SWATHExperimentReporting

This manual describes the steps to create quality control plots and result tables after having performed a SWATH proteomics experiment with the Spectronaut application. This manual assumes that you are working on the MCN lab data analysis workstation in room WN-C348. This computer has been configured to run the SWATH analysis. If you would like to run the analysis scripts on your own computer, please contact Pim van Nierop.

For this analysis you need:

  • The output file of Spectronaut
  • The fasta file that was used to produce the spectral library in MaxQuant
  • The R script Spectronaut_analysis.R

Step 1

Make a copy of the Spectronaut_analysis.R in the working directory of your experiment.

Step 2

Open the Spectronaut_analysis.R file in RStudio. In the top section you find several parameters that you need to configure for your analysis. The parameters are:
  • path_spectronaut_output: the path and filename of the Spectronaut output file
  • file_fasta: the path and filename of the fasta file that was used to produce the spectral library
  • filter.qvalue.minimum: the minimum quality value (q-value) for peptides to be included in the protein quantification calculations
  • regex: an R-list with the mapping of experimental groups. The names of the list elements will correspond to the names of the experimental conditions as they will appear in the generated reports. The values of the list elements correspond to regular expressions (regexp) that reflect the names of experimental groups as they actually appear in the column headers of the raw experimental output. For instance, when two experimental groups are labeled KO and WT in the headers of the raw experimental data and you would like to represent them as GluR5 KO and Wild type, respectively, the regexp parameter must look as follows:

regex = list("GluR5 KO"="KO", "Wild type"="WT")

Please note that more complicated selections are possible. If you have labeled your experiments inconsistently (for instance GluKO and KO for the same experimental group) more complicated regexp expressions are possible. In the example situation you would use:

regex = list("GluR5 KO"="KO|GluKO", "Wild type"="WT")

If you are unsure please consult Pim van Nierop for assistance.

Step 3

Run the Spectronaut_analysis.R file in RStudio. This will generate three files that start with the #file name# of the output file of Spectronaut:
  • #file name# _plot.pdf: this file contains plots that are useful of quality control.
  • #file name# _FG.tsv: this file contains the numerical data at the peptide (fragment group) level. It is a tab-separated file that can be imported into any spreadsheet software (e.g. Excel).
  • #file name# _PG.tsv: this file contains the numerical data at the protein (protein group) level. It is a tab-separated file that can be imported into any spreadsheet software (e.g. Excel).

-- PimVanNierop - 2015-03-16

Topic revision: r2 - 2015-03-17 - 10:49:49 - PimVanNierop
 
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