flippant

Description

A package for the analysis of data provided by dithionite scramblase assays.

Author(s)

Johannes Graumann

Extract the case study dataset

Description

Extracts the data files used by the case study from the zip archive.

Usage

extract_case_study_data(exdir = ".", files = NULL)

Arguments

Value

A character vector of the extracted file paths is invisibly returned.

Author(s)

Richard Cotton

See Also

unzip

Examples

extract_case_study_data(tempfile("flippant"))

parse_FluorS_Essence_3.8_output

Description

Parse spectra from files provided by a Horiba fluorimeter (HORIBA Europe GmbH, Oberursel, Germany) using FluorS Essence v3.8

Usage

parse_FluorS_Essence_3.8_output(x = NULL)

Arguments

Details

A helper function to parse_fluorimeter_output.

Value

See parse_fluorimeter_output.

Author(s)

Johannes Graumann

See Also

parse_fluorimeter_output, parse_felix_32_output, parse_felix_gx_output, parse_manual_output

parse_felix_32_output

Description

Parse spectra from files provided by a QuantaMaster fluorimeter (Photon Technology International, Inc., Edison, New Jersey) using Felix32 v1.20

Usage

parse_felix_32_output(x = NULL)

Arguments

Details

A helper function to parse_fluorimeter_output.

Value

See parse_fluorimeter_output.

Author(s)

Johannes Graumann

See Also

parse_fluorimeter_output, parse_felix_gx_output, parse_FluorS_Essence_3.8_output, parse_manual_output

parse_felix_gx_output

Description

Parse spectra from files provided by a QuantaMaster fluorimeter (Photon Technology International, Inc., Edison, New Jersey) using FelixGX v4.1

Usage

parse_felix_gx_output(x = NULL)

Arguments

Details

A helper function to parse_fluorimeter_output.

Value

See parse_fluorimeter_output.

Author(s)

Johannes Graumann

See Also

parse_fluorimeter_output, parse_felix_32_output, parse_FluorS_Essence_3.8_output, parse_manual_output

parse_fluorimeter_output

Description

Parse fluorimeter spectra

Usage

parse_fluorimeter_output(
  spec_file = NULL,
  timepoint_of_measurement = NULL,
  n_averaging = 10,
  determine_zero_time = TRUE,
  adjust = TRUE,
  file_type = c("auto", "FelixGXv4.1.0.3096", "Felix32v1.20", "FluorSEssencev3.8",
    "manual")
)

Arguments

Details

A function to read fluorimeter output directly. Intended as a helper function to scramblase activity determinations from dithionite assays.

The function is currently capable to deal with input derived from QuantaMaster instruments (Photon Technology International, Inc., Edison, New Jersey)running software versions FelixGX v4.1 (see parse_felix_gx_output), Felix32 v1.20 (see parse_felix_32_output) as well as Horiba fluorimeters (HORIBA Europe GmbH, Oberursel, Germany) using FluorS Essence v3.8. The format used in a given file is divined from the data structure and appropriate internal parsing functions are called.

If requested the time point of dithionite addition to a sample is determined using pracma-supplied methodology and the acquisition time reset accordingly (0 henceforth corresponds to the time of addition).

Value

A data frame with two columns:

Time.in.sec

Numeric. Number of seconds since the start of experiment.

Fluorescence.Intensity

Numeric. Intensity of fluorescence (relative scale, no official unit).

If determine_zero_time and/or adjust are set to TRUE, the return value will have an attribute ZeroTimePoint corresponding to the determined time point of dithionite addition (always 0 (zero) where adjust == TRUE).

For Felix GX, if the file contains the information, the return value will also have an attribute WavelengthsInNanometres, which contains the excitation and emission wavelengths.

Author(s)

Johannes Graumann

See Also

scramblase_assay_input_validation, parse_felix_gx_output, parse_felix_32_output, parse_FluorS_Essence_3.8_output, parse_manual_output

Examples

library(magrittr)
# Extract example data
analysis_dir <- file.path(tempdir(), "flippant-case-study")
fluor_file <- extract_case_study_data(analysis_dir, "wildtypeEx1_0.txt")
# Parse an exemplary file
parse_fluorimeter_output(fluor_file, timepoint_of_measurement = 350) %>%
  str()

parse_manual_output

Description

Parse spectra from files provided in a manually assembled format.

Usage

parse_manual_output(x = NULL)

Arguments

Details

A helper function to parse_fluorimeter_output. The file in question is supposed to contain a tab-delimited table with the columns Time (sec) and Fluorescense Intensity.

Value

See parse_fluorimeter_output.

Author(s)

Johannes Graumann

See Also

parse_fluorimeter_output, parse_felix_gx_output, parse_felix_32_output

scramblase_assay_plot

Description

Functions for the presentation and evaluaton of dithionite scramblase assays

Usage

scramblase_assay_input_template(
  path = "scramblase_assay_input_template.txt",
  input_directory = NULL,
  overwrite = FALSE
)

scramblase_assay_plot(
  x,
  scale_to = c("model", "data"),
  ppr_scale_factor = 0.65,
  force_through_origin = TRUE,
  generation_of_algorithm = c(2, 1),
  split_by_experiment = TRUE,
  r_bar = 88,
  sigma_r_bar = 28
)

scramblase_assay_stats(
  x,
  scale_to = c("model", "data"),
  ppr_scale_factor = 0.65,
  force_through_origin = TRUE,
  generation_of_algorithm = c(2, 1),
  split_by_experiment = TRUE,
  r_bar = 88,
  sigma_r_bar = 28
)

scramblase_assay_traces(
  x,
  ppr_scale_factor = 0.65,
  time_min_sec = NA_real_,
  time_max_sec = NA_real_,
  adjust = TRUE,
  timepoint_of_measurement = 400,
  n_averaging = 10,
  annotate_traces = FALSE
)

Arguments

Details

The data.frame accepted by the majority of the functions a an R object or path to a corresponding file (x) must have the following mandatory columns:

Path:

Paths to existing and readable ASCII output files of a fluorimeter. See parse_fluorimeter_output for details and supported formats.

Protein Reconstituted (mg):

Self-explanatory. In the case of scramblase_assay_traces ONLY this may be abused by taking character values rather than the usually required numerics. Handy when e.g. plotting traces for "Liposomes" and "Proteoliposomes", rather than defined PPRs.

Further (facultative) columns are:

Fluorescence Assay Vol. w/o DT (ul):

Volume of the fluorescence assay prior to addition of ditihionite (defaulting to 2000).

Fluorescence Assay Vol. with DT (ul):

Volume of the fluorescence assay after the addition ditihionite (defaulting to 2040).

Lipid in Reconstitution (mmol):

Self-explanatory. For the standard phospholipid experiment defaulting to 0.0045 (1 ml of a 4.5 mM solution).

Timepoint of Measurement (s):

The time to determine terminal fluorescence, calculated from the point when dithionite is added, in seconds, defaulting to 400).

Experiment:

Identifier for any given experiment. Used for facet_wrap during generation of ggplot output. All data with one Experiment identifier ends up on one plot/facet.

Experimental Series:

Identifier for a given series/graph (e.g. Extract and Depleted Extract). Used by color during generation of ggplot output to differentiate lines in the same plot/facet.

Based on Goren et al. (2014) and Ploier et al. (2016) data is processed as follows (the majority of the processing is split off into the internal function scramblase_assay_calculations):

• Input is format checked and defaults are injected for facultative parameters/columns as appropriate (see input data.frame format above). The internal function scramblase_assay_input_validation supplies this functionality.

• Fluorescence spectra are parsed using parse_fluorimeter_output. This includes automated determination of when dithionite was added to the sample using pracma-supplied methodology and resetting the acquisition time accordingly (0 henceforth corresponds to the time of addition).

• Pre-dithionite-addition Baseline Fluorescence is determined for each spectrum by averaging (median) over the 10 values preceding dithionite addition.

• Post-dithinonite-addition Minimum Fluorescence is determined for each spectrum by averaging (median) over the last ten datapoints \leq 400\,\mbox{s} (or Timepoint of Measurement (s), see above).

• The Minimum Fluorescence is volume-corrected based on Reaction Volume w/o DT (ul) and Reaction Volume with DT (ul) (see above).

• For each spectrum/datapoint a measured Fluorescence Reduction is calculated as

  1 - \left(\frac{\mbox{\small Minimum Fluorescence}}{\mbox{\small Baseline Fluorescence}}\right)

• A Relative Fluorescence Reduction is calculated in comparison to the liposomes-only/no-protein control).

• A Protein per Phospholipid (mg/mmol) ratio (PPR) is calculated. If ppr_scale_factor is not NULL, the value is scaled (divided) by that value to account for liposomes that remain inaccessible to reconstitution with scramblase molecules.

• Depending on split_by_experiment, data are split for parallel treatment using either Experimental Series (split_by_experiment = TRUE) or a combined Experimental Series/Experiment (split_by_experiment = FALSE) identifier (see above).

• A probability for a liposome holding \geq 1 scramblase molecules is calculated using

  \frac{y-y_0}{y_{\mbox{\scriptsize max}}-y_0}

  where y is the Relative Fluorescence Reduction and y_0 is the Relative Fluorescence Reduction in an experiment without addition of protein extract. Depending on the scale_to parameter, y_{\mbox{\scriptsize max}} is either the maximal Relative Fluorescence Reduction in the series (scale_to = "data") or derived from a mono-exponential fit to the data (scale_to = "model"). The latter (default) is a precaution for the case where the protein/phospholipid titration did not reach the plateau of the saturation curve.

• A monoexponential curve is fitted using nlsLM.

  If generation_of_algorithm is 1, the underlying formula is derived from Goren et al. (2014) and data is fitted to either

  p(\geq 1)=b\cdot(1-e^{-\frac{\mbox{\tiny PPR}}{a}})

  (if force_through_origin = TRUE; default) or

  p(\geq 1)=b-c\cdot e^{-\frac{\mbox{\tiny PPR}}{a}}

  (if force_through_origin = FALSE). The latter implies more degrees of freedom and occasionaly results in better fits to experimental data. Mechanistic implication, however, are unclear.

  If generation_of_algorithm is 2 (default), the more elaborate model put forth in Ploier et al. (2016) is employed, using either

  p(\geq 1)=b\cdot(\frac{1}{\sqrt{1+\sigma^2\cdot a \cdot x}})\cdot exp(\frac{-\bar{r}^2\cdot a \cdot x}{1+\sigma^2\cdot a\cdot x})

  (if force_through_origin = TRUE; default) or

  p(\geq 1)=b-c\cdot(\frac{1}{\sqrt{1+\sigma^2\cdot a \cdot x}})\cdot exp(\frac{-\bar{r}^2\cdot a \cdot x}{1+\sigma^2\cdot a\cdot x})

  (if force_through_origin = FALSE).

• Data split apart above are recombined and a ggplot object is assembled with the following layers:

  • Lines (geom_line) representing the monoexponential fit(s). color is used to differentiate Experimental Series.

  • If generation_of_algorithm is 1, segments (geom_segment) representing the PPR at which the fit constant a is equal to PPR. This \tau value has the implication that at this PPR all vesicles on average have one scramblase and 63% have one or more (i.e. are active). color is used to differentiate Experimental Series. Where generation_of_algorithm is 2, interpretation of a is less obvious and this layer is thus ommited in the plot.

  • Points (geom_point) representing the corresponding datapoints. color is used to differentiate Experimental Series.

  • Plots are finally facet_wraped by Experiment (if split_by_experiment = TRUE) and labels adjusted cosmetically.

Value

scramblase_assay_traces and scramblase_assay_plot return ggplot objects representing the raw fluorescence traces and a complete PPR plot, respectively. scramblase_assay_input_template generates a tab-delimited ASCII file in the file system and invisibly returns the path name. scramblase_assay_stats assembles (and prints) assay statistics as a data.frame.

Author(s)

Johannes Graumann

References

Menon, I., Huber, T., Sanyal, S., Banerjee, S., Barre, P., Canis, S., Warren, J.D., Hwa, J., Sakmar, T.P., and Menon, A.K. (2011) <DOI:10.1016/j.cub.2010.12.031>

Goren, M.A., Morizumi, T., Menon, I., Joseph, J.S., Dittman, J.S., Cherezov, V., Stevens, R.C., Ernst, O.P., and Menon, A.K. (2014) <DOI:10.1038/ncomms6115>

Ploier, B., Caro, L.N., Morizumi, T., Pandey, K., Pearring, J.N., Goren, M.A., Finnemann, S.C., Graumann, J., Arshavsky, V.Y., Dittman, J.S., Ernst, O.P., Menon, A.K. (2016). <DOI:10.1038/ncomms12832>

See Also

parse_fluorimeter_output nlsLM

Examples

library(magrittr)
library(ggplot2)
# Extract example data
analysis_dir <- file.path(tempdir(), "flippant-case-study")
extract_case_study_data(analysis_dir)
template_file <- file.path(analysis_dir, "inputTable.txt")
# Plot the spectral traces
scramblase_assay_traces(
  template_file,
  time_max_sec = 350,
  timepoint_of_measurement = 350)
# Plot the PPR plot(s) faceting by experiment
scramblase_assay_plot(template_file)
# Generate tabular results
scramblase_assay_stats(template_file)
# Plot the PPR plot(s) forgoing faceting by experiment
scramblase_assay_plot(template_file, split_by_experiment = FALSE)
# Generate tabular results
scramblase_assay_stats(template_file, split_by_experiment = FALSE)