Atmospheric pressure MALDI-FTMS of normal and chemically modified RNA.
Atmospheric pressure (AP) MALDI has been combined with Fourier transform mass spectrometry (FTMS) to obtain the unambiguous characterization of RNA samples modified by solvent accessibility reagents used in structural studies of RNA and protein-RNA complexes. The formation of cation adducts typical of MS analysis of nucleic acids was effectively reduced by extensive washing of the anionic analytes retained onto the probe surface by strong interactions with a cationic layer of poly(diallyldimethylammonium chloride) (PADMAC). This rapid desalting procedure allowed for the detection of DNA and RNA samples in high femtomole quantities distributed over a 4 x 4 mm sample well. AP MALDI-FTMS was shown to provide high-resolution spectra for analytes as large as approximately 6.4 kDa with little or no evidence of metastable decomposition. The absence of significant metastable decay observed for precursor ions selected for tandem experiments offered a further measure of the low energy content typical of ions generated by AP MALDI. This feature proved to be very beneficial in the characterization of chemically modified RNA samples, which become particularly prone to base losses upon alkylation. The high resolution offered by FTMS enabled the application of a data-reduction algorithm capable of rejecting any signal devoid of plausible isotopic distribution, thus facilitating the analysis of complex analyte mixtures produced by nuclease treatment of RNA substrates. Proper selection of nucleases and digestion conditions can ensure the production of hydrolytic fragments of manageable size, which could extend the range of applicability of this bottom-up strategy to the structural investigation of very large RNA and protein-RNA complexes.
Kellersberger
Katherine A
KA
University of Maryland, Baltimore Country, Baltimore, Maryland 21250, USA.
Yu
Eizadora T
ET
Merenbloom
Samuel I
SI
Fabris
Daniele
D
D001274:Atmospheric Pressure; D001483:Base Sequence; D005583:Fourier Analysis; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics; D012313:RNA / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D012313:RNA; D012260:Ribonucleases