Identification of aberrant 2'-5' RNA linkage isomers by pellicular anion exchange chromatography

During chemical RNA synthesis, many undesired products may be formed. In addition to the "n-x" sequences, depurination products, and incompletely deprotected oligonucleotides, linkage isomers may form during condensation and/or deprotection of the synthetic products. Under acidic conditions, bond migration may alter normal 3'-5' diesters to aberrant 2'-5' diesters. This results in isomers that are difficult to identify by MS and LC-MS techniques because the isomers have identical masses. HPLC methods for identification of these isomers have not advanced because the isomers are not expected to exhibit differences in hydrophobicity that allow resolution by reversed-phase columns. Neither are changes in ionic interactions anticipated for these isomers that would allow resolution by ion exchange methods. We observed that chromatography on pellicular anion exchange phases, but not on porous anion exchange phases, completely resolves oligonucleotides with very slight conformation differences (e.g., DNA vs. RNA of identical sequence). Because incorporation of 2'-5' linkages in RNA will alter solution conformation slightly, we considered that this pellicular ion exchanger might also allow resolution of identical RNA sequences harboring aberrant 2'-5' linkages from those lacking aberrant 2'-5' linkages. Using the nonporous DNAPac PA200 column, we demonstrated a chromatographic procedure for resolving synthetic RNA with aberrant linkages from their normally linked counterparts. Under certain conditions, aberrant isomers are not completely resolved from those containing only normal linkages. Therefore, we also developed an independent linkage-confirming method using a 5'-3' exonuclease. This enzyme produces incomplete digestion products during digestion of synthetic RNA containing aberrant 2'-5' linkages, and these are readily resolved by DNAPac PA200 chromatography.     

Identification of oxidized phospholipids by electrospray ionization mass spectrometry and LC-MS using a QQLIT instrument

Phospholipids are complex and varied biomolecules that are susceptible to lipid peroxidation after attack by free radicals or electrophilic oxidants and can yield a large number of different oxidation products. There are many available methods for detecting phospholipid oxidation products, but also various limitations and problems. Electrospray ionization mass spectrometry allows the simultaneous but specific analysis of multiple species with good sensitivity and has a further advantage that it can be coupled to liquid chromatography for separation of oxidation products. Here, we explain the principles of oxidized phospholipid analysis by electrospray mass spectrometry and describe fragmentation routines for surveying the structural properties of the analytes, in particular precursor ion and neutral loss scanning. These allow targeted detection of phospholipid headgroups and identification of phospholipids containing hydroperoxides and chlorine, as well as the detection of some individual oxidation products by their specific fragmentation patterns. We describe instrument protocols for carrying out these survey routines on a QTrap5500 mass spectrometer and also for interfacing with reverse-phase liquid chromatography. The article highlights critical aspects of the analysis as well as some limitations of the methodology.     

Simultaneous determination of 8-hydroxy-2′-deoxyguanosine and 5-methyl-2′-deoxycytidine in DNA sample by high performance liquid chromatography/positive electrospray ionization tandem mass spectrometry

8-Hydroxy-2′-deoxyguanosine (8-OHdG) and 5-methyl-2′-deoxycytidine (5-mdC) are utilized as useful biomarkers not only for early diagnosis but also for the detection and assessment of high-risk individuals. In the present study, a sensitive and specific method was developed for simultaneous determination of 8-OHdG and 5-mdC in DNA by high performance liquid chromatography/positive electrospray ionization tandem mass spectrometry. The limits of quantification for 8-OHdG and 5-mdC were 80 and 40 pg/ml, respectively. The calibration curves of 8-OHdG and 5-mdC were linear over the concentration range of 0.02–100 ng/ml and the correlation coefficients were higher than 0.9990. The intra-day and inter-day relative standard derivative values were in the range of 0.70–7.47% for 8-OHdG and 1.07–7.06% for 5-mdC, respectively. The recoveries were 93.4–108.5% for 8-OHdG and 87.4–104.9% for 5-mdC, respectively. This method was validated by determination of the background levels of 8-OHdG and 5-mdC in calf thymus DNA, and satisfactory results were obtained.