Mass spectrometry method cycle verification

mass-spectrometry-method-cycle-verification

Summary

Verify that parallel reaction monitoring (PRM) or targeted MS/MS acquisition maintains consistent scan spacing across all monitoring cycles by extracting scan indices from raw Orbitrap files and validating inter-scan deltas. This skill ensures that the mass spectrometry method is executing as designed without drift or missed cycles.

When to use

When you have a Thermo Fisher Scientific Orbitrap .raw file and need to confirm that a targeted acquisition method (e.g., PRM targeting a specific precursor m/z) is achieving uniform cycle timing. Use this skill if you suspect incomplete or irregular scan spacing, or when validating a new LC-MS method's consistency across a large number of targeted scans.

When NOT to use

• Input is already a feature table or peptide-level quantitation result — use this skill only on raw scan-level data.
• The raw file contains only full-scan (MS1) data without targeted MS/MS cycles.
• The LC-MS method does not use fixed-precursor-ion targeting (e.g., untargeted data-dependent acquisition).

Inputs

• Thermo Fisher Scientific .raw file (e.g., 20181113_010_autoQC01.raw)
• Target precursor m/z and scan type filter pattern
• Expected cycle length (number of scans per complete targeting cycle)

Outputs

• Filtered scan index data frame (rows matching target scanType)
• Array of inter-scan delta values
• Summary report: count of PRM scans, observed deltas, pass/fail status

How to apply

Load the raw file using rawrr::readIndex() to retrieve the complete scan index table, which contains metadata for every scan including scanType and precursor m/z. Filter the index to retain only rows matching your target acquisition pattern (e.g., 'FTMS + c NSI Full ms2' with a fixed precursor m/z and HCD energy). Extract the scan numbers from the filtered rows and compute the difference between consecutive scan indices. Validate that all inter-scan deltas equal the expected cycle length (e.g., 22 scans for one complete PRM cycle targeting LGGNEQVTR++ at 487.2567 m/z). Document the number of PRM scans detected, observed delta values, and pass/fail status; any deviation from the expected delta indicates method drift, missed scans, or acquisition irregularities.

Related tools

• rawrr (R package that wraps RawFileReader .NET assembly; provides readIndex() function to extract scan metadata and scanType information from .raw files) — https://github.com/fgcz/rawrr
• RawFileReader (.NET assembly (C#) underlying rawrr; implements low-level binary file parsing and data extraction from Thermo Fisher Orbitrap .raw files) — https://github.com/thermofisherlsms/RawFileReader
• rawDiag (R package for LC-MS method optimization and quality control; can visualize scan-level metrics and detect acquisition irregularities) — https://github.com/fgcz/rawDiag

Examples

rawrr::readIndex(rawfile = "20181113_010_autoQC01.raw") |> subset(scanType == "FTMS + c NSI Full ms2 [email protected] [100.0000-1015.0000]") |> {function(df) { deltas <- diff(df$scan); all(deltas == 22) }}

Evaluation signals

• All inter-scan deltas equal the expected cycle length (e.g., 22 scans); any delta ≠ expected value indicates acquisition disruption.
• The count of filtered PRM scans matches the expected number of monitoring cycles across the LC-MS run duration.
• scanType filter matches the intended acquisition method (e.g., 'FTMS + c NSI Full ms2 [email protected] [100.0000-1015.0000]' for PRM).
• No gaps or missing scans in the filtered scan sequence; consecutive scan numbers form a contiguous arithmetic progression with the expected common difference.
• Summary report passes the 22-scan-cycle (or applicable cycle length) criterion, confirming one complete targeting cycle between consecutive monitored precursor ions.

Limitations

• Requires installation of RawFileReader .NET runtime executable and the rawrr R package; on Windows, the decimal symbol must be configured as '.' for proper data extraction.
• Applicable only to Thermo Fisher Scientific Orbitrap instrument data in .raw format; cannot be used on other vendor formats (e.g., mzML, Bruker .d).
• Does not detect instrument hardware failures or transient acquisition anomalies that do not alter scan spacing; focuses only on temporal regularity of scans, not their spectral quality or intensity.

Evidence

• [other] The delta between consecutive PRM scans targeting LGGNEQVTR++ (487.2567) is consistently 22 scans, representing one complete PRM cycle.: "The delta between consecutive PRM scans targeting LGGNEQVTR++ (487.2567) is consistently 22 scans, representing one complete PRM cycle."
• [other] Call rawrr::readIndex() on the 20181113_010_autoQC01.raw file to retrieve the complete scan index table.: "Call rawrr::readIndex() on the 20181113_010_autoQC01.raw file to retrieve the complete scan index table."
• [other] Filter the index data frame to retain only rows where scanType matches the PRM acquisition method pattern (e.g., 'FTMS + c NSI Full ms2' with a fixed precursor m/z and HCD energy).: "Filter the index data frame to retain only rows where scanType matches the PRM acquisition method pattern (e.g., 'FTMS + c NSI Full ms2' with a fixed precursor m/z and HCD energy)."
• [other] Extract the scan numbers from the filtered rows and compute the difference between consecutive scan indices.: "Extract the scan numbers from the filtered rows and compute the difference between consecutive scan indices."
• [readme] rawrr wraps the functionality of the RawFileReader .NET assembly: "rawrr wraps the functionality of the RawFileReader .NET assembly"
• [other] On Windows, the decimal symbol has to be configured as a '.'!: "On Windows, the decimal symbol has to be configured as a '.'!"