get_conc_ECmax: Get FP concentrations using ECmax method

In ec363/fpcountr: Fluorescent Protein Calibration For Plate Readers

Get FP concentrations using ECmax method

Description

Get protein's concentration from a dilution series measured with an absorbance spectrum. Based on get_conc_a280(), but uses the FPbase-stated extinction coefficient (EC) for the FPbase-stated maximal excitation wavelength (which usually corresponds to its maximal absorbance wavelength), which we call the protein's 'ECmax', in order to convert absorbance values to concentrations. Expects 'processed' data such as that produced by process_absorbance_spectrum(), with a file name ending ⁠_processed.csv⁠, which contains values corrected for path length and normalised to blanks as a column called normalised_cm1_value, but retains replicate data containing positional (well) information required for exporting predicted concentrations at the end of this function. Uses get_fpbase_properties() to get FPbase EC in M-1cm-1 and wavelength, and converts it to an ECmax mass extinction coefficient in ⁠(mgml)-1cm-1⁠ using the MW (worked out from protein_seq and fpcountr::get_mw). Then the function uses the EC_max_mgml to work out the concentration of protein in each well, using three correction methods. Instead of using the normalised data directly, the values used are based on a LOESS fit through the absorption spectra to minimise fluctuations due to noise. Finally, linear models are fitted to each concentration prediction method, and a dataframe is built, returned and saved, containing predicted concentrations according to the user's chosen correction method. Plots showing each of the analytical steps are saved concurrently. Troubleshooting: for 'incompatible lengths' errors, adjust xrange to avoid noisy wavelengths.

Usage

get_conc_ecmax(
  protein_slug,
  protein_seq,
  processed_spectrum_csv,
  wells_to_remove = NULL,
  xrange = c(250, 800),
  corr_method = "none",
  wav_to_use1 = 340,
  wav_to_use2 = 333,
  outfolder,
  csv_only = FALSE
)

Arguments

protein_slug	character string of protein name in 'slug' form to match slug of FPbase entry.
protein_seq	character string of protein sequence using 1-letter code. Required for MW calculation.
processed_spectrum_csv	Path to CSV file of a processed absorbance spectrum. Processing should be done with process_absorbance_spectrum(), which corrects for path lengths and normalises to blank wells.
wells_to_remove	list of wells to remove before analysis. Defaults to NULL.
xrange	list of two numerical values corresponding to the wavelength range to keep when fitting the LOESS model across the absorbance spectrum. By default these values are 250nm and 800nm but where the data at the UV range is noisy, adjusting the xrange can prevent errors in the fitting.
corr_method	string corresponding to type of correction method to use for the data to remove contribution of light scatter. Options are none, baseline and scatter. Method none applies no correction. Method baseline subtracts the absorbance value of the wavelength supplied in wav_to_use1. Method scatter subtracts a fraction of the absorbance value of the wavelength supplied in wav_to_use2 according to scatter theory (details in section).
wav_to_use1	numerical value of wavelength (nm) to use for baseline correction. Defaults to 340nm.
wav_to_use2	numerical value of wavelength (nm) to use for scatter correction. Defaults to 333nm.
outfolder	path to folder where output files should be saved. Defaults to current working directory.
csv_only	logical. Saves only CSV files as outputs when TRUE. Defaults to FALSE.

Examples

## Not run: 
  ecmax_concs <- get_conc_ecmax(
    protein_slug = "mcherry", protein_seq = protein_seq,
    processed_spectrum_csv = "abs_parsed_processed.csv",
    corr_method = "scatter", wav_to_use1 = 700, wav_to_use2 = 315,
    outfolder = "protquant_ecmax/mCherry_T5N15pi"
  )

## End(Not run)

ec363/fpcountr documentation built on Nov. 29, 2024, 12:03 p.m.