Measurement of standards in different LC/MS configurations

This repository contains measurement of sets of standards on LC-MS systems with specific configurations. The aim is to provide approximate retention times (RTs) and ions for the standard compounds in the respective setups.

The first definition of retention times and ions for all standards was performed manually by Mar Garcia-Alloy. In the new workflow we aim to use the xcms and Spectra package to refine these and to determine also all additional adducts created/measurable from each compound. In the workflow we first:

• Identify chromatographic peaks in each file for one standard mix.
• Plot each chromatographic peak.
• Perform correspondence analysis to group each chromatographic peak to a feature.

Analysis workflow files

• match-standards-mix01.Rmd: matching and identifying signal from standards of mix 01. This includes preprocessing and ultimately defines the retention time, the measured ions and related MS/MS spectra of the standards.

Data tables

• internal_standards_files.txt: table indicating which mzML files correspond to the experiment in which internal standards (solved in water) were repeatedly measured (from September 2019).
• internal_standards.txt: table containing formulas (or neutral exact masses), highest ions in each mode (POS & NEG), and retention times for each standard.
• standards_dilution_files.txt: table indicating which mzML files correspond to the experiment Dynamic Range in which sets of standards (solved in water) were measured (in different concentrations).
• standards_dilution.txt: table containing formulas (or neutral exact masses), highest ions in each mode (POS & NEG), and retention times for each standard.
• exclusion_mz.txt: table containing the (exact) masses of mz values that probaly are noise / backgroun ions. This is a "dynamic" table that is being updated along the time.
• std_serum_files.txt: table containing the mzML files from the re-measurement of the standard mixes in January 2020. This contains:
  • measurement of standard mixes in water.
  • measurement of standard mixes in serum (QC CHRIS Pool samples). All mixes are added in a low and high concentration, for the high concentrations also DDA MS2 data was measured with two different collision energies.

Contributors

• Andrea Vicini
• Vinicius Verri Hernandes
• Mar Garcia-Aloy
• Johannes Rainer (contact)

Old R files

• preprocessing-standards.Rmd: preprocessing of LC-MS(/MS) data for the standards data set.
• EIC_superposed.Rmd: Markdown file for printing the EIC of one compound using all samples. The plots are saved in the folder plots.
• dinamic_range.Rmd: Markdown file for getting the calibration curves (and their corresponding plots) of each standard in each ionization mode (i.e., polarity). Plots are saved in the folder images/linearity and data in the text file dinamic_range_POL.txt.
• get_rt.Rmd: Markdown file for obtaining the RTs of the maximum peak corresponding to each compound (considering its exact mass). This serve as a starting point for define the RT of each compound. At the begining we have to specify the following parameters:
  • study: specify which of the 2 studies we want to focus (i.e., "internal_standards" or "standards_dilution").
  • mixnum: in case we specified study <- "standards_dilution", specify to which MIX we want to focus.
  • polarity: specify in which polarity ("POS" or "NEG") we want to focus.
• EIC_manually.R: R file for plot the EIC for a specific compound in both ionization modes.
• spectras_max_ions.Rmd: Markdown for printing the "cleaned" spectras of all compounds in a sample (i.e., after excluding the mz values specified in the file exclusion_mz.txt)
• spectra_manually.R: R file for plot the spectra for a specific compound.
• internal_standards_evaluation.Rmd: Markdown file that plot a graph with 1 boxplot / injection and all its
  measures, and that generates a pdf file with the trends of each compound along the samples.
• standard_dilution_dose_response.Rmd: Markdown file that generates a pdf file showing the linearity of each compound.
• exclusion_mz.R: R code for generate the table exclusion_mz.txt.

Matrix effects

In the folder std_serum there are the files used for analyse the experiments about IS and STDs (injected in different MIXs) at 2 different concentrations in water and in QC-serum samples.
The code RT_matrix_effect plots all the EICs present in the sample (ie, IS or MIX 01-20) in the folder images.
In that folder there is also the code MS2_plot to search a MS2 spectrum for a specific mz-rt value