Using immobilized enzymes to reduce RNase contamination in RNase mapping of transfer RNAs by mass spectrometry

Abstract

RNase (ribonuclease) mapping by nucleobase-specific endonucleases combined with mass spectrometry (MS) is a powerful analytical method for characterizing ribonucleic acids such as transfer RNAs. Typical free solution enzymatic digestion of RNA samples results in a significant amount of RNase being present in the sample solution analyzed by MS. In some cases, the RNase can lead to contamination of the high performance liquid chromatography and MS instrumentation. Here we investigate and compare several different approaches for reducing or eliminating contaminating RNase from the digested RNA sample before LC-MS analysis. Approaches using immobilized RNases were found to be most effective, with no enzyme carryover into the digested sample detected. Among the various options for immobilized RNases, we show that carbodiimide-based reactions can be used to couple RNases to carboxylic acid-terminated magnetic beads. The immobilized enzymes retain biological activity, are re-usable, and do not interfere with subsequent LC-MS analysis of the expected RNase digestion products. The use of immobilized RNases provides a simple approach for eliminating enzyme contamination in mass spectrometry-based RNase mapping experiments.

High resolution fingerprinting of single and double-stranded RNA using ion-pair reverse-phase chromatography

“…RNase mass mapping methods have been developed and used for the identification and quantification of RNA and RNA post transcriptional modifications [[1], [2], [3], [4], [5], [6], [7], [8], [9], [10]]. Typical workflows involve the purification of the RNA [[11], [12], [13], [14]], prior to RNase digestion into smaller oligoribonucleotides that are more amenable for chromatographic separation and intact mass measurements [[15], [16], [17], [18]]. Additional sequence information from the oligoribonucleotides can be obtained using tandem mass spectrometry (MS/MS) [19,20].…”

Characterisation and sequence mapping of large RNA and mRNA therapeutics using mass spectrometry

“…Utilising RNase T1 immobilised on magnetic beads also enables automation of the workflow which was performed both manually and further developed on an automated robotic liquid handling system which enabled automation of the RNase T1 digest and sample preparation for direct analysis using LC MS/MS. Furthermore, the use of RNase T1 immobilised on magnetic particles also prevents build-up of RNase T1 on the HPLC column, from standard in-solution digests, which can potentially further digest the RNA during chromatographic separation 18 which in this case would potentially further digest the oligoribonucleotides generated from the partial RNase digest. This is important to avoid, as the partial digestion is designed to release larger oligoribonucleotide fragments, which when sequenced will locate to a unique position within the mRNA.…”

“…RNase mapping methods have been developed previously and used in a wide variety of applications including RNA sequence mapping and identification of RNA post transcriptional modifications [4][5][6][7][8][9][10][11][12][13] . Enzymatic digestion of the RNA using ribonucleases such as RNase T1 or A generates smaller oligoribonucleotides that are more amenable for chromatographic separation and intact mass measurements [14][15][16][17][18] additional sequence information of the oligoribonucleotides can be obtained using tandem mass spectrometry (MS/MS) [19][20] . However, the use of high frequency RNase enzymes for RNA sequence mapping of long RNA and mRNA therapeutics results in the production of a large number of small oligoribonucleotides which map to multiple different locations throughout the RNA sequence and therefore do not generate unique sequences for sequence mapping.…”

Efficient tRNA degradation and quantification in Escherichia coli cell extract using RNase‐coated magnetic beads: A key step toward codon emancipation

“…To prevent dilution and facilitate precise RNase exposure optimization for both tRNA depletion and preservation of translation‐essential rRNA, RNase A molecules were covalently immobilized to epoxy‐functionalized superparamagnetic beads. This method was previously used for improving RNase mapping of tRNA by mass spectrometry . Using RNase A‐coated beads enables (1) control of RNase A concentration by adjusting bead concentrations, (2) control of treatment time, (3) rapid removal of RNase A by magnetic force, and (4) homogenous mixing to achieve uniform RNase A treatment of cell extract.…”