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Shot Noise

# load libraries
library(isoorbi) # for Orbitrap functions
library(dplyr) # for data wrangling

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
Isotopocule coverage

Visualize satellite peaks

Show peaks that were flagged as satellite peaks.

data |> orbi_plot_satellite_peaks()
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
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
Isotopocule ratios vs M0

Visualize shot noise

shot_noise |> orbi_plot_shot_noise()
amino acids shotnoise
amino acids shotnoise

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They may not be fully stable and should be used with caution. We make no claims about them.
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