An analytical platform for mass spectrometry-based identification and chemical analysis of RNA in ribonucleoprotein complexes

Abstract

We describe here a mass spectrometry (MS)-based analytical platform of RNA, which combines direct nano-flow reversed-phase liquid chromatography (RPLC) on a spray tip column and a high-resolution LTQ-Orbitrap mass spectrometer. Operating RPLC under a very low flow rate with volatile solvents and MS in the negative mode, we could estimate highly accurate mass values sufficient to predict the nucleotide composition of a ∼21-nucleotide small interfering RNA, detect post-transcriptional modifications in yeast tRNA, and perform collision-induced dissociation/tandem MS-based structural analysis of nucleolytic fragments of RNA at a sub-femtomole level. Importantly, the method allowed the identification and chemical analysis of small RNAs in ribonucleoprotein (RNP) complex, such as the pre-spliceosomal RNP complex, which was pulled down from cultured cells with a tagged protein cofactor as bait. We have recently developed a unique genome-oriented database search engine, Ariadne, which allows tandem MS-based identification of RNAs in biological samples. Thus, the method presented here has broad potential for automated analysis of RNA; it complements conventional molecular biology-based techniques and is particularly suited for simultaneous analysis of the composition, structure, interaction, and dynamics of RNA and protein components in various cellular RNP complexes.

Friend of Prmt1, FOP is a Novel Component of the Nuclear SMN Complex Isolated Using Biotin Affinity Purification

“…High voltage for ionization in negative mode (−1.4 kV) was applied to the metal union, and the eluate from RPLC was Affinity Proteomics J Proteomics Bioinform ISSN: 0974-276X JPB, an open access journal sprayed on-line to an LTQ-Orbitrap hybrid mass spectrometer (model XL, Thermo Fisher Scientific, San Jose, CA). Reverse phase separation of oligoribonucleotides was performed at flow rate of 100 nl/min using a 30-min linear gradient from 10 to 40% methanol in 10 mM triethylammonium acetate (pH 7.0).The mass spectrometer was operated in a data-dependent mode to automatically switch between Orbitrap-MS and linear ion trap-MS/MS acquisition as described [35]. Survey full scan MS spectra (from m/z 500 to 1,500) were acquired in the Orbitrap with resolution R=30,000 (after accumulation to a target value of 500,000 ions in the linear ion trap).…”

“…LC was performed at a flow rate of 100 nl/ min using a 30-min linear gradient from 10% to 40% methanol in 10 mM triethylammonium acetate (pH 7.0). The mass spectrometer was operated in a mode to automatically switch between Orbitrap-MS and linear ion trap-MS/MS acquisition as described [35].…”

“…We also recovered several small RNA components from the nuclear SMN complexes isolated under the high salt condition ( Figure 2D), and identified the RNAs by the LC-MS/MS system equipped with a genome-oriented database searching engine Ariadne [33,35,36]. This analysis identified a number of known RNA components of the SMN complexes, U1, U3, and U4 snRNAs ( Figure 2D, supplementary Table 2), showing that our preparation of the nuclear SMN complexes in fact contained UsnRNAs.…”

Mass Spectrometry of siRNA

“…Huber and coworkers have optimized the utility of monolithic capillary columns packed with octadecyl poly(styrene/divinylbenzene) particles as the stationary phase and gradients of acetonitrile in 25 mM TEAB as the mobile phase to analyze a diverse assortment of synthetic and biological oligonucleotides [67,71]. Recently Taoka et al reported the use of nanoelectrospray LC-MS for the analysis of synthetic siRNA [72]. They used a 50 mm × 150 µM i.d.…”

Subunit Composition of Ribosome in the <b><i>yqgF</i></b> Mutant Is Deficient in pre-16S rRNA Processing of <b><i>Escherichia coli</i></b>

“…We used nano-flow liquid chromatography (LC) and high-resolution hybrid mass spectrometry (MS) coupled with a data analysis system [Taoka et al, 2009] to measure the amount of individual r-proteins in the ribosome of the yqgF ts mutant and compared the data with those of an isogenic yqgF + strain. To prepare 70S ribosomes for this analysis, we subjected yqgF + cells to growth in M9 minimal medium containing 15 NH 4 Cl, and subsequently prepared 15 N-labeled 70S ribosomes (Fig.…”