Cross-Linking/mass spectrometry (CLMS) analysis of RNA polymerase II
The mass spectrometry data of a previous publication (Chen et.al. 2009 doi:10.1038/emboj.2009.401) were reprocesed and is used for method developing, teaching and training purpose.
Sample Processing Protocol
Endogenous complete Pol II was purified as described earlier (Sydow et al, 2009) except that the final gel filtration step was performed in presence of buffer B (10 mM HEPES pH 8.0, 200 mM potassium acetate, 1 mM EDTA, 1 mM DTT, 10% glycerol). Fractions that contained pure and stoichiometric Pol II were concentrated to 0.7 mg/ml and flash-frozen in liquid nitrogen in buffer B containing 10% glycerol. Protein cross-linking The mixing ratio of BS3 to complex was determined for Pol II using 2.5 µg aliquots and using a protein-to-cross-linker molar ratio of 1:200, 1:600, 1:1800, 1:5400, and 1:16 200, respectively (Supplementary Figure S1). As the best condition we chose the ratio that was sufficient to convert most of the individual Pol II subunits into a high molecular weight band as judged by SDS–PAGE. The purified Pol II complex (50 µl containing 35 µg) was mixed with 150 µg BS3 (Thermo Fisher Scientific) dissolved in 70 µl cross-link buffer (10 mM HEPES pH 8.0, 200 mM potassium acetate) and incubated on ice for 2 h. The reaction was stopped by adding 1 µl of 2.5 M ammonium bicarbonate for 45 min on ice. The reaction mix was separated on a NuPAGE 4–12% Bis–Tris gel using MES running buffer and Coomassie blue stain. Sample preparation for MS analysis Bands from the SDS–PAGE gel corresponding to cross-linked complexes were excised and the proteins reduced/alkylated and digested using trypsin following standard protocols. Pol II cross-linked peptides were fractionated using SCX-StageTips (Ishihama et al, 2006) following the published protocol for linear peptides (Rappsilber et al, 2007) and desalted using StageTips (Rappsilber et al, 2003) before MS analysis. Mass spectrometry Peptides were loaded directly onto the analytical column, packed with C18 material (ReproSil-Pur C18-AQ 3 µm; Dr Maisch GmbH, Ammerbuch-Entringen, Germany) using a self-assembled particle frit into the spray emitter (Ishihama et al, 2002), at a flow rate of 0.7 µl/min. A linear gradient going from 5% acetonitrile in 0.5% acetic acid to 23% acetonitrile in 0.5% acetic acid in 90 min eluted the peptides at 0.3 µl/min into an LTQ-Orbitrap classic. Peptides were analysed using a high/high strategy, detecting them at high resolution in the Orbitrap, and analysing their fragments also in the Orbitrap. FTMS spectra were recorded at 100 000 resolution. The three highest intensity peaks with a charge state of three or higher were selected in each cycle for iontrap fragmentation and Orbitrap detection of the fragments at 7500 resolution. Dynamic exclusion was set to 90 s and repeat count was 1. This resulted in a cycle time of up to 5 s and an average cycle time of 3 s.
Data Processing Protocol
Mass spectrometric raw files were processed into peak lists in .mgf format using MSconvert (Proteowizard version 3.0.6867) with “peak picking” filter enabled. Database search was performed using Xi (version 1.6.742, https://github.com/Rappsilber-Laboratory/XiSearch) for identification of cross-linked peptides. The peak lists were searched against a database containing sequences of all Pol II proteins. A decoy database containing inverting sequences of these proteins were generated by Xi automatedly as a part of the process. Search parameters were MS accuracy, 6 ppm; MS/MS accuracy, 20 ppm; enzyme, trypsin; specificity, fully tryptic; allowed number of missed cleavages, four; cross-linker, BS3; fixed modifications, carbamidomethylation on cysteine; variable modifications, oxidation on methionine, and modification hydrolysed and amidated BS3. The reactivity specificity for BS3 was assumed to be at lysine, serine, threonine, tyrosine and N-termini of polypeptidechains . FDR calculation was performed using XiFDR (Fischer, L., Rappsilber, J. 2017. Analytical Chemistry 89 (7) (version 1.1.27) with 5% link level FDR.