Infrared multiphoton dissociation of small-interfering RNA anions and cations.
Infrared multiphoton dissociation (IRMPD) on a linear ion trap mass spectrometer is applied for the sequencing of small interfering RNA (siRNA). Both single-strand siRNAs and duplex siRNA were characterized by IRMPD, and the results were compared with that obtained by traditional ion trap-based collision induced dissociation (CID). The single-strand siRNA anions were observed to dissociate via cleavage of the 5' P-O bonds yielding c- and y-type product ions as well as undergo neutral base loss. Full sequence coverage of the siRNA anions was obtained by both IRMPD and CID. While the CID mass spectra were dominated by base loss ions, accounting for approximately 25% to 40% of the product ion current, these ions were eliminated through secondary dissociation by increasing the irradiation time in the IRMPD mass spectra to produce higher abundances of informative sequence ions. With longer irradiation times, however, internal ions corresponding to cleavage of two 5' P-O bonds began to populate the product ion mass spectra as well as higher abundances of [a - Base] and w-type ions. IRMPD of siRNA cations predominantly produced c- and y-type ions with minimal contributions of [a - Base] and w-type ions to the product ion current; the presence of only two complementary series of product ions in the IRMPD mass spectra simplified spectral interpretation. In addition, IRMPD produced high abundances of protonated nucleobases, [G + H](+), [A + H](+), and [C + H](+), which were not detected in the CID mass spectra due to the low-mass cut-off associated with conventional CID in ion traps. CID and IRMPD using short irradiation times of duplex siRNA resulted in strand separation, similar to the dissociation trends observed for duplex DNA. With longer irradiation times, however, the individual single-strands underwent secondary dissociation to yield informative sequence ions not obtained by CID.
Department of Chemistry and Biochemistry, The University of Texas at Austin, Austin, Texas 78712-0165, USA.
Myles W
MW
Gardner
Na
N
Li
Andrew D
AD
Ellington
Jennifer S
JS
Brodbelt
D000838:Anions; D001483:Base Sequence; D002412:Cations; D008969:Molecular Sequence Data; D034741:RNA, Small Interfering / Q000737:chemistry* / Q000235:genetics*; D017423:Sequence Analysis, RNA / Q000379:methods*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013055:Spectrophotometry, Infrared / Q000379:methods*
D000838:Anions; D002412:Cations; D034741:RNA, Small Interfering