Get FP concentrations using ECmax method — get_conc

Get protein's concentration from a dilution series measured with an A200-1000 absorbance spectrum. Based on get_conc_A280, but uses "ECmax", the FPbase-stated extinction coefficient for the FPbase-stated maximal excitation wavelength (which usually corresponds to its maximal absorbance wavelength). ... Function expects an input such as the csv file exported from plot_absorbance_spectrum called '_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. ... Function uses fpcountR::get_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 csv file is built 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.

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,
  filename
)

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 `plot_absorbance_spectrum3`, 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.
filename	filename of csv file from fpbase

Examples

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", filename = "mCherry_properties.csv")