Model Peptide (MRFA)

This is an example for shot-noise analysis from Kantnerová et al. (2023).

Data

Load data

# load and process data
data <- 
  # load file included in isoorbi package
  system.file(package = "isoorbi", "extdata", "testfile_shotnoise.isox") |>
  orbi_read_isox() |>
  # check data for satellite peaks
  orbi_flag_satellite_peaks() |>
  # make sure isotopocules are present in (almost) all scans, otherwise
  # shot noise analyses can be inaccurate
  orbi_flag_weak_isotopocules(min_percent = 90) |> 
  # see if there are any AGC outliers
  orbi_flag_outliers(agc_fold_cutoff = 2)

This indicates that there were a few satellite peaks and weak isotopocules but no outliers.

Visualize isotopocule coverage

Show the isotopocule coverage for the different amino acids, thus highlighting which isotopocules were detected in which scans. Note that 2H was highlighted as weak for Alanine, Arginine, and Phenylalanine. This is because of the high cutoff (90%) in orbi_flag_weak_isotopocules() which is appropriate for shot-noise analyses but simply for inspecting ratios it would be reasonable to examine the 2H signal at least for Alanine.

data |> orbi_plot_isotopocule_coverage()

Isotopocule coverage

Visualize satellite peaks

Show peaks that were flagged as satellite peaks.

data |> orbi_plot_satellite_peaks()

Satellite peaks

Visualize intensity vs time

data |> orbi_plot_raw_data(
  isotopocules = "M0", 
  y = intensity,
  y_scale = "log"
)

Isotopocule intensity for M0 ion

Shot noise

Calculations

# calculate ratios vs basepeak
data_w_bp <- 
  data |>
  orbi_define_basepeak("M0")

# calculate shot noise
shot_noise <-
  data_w_bp |>
  orbi_analyze_shot_noise()

# export shot noise to an Excel file
shot_noise |>
  orbi_export_data_to_excel("shot_noise.xlsx")

Table

# example of the first few rows of the shot-noise calculations
shot_noise |>
  arrange(compound, isotopocule, scan.no) |>
  select(compound, scan.no, time.min, isotopocule,
         ratio, ratio_rel_se.permil, shot_noise.permil) |>
  head(10) |>
  knitr::kable()

compound	scan.no	time.min	isotopocule	ratio	ratio_rel_se.permil	shot_noise.permil
Alanine	1	0.059	15N	0.0062453	NaN	73.71127
Alanine	2	0.102	15N	0.0060919	12.43207	52.42764
Alanine	3	0.146	15N	0.0068807	37.74616	42.08467
Alanine	4	0.189	15N	0.0059526	32.61878	36.78579
Alanine	5	0.233	15N	0.0060549	26.57095	32.90351
Alanine	6	0.277	15N	0.0055040	29.99353	30.32862
Alanine	7	0.320	15N	0.0060244	25.50106	28.18264
Alanine	8	0.364	15N	0.0065182	23.43771	26.31067
Alanine	9	0.408	15N	0.0064846	21.36449	24.76418
Alanine	10	0.451	15N	0.0061980	19.10467	23.50447

Visualize ratios vs time

data_w_bp |> orbi_plot_raw_data(y = ratio)

Isotopocule ratios vs M0

Visualize shot noise

shot_noise |> orbi_plot_shot_noise()

amino acids shotnoise

Shot Noise