Stable isotope labeling quantification

stable-isotope-labeling-quantification

Summary

Automated extraction and quantification of isotopologue abundances from high-resolution mass spectrometry data (Orbitrap mzML files) using the autoQ function in isoSCAN. This skill enables measurement of 13C-labeled compound patterns with ppm mass error, peak area, and intensity metrics.

When to use

Use this skill when you have centroided high-resolution Orbitrap mzML files from stable isotope labeling experiments and need to measure isotopologue abundances (M+0, M+1, M+2, etc.) for a defined list of target compounds with 13C or other isotopic labels. Trigger conditions: (1) you have a formulaTable of target compounds with monoisotopic m/z, retention time, and molecular formula; (2) raw data has been converted to mzML format and centroided; (3) you need paired quantification outputs (abundance, ppm error, peak area, maxo intensity) for each isotopic form per sample.

When NOT to use

• Input is low-resolution MS data in profile format — use profile-format isoSCAN instead; autoQ requires centroided high-resolution input.
• Target compounds have not been converted to mzML format — autoQ depends on mzR, which requires mzML or mzXML; vendor-format files must be converted first.
• You need only peak detection without isotope quantification — use a general peak-picking tool instead; autoQ is optimized for multi-isotopologue extraction.

Inputs

• formulaTable data frame (columns: CompoundName, mz, RT, Formula, NumAtoms)
• High-resolution centroided Orbitrap mzML files
• enviPat isotopes object for isotope pattern definition

Outputs

• Quantification table with columns: CompoundName, m/z, abundance, Isotopologue designation (M+0, M+1, M+2), ppm mass error, maxo (maximum intensity), area (integrated peak area)

How to apply

Load a formulaTable data frame containing CompoundName, mz, RT, Formula, and NumAtoms columns using read.csv. Transform vendor-format raw data into centroided mzML format using Proteowizard MSconvert (essential for high-resolution data). Load the enviPat isotopes object for isotope pattern calculation. Call the autoQ function with parameters: labelatom='13C', mzerror or maxppm tolerance (typically 5–10 ppm for Orbitrap), RTwin (retention time window in seconds), minscans, SNR (signal-to-noise ratio threshold), and minwidth/maxwidth (peak width constraints). The function returns a table with columns for CompoundName, m/z, abundance, Isotopologue designation (M+0, M+1, M+2), ppm mass error, and paired maxo and area values for each isotopic form. Rationale: isoSCAN's autoQ is specifically designed to handle the complexity of high-resolution isotope pattern deconvolution using enviPat and enforces quality thresholds (SNR, peak width, scan count) to filter noise and false positives.

Related tools

• isoSCAN (R package that implements autoQ function for automated isotopologue extraction and quantification) — github.com/jcapelladesto/isoSCAN
• mzR (Reads centroided mzML/mzXML files into memory for isoSCAN processing)
• enviPat (Generates theoretical isotope patterns for targeted compound formulas to enable isotopologue deconvolution)
• Proteowizard MSconvert (Converts vendor-format raw MS data to centroided mzML format required by autoQ)

Examples

library(isoSCAN); formulaTable <- read.csv('targets.csv'); autoQ(formulaTable, mzmldir='./centroided_mzML/', labelatom='13C', maxppm=5, RTwin=30, SNR=3, minscans=10, minwidth=0.1, maxwidth=0.5)

Evaluation signals

• Quantification table contains all expected compounds from formulaTable without missing rows (100% matching rate).
• ppm mass error values fall within specified tolerance (e.g., all ≤ 5 ppm for Orbitrap high-resolution).
• Isotopologue designations (M+0, M+1, M+2) are present and correctly ordered by increasing mass; abundance values sum to ~100% or documented baseline.
• Peak area and maxo (maximum intensity) values are positive, non-zero, and consistent with SNR threshold applied (SNR ≥ user-specified minimum).
• Quality control plots (rawPlot, meanRawPlot) show no saturated peaks, excessive noise, or moving peaks within retention time window.

Limitations

• autoQ requires formulaTable Formula column to match the derivatized form of the compound; incorrect or incomplete formulas will fail isotope pattern matching.
• High-resolution data must be centroided before input; profile-format Orbitrap files will produce incorrect quantification.
• SNR and peak-width thresholds (minscans, minwidth, maxwidth) are user-configurable but lack default guidance; suboptimal choices may miss low-abundance isotopologues or include noise.
• enviPat isotope patterns are calculated assuming natural or specified labeling; non-uniform labeling or enrichment isotopes not in enviPat's database may not be handled correctly.

Evidence

• [intro] The package is designed to automatically extract the abundances of isotopologues of a targeted list of compounds.: "The package is designed to automatically extract the abundances of isotopologues of a targeted list of compounds."
• [intro] For high-resolution data, centroiding should be used.: "In the case of High-resolution, please use centroiding (e.g. peakPicking= True in MSconvert)"
• [intro] formulaTable must contain specific columns in no particular order.: "formulaTable must contain the following column names in no specific order: * CompoundName * mz * RT * Formula * NumAtoms"
• [intro] autoQ function parameters and their meaning.: "This parameters refer to peak width and number of scans recorded, together with signal-to-noise ratio and mass error."
• [other] Output structure of autoQ for high-resolution Orbitrap data.: "The autoQ function returns for high-resolution Orbitrap data a table with columns for CompoundName, m.z, abundance, Isotopologue designation (M+0, M+1, M+2), ppm mass error, and paired maxo and area"