Reductive methylation: A method for preparing functionally active radioactive ribosomes

Moore, Graham J.; Crichton, Robert R.

doi:10.1016/0014-5793(73)80429-6

Cited by 47 publications

(9 citation statements)

References 17 publications

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“…To this end, 50 μg of a HEK whole cell lysate was digested with the enzyme LysC, either light or heavy dimethyl labeled, and mixed in a ratio of 1:1 (Figure 1A). The robust protocol for reductive methylation of peptides and proteins has a long-standing tradition 9 and drew recent attention for isotope-based quantification in MS survey scans. 10 To dimethyl label peptides with light isobaric isotopologues at primary amines of N-termini and C-terminal lysine side chains, 13 C formaldehyde introduced a Schiff’s base that was subsequently reduced with trihydrogen borocyanate.…”

Section: Resultsmentioning

confidence: 99%

Interference-Free Proteome Quantification with MS/MS-based Isobaric Isotopologue Detection

et al. 2014

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Chemical labeling of peptides prior to shotgun proteomics allows relative quantification of proteins in biological samples independent of sample origin. Current strategies utilize isobaric labels that fragment into reporter ions. However, quantification of reporter ions results in distorted ratio measurements due to contaminating peptides that are co-selected in the same precursor isolation window. Here, we show that quantitation of isobaric peptide fragment isotopologues in tandem mass spectra reduces precursor interference. The method is based on the relative quantitation of isobaric isotopologues of di-methylated peptide fragments in tandem mass spectra following higher energy collisional dissociation (HCD). The approach enables precise quantification of a proteome down to single spectra per protein and quantifies >90 % of proteins in a MudPIT 1 experiment and accurately measures proteins in a model cell line for Cystic Fibrosis.

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Section: Resultsmentioning

confidence: 99%

Interference-Free Proteome Quantification with MS/MS-based Isobaric Isotopologue Detection

et al. 2014

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“…The third column on the gel corresponds to the 30s subunits treated with borohydride in 25 mM concentration in the presence of methyl-RNA. According to the data of Moore and Crichton [15], incubation of the E. coli ribosomes with such a concentration of sodium borohydride does not lead to a loss of their biological activity. Indirect proof thereof is that upon the borohydride treatment in the presence of methyl-RNA, the sedimentation profile of the 30s subunits in sucrose gradient did not change (not shown).…”

Section: Localization Of the 16s Rrna Cleavage Pointmentioning

confidence: 99%

Specific fragmentation of tRNA and rRNA at a 7-methylguanine residue in the presence of methylated carrier RNA

et al. 1985

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1. The reaction of site-specific cleavage of tRNA at a 7-methylguanine residue, including subsequent treatment with sodium borohydride and aniline [Wintermeyer, W. and Zachau, H. G. (1975) FEBS Lett. 58,306-3091, was shown to work only within a certain range of tRNA concentrations (higher than 30 pM). The Escherichia coli 16s rRNA, which contained a unique m7G (position 527), could not be split by this method when taken at any concentration.2. It was found that the presence of statistically methylated carrier RNA in the reaction mixture at the borohydride stage significantly stimulates site-specific fragmentation of 16s rRNA and 32P-labeled tRNAs.3. Direct sequencing proved that 16s rRNA and tRNAs are cleaved by this procedure successfully at the m7G residue.4. The E. coli 16s rRNA was preparatively cleaved by the described procedure into two fragments. The 5'-terminal fragment (1 -526) and the 3'-terminal fragment (528 -1542) were isolated in the pure form and their secondary structure investigated by the circular dichroism method. The results of this study showed that the secondary and tertiary structures of the 5'-terminal one-third of the 16s rRNA are at least as ordered as those of intact 16s rRNA or its 3'4erminal two-thirds.The site-specific fragmentation of a polynucleotide chain is widely used for the study of the structure and functions of RNA and ribonucleoprotein complexes. Apart from the enzymatic approaches El-41, the chemical methods of splitting native RNAs at modified bases hold promise. Previously Zachau and co-workers described the procedure for splitting the yeast tRNAPhe at some minor nucleotides [5-71. The procedure of tRNA cleavage at a 7-methylguanine residue was based on reducing this base with sodium borohydride and subsequently splitting the polynucleotide chain at the reduced base with aniline.However, we have not been successful in our attempts to make direct use of this method for splitting the Escherichiu coli 16s rRNA at the unique 7-methylguanine residue or the 3'-terminally labeled yeast [32P]tRNAPhe. A closer study of the reaction has shown that the addition of the methylated RNA-carrier (methyl-RNA) at the borohydride reduction stage considerably enhances the efficiency of the reaction and makes it possible to cleave site-specifically 16s rRNA or tRNA with a 50-70% yield. Using this method we could cleave relatively large amounts of E. coli 16s rRNA at the unique residue of 7-methylguanine at position 527. A 526-nucleotide-long 5'-terminal fragment and a 1 01 5-nucleotidelong 3'-terminal fragment have been isolated and their secondary structure investigated by the circular dichroism (CD) method. MATERIALS AND METHODSThe 16s rRNA was isolated by phenol extraction from the 30s ribosomal subunits of Escherichia coli (strain MRE600).Abbreviations. Methyl-RNA, carrier RNA statistically methylated with dimethylsulfate; CD, circular dichroism.The phenylalanine and valine tRNA from yeast were kindly provided by Dr T.V. Venkstern. The RNase H preparation from E. coli was furnished by...

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“…The use of tritiated internal protein standards permitted a more reliable and accurate estimate of molecular weight than the conventional use of externally calibrated systems; reductive methylation does not significantly affect the molecular weight and charge properties of a protein or its mobility in polyacrylamide gels (11). two peaks, a large peak eluting at the void volume and a small peak eluting at a volume of about 38 ml.…”

Section: Resultsmentioning

confidence: 99%

Putative Precursors of Vasopressin, Oxytocin, and Neurophysins in the Rat Hypothalamus*

Rosenior

North

Moore³

1981

Endocrinology

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The biosynthesis of [arginine8]vasopressin (AVP) and oxytocin (OT) was studied, and the results obtained have been compared with a study of their associated neurophysins (NP), AVP-NP and OT-NP. Rat hypothalamic extracts obtained at acid pH were subjected to Sephadex G-75 gel filtration chromatography and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Fractions of gel chromatography effluent and extract of SDS-PAGE gel slices were subjected to RIA for immunoreactive precursors of AVP, AVP-NP, OT, and OT-NP using specific antisera fro each molecule. Tritiated bovine serum albumin, ovalbumin, and cytochrome c were used as internal standards during gel filtration chromatography and SDS-PAGE. Molecular weight estimates obtained for precursors of AVP were more than 70K, 31K, 13K, 5K, and less than 5K (AVP) by G-75 chromatography and more than 70K, 39K, 15K, and less than 5K (AVP) by SDS-PAGE. Molecular weight estimates obtained for precursors of AVP-NP were more than 70K, 35K, 24K, and 12K (AVP-NP) by G-75 chromatography and 19K and 10K (AVP-NP) by SDS-PAGE. Molecular weight estimates of OT were more than 70K, 35K, 19K, 8K, and less than 5K (OT) by G-75 chromatography and 60K, 35K, 19K, 9K, and less than 5K (OT) by SDS-PAGE. Precursors of OT-NP were estimated to be more than 70K, 17-18K, and 10K (OT-NP) by G-75 chromatography and 28K, 18K and 10K (Ot-NP) by SDS-PAGE. These studies show that there are small differences in precursor processing among AVP, AVP-NP, and OT, OT-NP, but allow for the existence of common precursors for AVP and AVP-NP and for OT and OT-NP.

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Reductive methylation: A method for preparing functionally active radioactive ribosomes

Cited by 47 publications

References 17 publications

Interference-Free Proteome Quantification with MS/MS-based Isobaric Isotopologue Detection

Interference-Free Proteome Quantification with MS/MS-based Isobaric Isotopologue Detection

Specific fragmentation of tRNA and rRNA at a 7-methylguanine residue in the presence of methylated carrier RNA

Putative Precursors of Vasopressin, Oxytocin, and Neurophysins in the Rat Hypothalamus*

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