Microas autosampler

Digestion products of the RNase U2 mutant or codon-optimized control were separated on a Waters Xbridge C18 column (3.5 μm, 1.0 × 150 mm) with MPA and mobile phase B (MPB: 50% MPA and 50% methanol) at a flow rate of 30 μL/min. The gradient started from 5% to 40% MPB in 5 min, followed by an increase until 95% MPB in 30 min. Samples were injected using a Thermo Finnigan micro AS autosampler and all separations were done through a Thermo Finnigan Surveyor MS Pump. A Thermo LTQ-XL mass spectrometer with an electrospray ionization source was used. Mass spectra were acquired in negative polarity. The capillary temperature was set at 275 °C while the spray voltage was set at 4 kV.

After in-gel trypsin digestion, we analyzed peptide mixtures by capillary liquid chromatography-mass spectrometry (LC-MS) on an Eksigent 1D plus LC with a MicroAS autosampler (Dublin) online with an Orbitrap Velos MS system (Thermo Fisher Scientific, Inc.). We analyzed four sample types by 1D LC-MS for each quantification measurement consecutively on the same analytical column. The four sample types were: sample 1) WT expressing untagged SUMO; sample 2) WT expressing His₈SUMO; sample 3) mcm10-1 mutants expressing untagged SUMO; sample 4) mcm10-1 mutants expressing His₈SUMO. The quantitative analysis strategy included the following sample sets: A) triplicate injections of samples 1 to 4 analyzed in random order in MS1 (survey scan) only; B) directed MS/MS with inclusion lists for analytes differentially quantified between samples 2 and 4 that were undetected in control samples 1 and 3. Details of the trypsin digestion, LC-MS/MS, generetaion of an inclusion list, database searching, matching quantification and identification runs, optimization of directed MS runs and DDA analysis are in Supplemental Experimental Procedures. The proteomics data have been deposited to the ProteomeXchange Consortium (Vizcaino et al., 2014 (link)) via the PRIDE partner repository (PRIDE: PXD002607).

Oligonucleotide separations were performed on a Thermo Finnigan Surveyor MS Pump. Samples were injected using a Thermo Finnigan micro AS auto sampler. RNase digestion products were separated on an Waters XBridge™ C18 column (3.5 um, 1.0 × 150 mm) with MPA of 200 mM HFIP, 8 mM triethylamine (TEA) in water, pH 7.0 and MPB of 50% MPA and 50% methanol at a flow rate of 30 μL/min. RNase digestion products were eluted using a gradient from 5 %B to 20 %B in 5 min, followed by 1.7% increase of B/min until 95 %B. NOTE: HFIP and TEA will contaminate the HPLC and mass spectrometer ionization source. Removing this contamination is time consuming, so the use of a dedicated platform for RNA modification mapping is recommended.