Nuclear Structure and Function Research Group

All files mentioned below are available at https://proteomecommons.org/tranche/. The same set of files are available at http://users.path.ox.ac.uk/~pcook/Data/ContentOfFactories.html .

Three experiments were conducted, and data for all three are presented here. Data from experiment 1 was mainly discussed in the paper, as results from experiment 2 (where spectral indices were not analyzed) and experiment 3 (where complex I was not isolated) were similar.

EXPERIMENT 1
I. "RAW files" from the mass spectrometer
(i) "pol I"
After fractionation on 2D gels, the region rich in polymerase I was excised, loaded on a 4-15% SDS-acrylamide gel, and subjected to electrophoresis. The whole lane was excised, cut into 10 pieces, and each piece treated with trypsin; the resulting peptides were then extracted, vacuum dried, and injected (1 injection/sample) into a nanoHPLC system coupled to a Thermo LTQ Orbitrap mass spectrometer. Therefore, the 10 resulting raw data files are derived from one biological sample. They were converted to .mzXML format.
(ii) "pol II"
As for "pol I", except that (i) proteins in the piece from the 2D gel were subjected briefly to electrophoresis so that all denatured proteins just entered the resolving gel, (ii) then the whole sample was excised as one gel piece, and (iii) there were 2 injections/sample into the nanoHPLC system; therefore, there are two resulting raw data files.
(iii) "pol III"
As for "pol I", except that there were 3 injections/sample into the nanoHPLC system; therefore, there are three raw data files.
II. Output files from the CPFP
Raw data files (see "RAW files") in .mzXML format were submitted to the Central Proteomics Facilities Pipeline (CPFP). CPFP uses multiple search engines, modeling tools, and target-decoy validation to provide peptide/protein identifications with high confidence. One file for each of the 3 complexes is presented in 3 formats: (i) 3 files in the "protein XML" folder, (ii) 3 files in the "peptide XML" folder, and (iii) 3 files in the "protein Excel" folder.
III. Output from "ProteinCentre"
To analyze complex content, protein identifications were exported from the CPFP into "ProteinCenter" (Proxeon); FDR filters of 0.82%, 0.8%, and 0.84% (the average FDRs of each dataset) were maintained throughout analysis. The resulting list of proteins for each complex (pol I, pol II and pol III) is presented here, together with a comparison file.
IV. Quantitative analysis using the spectral index method
Label-free relative quantitation of proteins within samples was performed using the normalized spectral index (SI) method which combines three MS abundance features (peptide count, spectral count, and fragment-ion intensity. SIs were calculated using the output from one search engine (i.e., "Mascot", using the default significance setting of <0.05) and a script available on request. Lists of abundancies in complexes I, II, and III are presented here.

EXPERIMENT 2
I. "RAW files" from the mass spectrometer
After fractionation on 2D gels, the region rich in polymerase I, II, or III was excised, loaded on a 7% SDS-acrylamide gel, and subjected briefly to electrophoresis so that all denatured proteins just entered the resolving gel; then the whole sample was excised as one gel piece, and there were 3 injections/sample into the nanoHPLC system. Therefore, there are three resulting raw data files for each of complexes I, II, and III.
II. Output files from the CPFP
Raw data files (see "RAW files") in .mzXML format were submitted to the CPFP. One file for each of the 3 complexes is presented in 2 formats: (i) 3 files in the "peptide XML" folder, and (ii) 3 files in the "protein Excel" folder.
III. Output from "ProteinCentre"
To analyze complex content, protein identifications were exported from the CPFP into "ProteinCenter"; FDR filters of 0.8% for each dataset were maintained throughout analysis. The resulting list of proteins for each complex (pol I, pol II and pol III) is presented here.

EXPERIMENT 3
I. "RAW files" from the mass spectrometer
After fractionation on 2D gels, the region rich in polymerase II or III was excised, loaded on a 7% SDS-acrylamide gel, and subjected briefly to electrophoresis so that all denatured proteins just entered the resolving gel; then the whole sample from one complex was excised as two gel pieces, and there were 3 injections/sample into the nanoHPLC system per gel piece. Therefore, there are six resulting raw data files for complex II (and the same for complex III).
II. Output files from the CPFP
Raw data files (see "RAW files") in .mzXML format were submitted to the CPFP. One file for each of the 2 complexes is presented in 2 formats: (i) 2 files in the "peptide XML" folder, and (ii) 2 files in the "protein Excel" folder.
III. Output from "ProteinCentre"
To analyze complex content, protein identifications were exported from the CPFP into "ProteinCenter"; FDR filters of 0.75% for complex II and 0.65% for complex III were maintained throughout analysis. The resulting list of proteins for each complex (pol I, pol II and pol III) is presented here.
IV. Quantitative analysis using the spectral index method
Label-free relative quantitation of proteins within samples was performed using the normalized spectral index (SI) as above. Lists of abundancies in complexes II and III are presented here.

Top | Home | Maintained by Peter Cook |