Identification of RNA sequence isomer by isotope labeling and LC-MS/MS

Abstract

Recently, we developed a method for modified ribonucleic acid (RNA) analysis based on the comparative analysis of RNA digests (CARD). Within this CARD approach, sequence or modification differences between two samples are identified through differential isotopic labeling of two samples. Components present in both samples will each be labeled, yielding doublets in the CARD mass spectrum. Components unique to only one sample should be detected as singlets. A limitation of the prior singlet identification strategy occurs when the two samples contain components of unique sequence but identical base composition. At the first stage of mass spectrometry, these sequence isomers cannot be differentiated and would appear as doublets rather than singlets. However, underlying sequence differences should be detectable by collision-induced dissociation tandem mass spectrometry (CID MS/MS), as y-type product ions will retain the original enzymatically incorporated isotope label. Here, we determine appropriate instrumental conditions that enable CID MS/MS of isotopically labeled ribonuclease T1 (RNase T1) digestion products such that the original isotope label is maintained in the product ion mass spectrum. Next, we demonstrate how y-type product ions can be used to differentiate singlets and doublets from isomer sequences. We were then able to extend the utility of this approach by using CID MS/MS for the confirmation of an expected RNase T1 digestion product within the CARD analysis of an Escherichia coli mutant strain even in the presence of interfering and overlapping digestion products from other transfer RNAs.

High Percentage of Isomeric Human MicroRNA and Their Analytical Challenges

“…For determining the size of a specific RNA molecule, the conventional or chip-based gel electrophoresis methods are commonly used [ 32 , 33 ]. Alternatively, sequencing methods can be used to determine the size of an RNA molecule as well as its RNA sequence, the latter information is particularly important to the identification of a specific RNA molecule including miR [ 34 , 35 ]. Until the recent development on the technology for next generation sequencing, the use of complementary nucleic acid probe(s) to detect specific RNA target has been the preferred method to achieve fast turnaround time and multiplexing for both RNA identification and quantitation [ 36 ].…”

Gold-Coated Nanoelectrospray Emitters Fabricated by Gravity-Assisted Etching Self-Termination and Electroless Deposition

“…Nanoelectrospray ionization–mass spectrometry (nanoESI-MS) has become a powerful tool in the sensitive detection of various samples. , The emitter is of great significance to ensure the stability and sensitivity of mass spectrometry (MS) analysis by providing a stable Taylor cone . Usually, the emitter with a small orifice is fabricated by the microelectrode puller; however, with both the inner and outer surface tapered, , the emitter is prone to clogging, resulting in poor durability. , Although emitters without an internal taper could be fabricated by physically grinding, , the stable spray could hardly be obtained at low flow rates, because of the large outer diameter .…”