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The use of liquid chromatography coupled with mass spectrometry (LC-MS) for the characterization of oligonucleotides and nucleic acids is a powerful analytical method. Recently, hydrophilic interaction chromatography (HILIC) has been proposed as a reasonable alternative to ion-pair reversed phase separations of oligonucleotides prior to MS. A wide variety of HILIC stationary phase surface chemistries are currently available. Although their selectivity can be considerably different, few studies have compared these chemistries for LC-MS analysis of oligonucleotides. We evaluated ten different HILIC column chemistries to understand their capabilities for separating a variety of oligonucleotides. In general, we found that most columns were ineffective at separating larger ( n > 15-mer) oligonucleotides under the mobile phase and gradient conditions evaluated here. However, several stationary phases were found to be effective for separating smaller oligonucleotides such as endonuclease digestion products. Given that early eluting oligonucleotides were found to be compatible with standard electrospray ionization conditions, several different HILIC stationary phase options are available for LC-MS studies of smaller oligonucleotides including those generated in RNA modification mapping experiments.

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., (© 2022. The Author(s).)

Background & Aims: The liver has a unique capacity to regenerate after injury in a highly orchestrated and regulated manner. Here, we report that O-GlcNAcylation, an intracellular post-translational modification regulated by 2 enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), is a critical termination signal for liver regeneration following partial hepatectomy (PHX)., Methods: We studied liver regeneration after PHX on hepatocyte specific OGT and OGA knockout mice (OGT-KO and OGA-KO), which caused a significant decrease (OGT-KO) and increase (OGA-KO) in hepatic O-GlcNAcylation, respectively., Results: OGA-KO mice had normal regeneration, but the OGT-KO mice exhibited substantial defects in termination of liver regeneration with increased liver injury, sustained cell proliferation resulting in significant hepatomegaly, hepatic dysplasia, and appearance of small nodules at 28 days after PHX. This was accompanied by a sustained increase in expression of cyclins along with significant induction in pro-inflammatory and pro-fibrotic gene expression in the OGT-KO livers. RNA-sequencing studies revealed inactivation of hepatocyte nuclear 4 alpha (HNF4α), the master regulator of hepatic differentiation and a known termination signal, in OGT-KO mice at 28 days after PHX, which was confirmed by both Western blot and immunohistochemistry analysis. Furthermore, a significant decrease in HNFα target genes was observed in OGT-KO mice, indicating a lack of hepatocyte differentiation following decreased hepatic O-GlcNAcylation. Immunoprecipitation experiments revealed HNF4α is O-GlcNAcylated in normal differentiated hepatocytes., Conclusions: These studies show that O-GlcNAcylation plays a critical role in the termination of liver regeneration via regulation of HNF4α in hepatocytes., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)