications of these epitranscriptome marks.

Keywords:
RNA PTRNA modificationdigestion artifactmass spectrometrynucleoside analysis

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Epitranscriptomics

Background:

  • Recent reports suggested the presence of RNA phosphorothioate (PT) modifications, a novel class of epitranscriptome marks, in various organisms including humans.
  • These proposed modifications, if confirmed, would significantly expand our understanding of RNA regulation and function.

Purpose of the Study:

  • To rigorously validate the structural integrity and existence of reported RNA phosphorothioate (PT) modifications in biological samples.
  • To investigate the potential causes for the initial misidentification of these RNA modifications.

Main Methods:

  • Structure validation using high-resolution mass spectrometry (MS) on synthetic and native RNA hydrolysates.
  • Metabolic stable isotope labeling to trace and confirm modification presence.
  • PT-specific iodine-desulfurization assays.
  • Analysis of RNA hydrolysates from E. coli, S. cerevisiae, human cell lines, and mouse brain.

Main Results:

  • The existence of RNA phosphorothioate (PT) modifications in E. coli, S. cerevisiae, human cell lines, and mouse brain RNA was disproven.
  • An MS artifact involving 2'-O-methylated diribonucleotides was identified as the source of the initial misidentification of PT modifications.
  • The study highlights the critical role of RNA hydrolysis protocols in the accurate detection and quantification of RNA modifications.

Conclusions:

  • Reported RNA phosphorothioate (PT) modifications are not present in the analyzed biological samples.
  • A detailed guideline for MS-based structure validation of novel nucleic acid modifications is provided to prevent future misidentifications.
  • The findings necessitate a re-evaluation of epitranscriptomic studies relying on the presence of RNA PT modifications.