The Separation And Analysis Of Oligonucleotides By Mass Spectrometry

Oligonucleotides with short sequence are the basic tools for regulating gene expression in life science and biomedical research.Synthetic oligonucleotides can increase their stability in vivo by chemical modifications and they have been developed as gene therapy targeting drugs for the treatment of many gene-related diseases such as viruses,tumors and genetic diseases.Nevertheless,it has brought challenges as well to develop suitable methods to identify the type of modifications carried by the oligonucleotide and the modification sites quickly,effectively and accurately identify.Nucleic acids and proteins are two closely related biological macromolecules that regulate their respective life cycles and biological functions through interactions with each other.The interaction between nucleic acid proteins is a key factor in determining intracellular balance.The disruption of nucleic acid protein interactions will lead to cell dysfunction and related diseases.The study of nucleic acid-protein interactions involves LC-MS analysis of oligonucleotides and peptides,while the property difference makes it difficult to achieve simultaneous online analyze the oligonucleotides and peptides.Based on the requirements and difficulties in the MS analysis of oligonucleotides,including the MS signal inhibition of oligonucleotide caused by traditional ion-pairing reagents,the low sensitivity of oligonucleotides in mass spectrometry positive ion mode,the difficulty in simultaneous LC-MS analysis of oligonucleotides and peptides in positive ion mode.The details,including three parts,are as follows:Enhancing the mass spectrometry sensitivity for oligonucleotides detection by organic vapor assisted electrospray:There are two challenges in oligonucleotides detection by liquid chromatography coupled with mass spectrometry(LC-MS),the serious ion suppression effects caused by ion-pair reagents and the low detection sensitivity in positive mode MS.In this study,highly concentrated alcohol vapors were introduced into an enclosed electrospray ionization chamber and oligonucleotides could be well detected in negative mode MS even with 100 mM triethylammonium acetate(TEAA)as ion-pair reagent.The MS signal intensity was improved 600 folds(for standard oligonucleotide dT15)by the isopropanol vapor assisted electrospray and effective ion-pair LC separation was feasibly coupled with high-sensitive MS detection.Then,oligonucleotides were successfully detected in positive mode MS with little adducts by propanoic acid vapor assisted electrospray.The signal intensity was enhanced more than 10 folds on average compared with adding acids into the electrospray solution.Finally,oligonucleotides and peptides or histones were simultaneously detected in MS with little interference to each other.Our strategy provides a useful alternative for investigating the biological functions of oligonucleotides.Analysis of Oligonucleotides by Ion-Pair Reversed-Phase Liquid Chromatography Coupled with Positive Mode Electrospray Ionization Mass Spectrometry:Oligonucleotides are usually analyzed by ion-pair reversed-phase liquid chromatography(IP-RPLC)coupled with negative mode electrospray ionization mass spectrometry(ESI-MS)due to their highly negative charged phosphodiester backbones.Herein,the signal suppression effect of TEA adducts caused by ion-pair reagent TEA/HFIP was greatly alleviated after improving the in-source energy in positive mode ESI-MS.This strategy was applied for different RNAs sequencing through analyzing their formic acid hydrolysates via IP-RPLC-MS.Comparing with negative ion mode,we demonstrated the IP-RPLC MS analysis in positive ion mode was more suitable for RNA sequencing with fewer contaminants interferences.Finally,simultaneous online separation and detection of oligonucleotides and protein digests were achieved in positive ion mode IP-RPLC MS analysis with little interference to each other.Characterization of Small Interfering RNA Using In-Source Decay in MALDI-TOF Mass Spectrometry with Different Matrices:The sequence characterization of synthetic therapeutic siRNA with tandem mass spectrometry used to be complex and difficult due to the multiple chemical modifications and the multiply charged fragments produced by ESI.In this work,a model single-stranded modified siRNA sample SR16-2M2-AS has been analysed by in-source decay(ISD)in MALDI-TOF mass spectrometry using matrix sDHB(a mixture of 2,5-dihydroxybenzoic and 2-hydroxy-5-methoxybenzoic acid)and 3-HPA(3-hydroxypicolinic acid)respectively.Intriguing results indicated that the main fragment types produced in the ISD spectrum were close related to the applied matrix for d/y-ions in matrix sDHB while a/w-ions in 3-HPA.In addition,the results illustrated that 2'-F modification inhibited the fragmentation pathway of all fragment ion types with corresponding mechanisms.After grasping the fragmentation regularity,conjoint analysis of the ISD results obtained with different matrices could be a quick,easy,accurate and efficient method to sequence and determine the modified sites of the synthetic siRNA.