PEPTIDE

an interactive interpreter for reasoning about peptide MS/MS fragmentation. PEPTIDE allows users to inspect a tandem mass (MS/MS) spectrum and reason about whether a proposed peptide identification should be trusted or distrusted. it calculates theoretical b/y fragment ions and matches them to an observed spectrum. Rather than stopping at annotation, it explains why each ion exists, weighs the evidence a reviewer would demand, and suggests what experiment to run next.

---

Overview

most fragment-ion tools answer “what is this peak?” PEPTIDE is built around a different question: “how much should I believe this identification, and why?”

the application is a single, self-contained web page. given a peptide sequence and an observed peak list, it:

• computes theoretical fragment ions from monoisotopic amino-acid masses
• matches them to observed peaks within a user-defined tolerance
• draws an interactive, annotated spectrum and a sequence-coverage map

the interface follows the scientific method as a workflow: Hypothesis → Observation → Evidence → Interpretation → Confidence.

---

workflow

flowchart LR
    A[Hypothesis<br/>sequence · charge · method · tolerance] --> B[Observation<br/>annotated MS/MS spectrum]
    B --> C[Evidence<br/>coverage map · ion ladders]
    C --> D[Interpretation<br/>reviewer questions · chemistry]
    D --> E[Confidence<br/>trust / distrust judgement]
    E --> F[Design a better experiment]

Loading

---

installation

PEPTIDE is a single HTML file with no build step.

git clone https://github.com/sprudhvi728/peptide.git
cd peptide
# open the file in any modern browser:
open peptide.html      # macOS
# or: xdg-open peptide.html   (Linux) / start peptide.html (Windows)

alternatively, download peptide.html and open it directly, or serve the folder locally (e.g. python3 -m http.server) and visit the page.

note: the page loads React, Plotly, and Babel from a public CDN, so an internet connection is required the first time it is opened. No data leaves the browser.

---

required input format

PEPTIDE currently accepts two inputs, entered directly in the application:

1. peptide sequence — one-letter amino-acid codes (the 20 standard residues), e.g. PEPTIDE.

2. observed spectrum (optional) — a pasted peak list, one peak per line, as m/z,intensity pairs:

   148.06,45
   263.09,80
   376.17,100
   

   whitespace-separated values are also accepted. If no spectrum is provided, PEPTIDE displays only the theoretical prediction.

---

analysis pipeline

1. parse the peptide sequence into residues (parsePeptide).
2. enumerate theoretical b and y ions at the requested charge states, including licensed neutral losses (calculateBIons, calculateYIons, calculateNeutralLosses, calculateMz).
3. match theoretical ions to observed peaks within the selected tolerance, retaining all candidate annotations per peak (matchObservedPeaks).
4. score the match: coverage, ladder length, explained intensity, and mass-error statistics (calculateCoverage, and metric helpers).
5. reason over the result: generate the reviewer questions, chemistry explanations, and experiment suggestions, and synthesise a confidence judgement.

all mass constants and formulas are defined transparently and commented in the source (peptide.html), including the proton, water, and ammonia masses and the monoisotopic residue table.

fragment ion formulas (monoisotopic)

• b ion neutral mass = Σ(residue masses of the N-terminal fragment)
• y ion neutral mass = Σ(residue masses of the C-terminal fragment) + H₂O
• observed m/z = (neutral mass + z · proton) / z

---

reference data

PEPTIDE does not query external sequence or spectral databases. all calculations use built-in tables:

• monoisotopic residue masses for the 20 standard amino acids,
• monoisotopic masses of the proton, water (H₂O), and ammonia (NH₃),
• a reserved constant for phosphoric acid (H₃PO₄) for planned phosphorylation support.

these values follow standard elemental monoisotopic masses as used throughout proteomics. because nothing is looked up remotely, results are fully reproducible offline once the page has loaded.

---

Region	Stage	Contents
left	hypothesis	sequence, precursor charge, fragmentation method, ion/charge selection, tolerance, spectrum input
center	observation → evidence	annotated interactive spectrum, coverage map, cleavage inspector, fragment ledger
right	interpretation → confidence	conclusions, reviewer questions, chemistry, confidence, experiment suggestions

---

biological interpretation

a fragment spectrum is only useful if it lets you make, or withhold, a biological claim with the right level of confidence. PEPTIDE frames its output accordingly:

• continuous, bilateral coverage (b and y ions bracketing each residue) is the basis for reading a sequence with confidence and for beginning to localize a modification, which additionally requires matched ions flanking the modified residue.
• partial or one-sided coverage may be enough to shortlist a peptide but not to attribute a function, variant, or modification to it — ambiguous regions are where a wrong residue or misplaced modification hides.
• sparse coverage or unexplained intense peaks should prompt caution: near-isobaric sequences and co-isolated precursors can mimic weak evidence.

the tool makes these consequences explicit so interpretation is tied to evidence rather than to a single score.

downloadable outputs

• annotated spectrum (PNG) — the interactive plot includes a toolbar (top-right) with a download as PNG option.
• fragment table and reasoning export (CSV / report)

---

intended use

PEPTIDE is intended for:

• hypothesis generation and manual inspection of MS/MS spectra,
• critical review of a proposed identification before or alongside a formal search.

not intended as a replacement for validated database search engines.

This tool was developed with Claude Code; the tool design, analysis workflows, and statistical logic are my own.

---

references

1. Roepstorff, P. & Fohlman, J. (1984). Proposal for a common nomenclature for sequence ions in mass spectra of peptides. Biomedical Mass Spectrometry, 11(11), 601.
2. Biemann, K. (1988). Contributions of mass spectrometry to peptide and protein structure. Biomedical & Environmental Mass Spectrometry, 16(1–12), 99–111.
3. Steen, H. & Mann, M. (2004). The ABC's (and XYZ's) of peptide sequencing. Nature Reviews Molecular Cell Biology, 5(9), 699–711.
4. Wysocki, V. H., Tsaprailis, G., Smith, L. L. & Breci, L. A. (2000). Mobile and localized protons: a framework for understanding peptide dissociation. Journal of Mass Spectrometry, 35(12), 1399–1406.
5. Breci, L. A., Tabb, D. L., Yates, J. R. & Wysocki, V. H. (2003). Cleavage N-terminal to proline: analysis of a database of peptide tandem mass spectra. Analytical Chemistry, 75(9), 1963–1971.
6. Syka, J. E. P., Coon, J. J., Schroeder, M. J., Shabanowitz, J. & Hunt, D. F. (2004). Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry. PNAS, 101(26), 9528–9533.
7. Olsen, J. V., Macek, B., Lange, O., Makarov, A., Horning, S. & Mann, M. (2007). Higher-energy C-trap dissociation for peptide modification analysis. Nature Methods, 4(9), 709–712.

---