Application of high-resolution ESI and MALDI mass spectrometry to metabolite profiling of small interfering RNA duplex.
We investigated the application of a high-resolution Orbitrap mass spectrometer equipped with an electrospray ionization (ESI) source and a matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometer to the metabolite profiling of a model small interfering RNA (siRNA) duplex TSR#34 and compared their functions and capabilities. TSR#34 duplex was incubated in human serum in vitro, and the duplex and its metabolites were then purified by ion exchange chromatography in order to remove the biological matrices. The fraction containing the siRNA duplex and its metabolites was collected and desalted and then subjected to high-performance liquid chromatography (HPLC) equipped with a reversed phase column. The siRNA and its metabolites were separated into single strands by elevated chromatographic temperature and analyzed using the ESI-Orbitrap or the MALDI-TOF mass spectrometer. Using this method, the 5' and/or 3' truncated metabolites of each strand were detected in the human serum samples. The ESI-Orbitrap mass spectrometer enabled differentiation between two possible RNA-based sequences, a monoisotopic molecular mass difference which was less than 2 Da, with an intrinsic mass resolving power. In-source decay (ISD) analysis using a MALDI-TOF mass spectrometer allowed the sequencing of the RNA metabolite with characteristic fragment ions, using 2,4-dihydroxyacetophenone (2,4-DHAP) as a matrix. The ESI-Orbitrap mass spectrometer provided the highest mass accuracy and the benefit of on-line coupling with HPLC for metabolite profiling. Meanwhile, the MALDI-TOF mass spectrometer, in combination with 2,4-DHAP, has the potential for the sequencing of RNA by ISD analysis. The combined use of these methods will be beneficial to characterize the metabolites of therapeutic siRNA compounds.
Drug Metabolism and Pharmacokinetics Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, Kanagawa, Japan. hisao.shimizu@takeda.com
Hisao
H
Shimizu
Fumihiro
F
Jinno
Akio
A
Morohashi
Yuzo
Y
Yamazaki
Masaki
M
Yamada
Takahiro
T
Kondo
Satoru
S
Asahi
D000098:Acetophenones / Q000737:chemistry; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D006801:Humans; D008956:Models, Chemical; D008969:Molecular Sequence Data; D034741:RNA, Small Interfering / Q000097:blood / Q000737:chemistry*; D017423:Sequence Analysis, RNA; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D000098:Acetophenones; D034741:RNA, Small Interfering; C078634:2,4-dihydroxyacetophenone