Psm Annotation And Ion Matching

PSM Annotation and Ion Matching

Summary

Annotate peptide-spectrum matches (PSMs) by matching observed MS2 fragment ions (m/z, intensity, charge) to theoretical B and Y ion series calculated for a peptide sequence at specified isotopic enrichment. This skill enables validation and interpretation of fragment assignments in both unlabeled and stable-isotope-labeled proteomics data.

When to use

When you have an MS2 scan with observed peak data (m/z, intensity, charge state), a known peptide sequence, and need to determine which observed ions correspond to B and Y fragment ions. Apply this skill particularly when working with natural abundance (1.07% 13C) or enriched stable-isotope labeled samples where isotopic envelope matching is critical for accurate PSM interpretation.

When NOT to use

• Input is an already-validated PSM table from a database search engine (e.g., Sipros or Percolator output); use only if manual re-annotation or isotopic validation is required.
• The observed spectrum contains only MS1 data or precursor-level information; B and Y ion annotation requires MS2 spectra.
• Peptide sequence is unknown or ambiguous; annotatePSM requires a definite sequence to compute theoretical fragment envelopes.

Inputs

• MS2 scan data (m/z, intensity, charge state per peak)
• Peptide amino acid sequence
• Precursor m/z and charge state
• Isolation window width (Da)
• Isotope type and natural abundance or enrichment level (e.g., 0.0107 for natural 13C)

Outputs

• ExpectedBYions table with matchedIndices (non-negative when matched, negative when unmatched)
• residuePositions indicating amino acid positions contributing to each fragment ion
• Annotated fragment ion list with B/Y ion type and theoretical m/z assignments
• Tabular export of matched ions with intensity and residue position information

How to apply

Load the experimental MS2 scan data (m/z, intensity, charge) and peptide sequence using Aerith's file parsing interface (supporting FT2, mzML, or MGF formats via mzR integration). Calculate theoretical B and Y ion isotopic envelopes for the peptide using sequential convolution, specifying the isotope type (C13), natural abundance probability (0.0107 for unlabeled; adjust for labeled samples), fragment charge states (typically 1–2), precursor m/z, and isolation window width (e.g., 4.0 Da). Run annotatePSM to match experimental ions against the theoretical envelopes. Extract the matched-ion subset where matchedIndices are non-negative and residuePositions indicate which amino acids contribute to each observed peak. Validate results by confirming coverage of expected B and Y series and checking for chimeric or noise peaks outside the isolation window.

Related tools

• Aerith (R package for reading MS scans, calculating theoretical isotopic envelopes for peptide B/Y ions, executing annotatePSM annotation, and visualizing PSM results) — https://github.com/xyz1396/Aerith
• Raxport (Converts Thermo RAW files to FT2 format for input to Aerith's file parsing interface) — https://github.com/xyz1396/Raxport.net
• mzR (Bioconductor package integrated with Aerith to parse mzML and MGF spectral files)
• MSnbase (Bioconductor package integrated with Aerith to parse pepXML PSM files)
• Sipros (Peptide search engine that generates TSV PSM input files readable by Aerith for annotation and validation) — https://github.com/thepanlab/Sipros4
• R (Language and runtime for executing Aerith and dependent packages)

Examples

Load scan 107728 from FT2 file and peptide HSQVFSTAEDNQSAVTIHVLQGER; calculate B/Y ion envelopes at 1.07% 13C using Aerith; run annotatePSM with precursor m/z, 4.0 Da isolation window, and fragment charges 1–2; export matched ions with residue positions to TSV.

Evaluation signals

• Matched ions have non-negative matchedIndices and residuePositions corresponding to valid amino acid positions in the peptide sequence.
• B and Y ion series coverage is contiguous or near-contiguous (no unexplained gaps in expected fragment positions).
• Theoretical m/z values of matched fragments agree with observed m/z within the instrument's mass accuracy tolerance (e.g., <5 ppm for Orbitrap).
• Isotopic envelope fine structure (if present) aligns with expected 13C multiplet patterns for the specified enrichment level.
• Unmatched peaks fall outside the isolation window, are below noise threshold, or correspond to neutral loss or immonium ions not captured by B/Y ion model.

Limitations

• annotatePSM matches only B and Y ion series; neutral loss ions (e.g., -H2O, -NH3), immonium ions, or internal fragments are not annotated.
• Matching accuracy depends on accurate precursor m/z and isolation window specification; incorrect window width can exclude or include off-target ions.
• Sequential convolution algorithm for isotopic envelopes may lose fine-structure detail in highly enriched samples; Monte Carlo simulation is available for fine isotopic structures but at higher computational cost.
• Performance on very large peptides (>40 amino acids) or highly complex spectra with many overlapping isotopic envelopes has not been detailed in the article.

Evidence

• [other] annotatePSM accepts observed peak data (m/z, intensity, charge), peptide sequence, fragment charge states (1:2), isotope type (C13), isotope probability (0.0107 for natural abundance): "annotatePSM accepts observed peak data (m/z, intensity, charge), peptide sequence, fragment charge states (1:2), isotope type (C13), isotope probability (0.0107 for natural abundance)"
• [other] returning an ExpectedBYions table where matched fragments have non-negative matchedIndices and residuePositions indicating which amino acid positions contribute to observed peaks: "returning an ExpectedBYions table where matched fragments have non-negative matchedIndices and residuePositions indicating which amino acid positions contribute to observed peaks"
• [other] Calculate theoretical B and Y ion isotopic envelopes for the peptide at 1.07% 13C enrichment using sequential convolution algorithm for peptide fragments: "Calculate theoretical B and Y ion isotopic envelopes for the peptide at 1.07% 13C enrichment using sequential convolution algorithm for peptide fragments"
• [abstract] employs efficient algorithms—including sequential convolution for peptide fragments, Monte Carlo simulation for fine isotopic structures, and FFT-based methods for metabolites—to accurately generate: "employs efficient algorithms—including sequential convolution for peptide fragments, Monte Carlo simulation for fine isotopic structures, and FFT-based methods for metabolites"
• [intro] Aerith accepts spectral data files in multiple formats, including Raxport-processed FT2, mzML, and MGF files, as well as pepXML and PIN files (Percolator outputs), and TSV files from the Sipros: "Aerith accepts spectral data files in multiple formats, including Raxport-processed FT2, mzML, and MGF files, as well as pepXML and PIN files (Percolator outputs), and TSV files from the Sipros"
• [intro] calculate the theoretical isotopic peak envelope of peptide precursors and their B Y ions: "calculate the theoretical isotopic peak envelope of peptide precursors and their B Y ions"
• [intro] Integration with the mzR package from Bioconductor allows direct parsing of mzML and MGF files: "Integration with the mzR package from Bioconductor allows direct parsing of mzML and MGF files"
• [intro] Aerith enables visualization and manual validation of both unlabeled and SIP-labeled peptide-spectrum matches (PSMs), facilitating interpretation of complex mass spectrometry data: "Aerith enables visualization and manual validation of both unlabeled and SIP-labeled peptide-spectrum matches (PSMs), facilitating interpretation of complex mass spectrometry data"