title: In-gel digestion for mass spectrometric characterization of RNA from fluorescently stained polyacrylamide gels.
abstract: Although current mass spectrometry-based proteomics technology allows for high-throughput analysis of protein components in functional ribonucleoprotein complexes, this technology has had limited application to studies of RNA itself. Here we present a protocol for RNA analysis using polyacrylamide gel electrophoresis coupled with liquid chromatography-tandem mass spectrometry. Specifically, RNAs of interest are subjected to polyacrylamide gel electrophoresis and stained with a fluorescent dye, and RNAs in gel bands are digested with nuclease and then analyzed directly liquid chromatography-mass spectrometry, resulting in highly accurate mass values and reliable information on post-transcriptional modifications. We demonstrate that the method can be applied to the identification and chemical analysis of small RNAs in mouse embryonic stem cell extracts and of small RNAs in the spliceosomal ribonucleoprotein complex pulled down from yeast cells using a tagged protein cofactor as bait. The protocol is relatively simple and allowed us to identify not only three novel methylated nucleotide residues of RNase P RNA, U6 snRNA, and 7SL RNA prepared from mouse ES cells but also various 3'-end forms of U4, U5S, and U6 snRNAs isolated from the yeast spliceosome at the femtomole level. The method is thus a convenient tool for direct analysis of RNAs in various cellular ribonucleoprotein complexes, particularly for the analysis of post-transcriptional modifications and metabolic processing of RNA.
authors.affiliation: Department of Chemistry, Graduate School of Sciences and Engineering, Tokyo Metropolitan University, Minamiosawa 1-1, Hachioji-shi, Tokyo 192-0397, Japan. mango@tmu.ac.jp
authors.forename: Masato
authors.initials: M
authors.lastname: Taoka
authors.forename: Maki
authors.initials: M
authors.lastname: Ikumi
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nakayama
authors.forename: Shunpei
authors.initials: S
authors.lastname: Masaki
authors.forename: Ryozo
authors.initials: R
authors.lastname: Matsuda
authors.forename: Yuko
authors.initials: Y
authors.lastname: Nobe
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Yamauchi
authors.forename: Jun
authors.initials: J
authors.lastname: Takeda
authors.forename: Nobuhiro
authors.initials: N
authors.lastname: Takahashi
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Isobe
mesh_terms: D000180:Acrylic Resins / Q000737:chemistry*; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D057225:Data Mining; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D053595:Embryonic Stem Cells; D005456:Fluorescent Dyes / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D012313:RNA / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D012331:RNA, Fungal / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D012342:RNA, Small Nuclear / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae; D053719:Tandem Mass Spectrometry
chemical_list: D000180:Acrylic Resins; D005456:Fluorescent Dyes; D012331:RNA, Fungal; D012342:RNA, Small Nuclear; C016680:polyacrylamide gels; D012313:RNA