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1. Direct sequencing of total S. cerevisiae tRNAs by LC-MS/MS

   https://doi.org/10.1261/rna.079656.123  

   Jones, Joshua D; Simcox, Kaley M; Kennedy, Robert T; Koutmou, Kristin S (May 2023, RNA)

   Among RNAs, transfer RNAs (tRNAs) contain the widest variety of abundant post-transcriptional chemical modifications. These modifications are crucial for tRNAs to participate in protein synthesis, promoting proper tRNA structure and aminoacylation, facilitating anticodon:codon recognition, and ensuring the reading frame maintenance of the ribosome. While tRNA modifications were long thought to be stoichiometric, it is becoming increasingly apparent that these modifications can change dynamically in response to the cellular environment. The ability to broadly characterize the fluctuating tRNA modification landscape will be essential for establishing the molecular level contributions of individual sites of tRNA modification. The locations of modifications within individual tRNA sequences can be mapped using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). In this approach, a single tRNA species is purified, treated with ribonucleases and the resulting single-stranded RNA products are subject to LC-MS/MS analysis. The application of LC-MS/MS to study tRNAs is limited by the necessity of analyzing one tRNA at a time because the digestion of total tRNA mixtures by commercially available ribonucleases produces many short digestion products unable to be uniquely mapped back to a single site within a tRNA. We overcame these limitations by taking advantage of the highly structured nature of tRNAs to prevent the full digestion by single-stranded RNA specific ribonucleases. Folding total tRNA prior to digestion allowed us to sequence S. cerevisiae tRNAs with up to 97% sequence coverage for individual tRNA species by LC-MS/MS. This method presents a robust avenue for directly detecting the distribution of modifications in total tRNAs. 

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2. Separation, identification, and confirmation of cyclic and tadpole macromolecules via UPLC-MS/MS

   https://doi.org/10.1039/d2an00208f  

   O'Neill, Jason M.; Mao, Jialin; Haque, Farihah M.; Barroso-Bujans, Fabienne; Grayson, Scott M.; Wesdemiotis, Chrys (May 2022, The Analyst)

   Macrocyclic poly(glycidyl phenyl ether) (pGPE) synthesized via zwitterionic ring opening polymerization is typically contaminated by chains with linear and tadpole architecture. Although mass spectrometry (MS) analysis can readily confirm the presence of the linear byproduct, due to its unique mass, it is unable to differentiate between the cyclic and tadpole structures, which are constitutional isomers produced by backbiting reactions in monomeric or dimeric chains, respectively. To overcome this problem, ultraperformance reversed-phase liquid chromatography interfaced with electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) was employed. The separation achieved by UPLC revealed that the tadpole isomer elutes before the cyclic structure because of the increased polarity afforded by its distinctive substituents. The ratio of tadpole to cyclic species increased with the degree of polymerization, in agreement with the synthetic method used, as the potential for forming tadpole structures by backbiting is entropically favored in longer polymer chains. Once separated, the two isomers could be independently characterized by tandem mass spectrometry. The macrocyclic and tadpole species exhibit unique fragmentation patterns, including structurally diagnostic fragments for each structure. 

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3. IsoForma: An R Package for Quantifying and Visualizing Positional Isomers in Top-Down LC-MS/MS Data

   https://doi.org/10.1021/acs.jproteome.3c00681  

   Degnan, David J.; Lewis, Logan A.; Bramer, Lisa M.; McCue, Lee Ann; Pesavento, James J.; Zhou, Mowei; Bilbao, Aivett (February 2024, Journal of Proteome Research)

   Proteoforms, the different forms of a protein with sequence variations including post-translational modifications (PTMs), execute vital functions in biological systems, such as cell signaling and epigenetic regulation. Advances in top-down mass spectrometry (MS) technology have permitted the direct characterization of intact proteoforms and their exact number of modification sites, allowing for the relative quantification of positional isomers (PI). Protein positional isomers refer to a set of proteoforms with identical total mass and set of modifications, but varying PTM site combinations. The relative abundance of PI can be estimated by matching proteoform-specific fragment ions to top-down tandem MS (MS2) data to localize and quantify modifications. However, the current approaches heavily rely on manual annotation. Here, we present IsoForma, an open-source R package for the relative quantification of PI within a single tool. Benchmarking IsoForma's performance against two existing workflows produced comparable results and improvements in speed. Overall, IsoForma provides a streamlined process for quantifying PI, reduces the analysis time, and offers an essential framework for developing customized proteoform analysis workflows. The software is open source and available at https://github.com/EMSL-Computing/isoforma-lib. 

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4. MS-based proteomics for comprehensive investigation of protein O -GlcNAcylation

   https://doi.org/10.1039/d1mo00025j  

   Xu, Senhan; Sun, Fangxu; Tong, Ming; Wu, Ronghu (April 2021, Molecular Omics)

   null (Ed.)

   Protein O -GlcNAcylation refers to the covalent binding of a single N -acetylglucosamine (GlcNAc) to the serine or threonine residue. This modification primarily occurs on proteins in the nucleus and the cytosol, and plays critical roles in many cellular events, including regulation of gene expression and signal transduction. Aberrant protein O -GlcNAcylation is directly related to human diseases such as cancers, diabetes and neurodegenerative diseases. In the past decades, considerable progress has been made for global and site-specific analysis of O -GlcNAcylation in complex biological samples using mass spectrometry (MS)-based proteomics. In this review, we summarized previous efforts on comprehensive investigation of protein O -GlcNAcylation by MS. Specifically, the review is focused on methods for enriching and site-specifically mapping O -GlcNAcylated peptides, and applications for quantifying protein O -GlcNAcylation in different biological systems. As O -GlcNAcylation is an important protein modification for cell survival, effective methods are essential for advancing our understanding of glycoprotein functions and cellular events. 

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5. Methods of Metabolite Identification Using MS/MS Data

   https://doi.org/10.1080/08874417.2019.1681328  

   Kwak, Myungjae; Kang, Kyungwoo; Wang, Yingfeng (January 2022, Journal of Computer Information Systems)