Ltq xl ion trap mass spectrometer

The digestate obtained from the SDS-PAGE gel slices were analyzed by nanoflow liquid chromatography tandem mass spectrometry (nanoLC-MS/MS) using an LTQ-XL ion-trap mass spectrometer (Thermo, Fremont, CA, USA). Reversed phase columns were packed in-house to approximately 7 cm (100 μmi.d.) using 100 Å, 5 μM Zorbax C18 resin (Agilent Technologies, Santa Clara, CA, USA) in a fused silica capillary with an integrated electrospray tip. A 1.8 kV electrospray voltage was applied via a liquid junction upstream of the C18 column. Samples were then injected into the C18 column using a Surveyor autosampler (Thermo, Fremont, CA, USA). The column was washed with buffer A [5% (v/v) ACN, 0.1% (v/v) formic acid] for 10 min at 1 μL/min before each loading. Peptides were subsequently eluted from the C18 column with 0–50% Buffer B [95% (v/v) ACN, 0.1% (v/v) formic acid] over 58 min at 500 nL per min followed by 50–95% Buffer B over 5 min at 500 nL per min. The column eluate was then directed into a nanospray ionization source of the mass spectrometer. Spectra were scanned over the range 400–1500 amu. Automated peak recognition, dynamic exclusion window set to 90s38 (link) and tandem MS of the top six most intense precursor ions at 35% normalization collision energy were performed using Xcalibur™ software (version 2.06) (Thermo, Fremont, CA, USA).

Purified 30-nucleotide and 60-nucleotide RNA with different uridine modifications were analyzed at Novatia, LLC using on-line desalting, flow injection electrospray ionization on an LTQ-XL ion trap mass spectrometer (Thermo Fisher Scientific) and analyzed with ProMass Deconvolution software from Novatia, LLC (https://www.enovatia.com/our-products/promass/?gclid=EAIaIQobChMIoZS6xKDn-AIViNrICh3peATrEAAYASAAEgLRNPD_BwE).

After co-immunoprecipitation, samples were resolved using a 10% Tris-Glycine SDS-PAGE and proteins were visualized using PageBlue (Thermo Scientific, 24620). Bands of interest were cut out from the gel and digested with trypsin (50ng/μl in 50 mM NH4HCO3 buffer, PH 8.0). The peptides were analyzed, as previously described [40 (link)], by capillary LC-tandem mass spectrometry in a LTQ XL ion trap mass spectrometer (ThermoScientific, San Jose, CA) fitted with a microelectrospray probe. The data were analyzed with the ProteomeDiscoverer software (ThermoScientific, version 1.4.1), and the proteins were identified with SequestHT against a target-decoy nonredundant human or mouse proteins database obtained from Uniprot. The following parameters were used: trypsin was selected with proteolytic cleavage only after arginine and lysine, number of internal cleavage sites was set to 1, mass tolerance for precursors and fragment ions was 1.0 Da, considered dynamic modifications were + 15.99 Da for oxidized methionine. Peptide matches were filtered using the q-value and Posterior Error Probability calculated by the Percolator algorithm ensuring an estimated false positive rate below 5%. The filtered Sequest HT output files for each peptide were grouped according to the protein from which they were derived.