Bio Proteomics Spectral Libraries

Version Compatibility

Reference examples tested with: koinapy 0.0.5+, ms2pip 4.0+, deeplc 3.0+, pandas 2.2+

Before using code patterns, verify installed versions match. If versions differ:

• Python: pip show <package> then help(module.function) to check signatures
• CLI: <tool> --version then <tool> --help to confirm flags

If code throws ImportError, AttributeError, or TypeError, introspect the installed package and adapt the example to match the actual API rather than retrying.

DIA Spectral Libraries -- Query Parameters That Are Only as Good as Their Empirical Calibration

"Build a spectral library for my DIA search" -> Assemble a table of peptide query parameters (precursor m/z, top fragment m/z plus relative intensities, normalized RT, optional CCS), then calibrate the predicted RT/CCS to the actual gradient/instrument -- because a DIA library is not whole spectra and an uncalibrated prediction extracts every peak group at the wrong time.

• Python: koinapy.Koina(...).predict(df) for Prosit/AlphaPeptDeep/MS2PIP/UniSpec fragment intensities and iRT served from Koina
• Python: deeplc.DeepLC().calibrate_preds(); .make_preds() for RT prediction of any modification
• Python: ms2pip.predict_batch(psms, model='HCD') for local fragment-intensity prediction
• CLI: EncyclopeDIA for empirical chromatogram libraries; EasyPQP/FragPipe for DDA-based libraries

Scope: this skill owns library generation (experimental, chromatogram, predicted, empirically-corrected), RT/CCS/NCE calibration, format conversion, and library QC/merge. Running the DIA search against the library is dia-analysis. Generating the DDA peptide identifications a DDA library is built from depends on peptide-identification. Modified-peptide and PTM-resolved library design routes to ptm-analysis. Quantifying and rolling up the search output is quantification. OUT OF SCOPE: acquisition-window design (fixed/variable/staggered/diaPASEF) and demultiplexing -- those belong to dia-analysis.

The Single Most Important Modern Insight -- A Predicted Library Is Only as Good as Its Empirical Calibration

1. A DIA library is peptide QUERY PARAMETERS, not spectra. Each entry is a precursor m/z, a handful of fragment m/z with RELATIVE intensities, a normalized RT, and optionally CCS -- the inputs to extract and score a co-eluting fragment-chromatogram peak group, not a lookup spectrum to match (Gillet 2012). The fragments and their relative intensities are the discriminating content; absolute intensity is irrelevant.

2. Fragment-intensity prediction is robust; predicted RT and CCS are in ARBITRARY model units and MUST be calibrated. HCD fragmentation is reproducible at matched collision energy, so predicted relative intensities transfer across instruments. But predicted iRT is an arbitrary scale and real RT depends on the exact column, gradient, temperature, and mobile phase. The catastrophic error: drop a predicted library straight into a search without anchoring its RT to observed RT (via iRT/CiRT spike-in peptides or a GPF-DIA empirical pass). Every peak group is then extracted at the wrong time, selectivity collapses, and IDs silently disappear with no error. The same holds for AlphaPeptDeep CCS against the timsTOF's measured 1/K0.

3. NCE (normalized collision energy) must match the predictor's training or intensities mismatch the real spectra. Intensity predictors are conditioned on NCE. Feed a value that differs from the instrument's effective NCE and predicted intensities diverge from reality, losing sensitivity with no error. Do not blindly use NCE=30 from a tutorial -- scan candidate NCE values, predict, and pick the one maximizing spectral contrast/correlation against a few real spectra.

Tool Taxonomy

Decision Tree by Scenario

Default when uncertain: a Koina-served predicted library (Prosit intensity + iRT) with explicit iRT/CiRT RT calibration and an NCE scan, exported to the search engine's native format.

Generate a Predicted Library via Koina

Goal: Produce fragment intensities and iRT for a peptide list without a local GPU or wet-lab library.

Approach: Send a DataFrame of peptide sequences, charges, and collision energies to a Koina-hosted model; the dead proteomicsdb endpoint is replaced by the Koina server. Network calls are shown; the runnable example operates on an in-memory table so it needs no network.

# Koina serves Prosit/AlphaPeptDeep/MS2PIP/UniSpec predictions; verify the
# koinapy constructor signature and input column names at runtime with help(Koina).
from koinapy import Koina
import pandas as pd

inputs = pd.DataFrame({
    'peptide_sequences': ['LGGNEQVTR', 'VEATFGVDESNAK'],
    'precursor_charges': [2, 2],
    'collision_energies': [30, 30]  # NCE; scan candidates and pick max spectral contrast
})

intensity_model = Koina('Prosit_2019_intensity', 'koina.wilhelmlab.org:443')
fragments = intensity_model.predict(inputs)  # mz, intensities, annotation per fragment

irt_model = Koina('Prosit_2019_irt', 'koina.wilhelmlab.org:443')
irt = irt_model.predict(inputs[['peptide_sequences']])  # arbitrary iRT units -- calibrate before use

Calibrate iRT to Observed RT

Goal: Map arbitrary-unit predicted iRT onto the run's real RT so peak groups extract at the right time.

Approach: Spike or detect anchor peptides (11 Biognosys iRT peptides, or CiRT endogenous peptides when no spike-in exists), fit a regression from library iRT to observed RT, and require a tight fit before trusting it. A global linear fit fails on nonlinear gradients -- fall back to LOWESS.

import numpy as np
from scipy import stats

IRT_PEPTIDES = {'LGGNEQVTR': -24.92, 'GAGSSEPVTGLDAK': 0.00, 'VEATFGVDESNAK': 12.39,
                'YILAGVENSK': 19.79, 'TPVISGGPYEYR': 28.71, 'TPVITGAPYEYR': 33.38,
                'DGLDAASYYAPVR': 42.26, 'ADVTPADFSEWSK': 54.62, 'GTFIIDPGGVIR': 70.52,
                'GTFIIDPAAVIR': 87.23, 'LFLQFGAQGSPFLK': 100.00}

R2_MIN = 0.95  # below this the RT alignment is untrustworthy and extraction windows are misplaced

def fit_irt_to_rt(anchor_irt, observed_rt):
    slope, intercept, r, _, _ = stats.linregress(anchor_irt, observed_rt)
    if r ** 2 < R2_MIN:
        raise ValueError(f'iRT fit R^2={r**2:.3f} < {R2_MIN}; gradient may be nonlinear, use LOWESS')
    return lambda irt: slope * irt + intercept

Convert Library Formats

Goal: Move a library between DIA-NN, OpenSWATH, and Spectronaut conventions without silently corrupting RT, intensity, modification, or decoy content.

Approach: Conversion is renaming columns AND reconciling units, not a copy. Check RT units (iRT ~ -25..150 vs normalized 0-1 vs minutes), intensity scaling (relative vs absolute), and modification notation (UniMod:35 vs +15.9949 vs Oxidation). For OpenSWATH, generate decoys with OpenSwathDecoyGenerator -- a target-only library has no null.

import pandas as pd

# Spectronaut -> DIA-NN column mapping; iRT and RelativeIntensity are renamed, not recomputed.
SPECTRONAUT_TO_DIANN = {'ModifiedPeptide': 'ModifiedPeptide', 'iRT': 'iRT',
                        'RelativeIntensity': 'LibraryIntensity', 'FragmentMz': 'ProductMz',
                        'FragmentNumber': 'FragmentSeriesNumber', 'PrecursorMz': 'PrecursorMz',
                        'PrecursorCharge': 'PrecursorCharge', 'FragmentCharge': 'FragmentCharge',
                        'FragmentType': 'FragmentType', 'Genes': 'Genes'}

def spectronaut_to_diann(lib):
    out = lib.rename(columns=SPECTRONAUT_TO_DIANN)
    assert out['iRT'].between(-50, 200).all(), 'RT not in iRT units; check column before converting'
    return out

QC and Merge Libraries

Goal: Summarize a library and combine multiple libraries without dropping legitimate distinct transitions.

Approach: Report precursor/protein counts and transitions-per-precursor, then dedup on the FULL transition key. Deduping on (sequence, fragment-type, fragment-number) alone drops real transitions that differ only in precursor charge or fragment charge -- key on all five.

import pandas as pd

TRANSITION_KEY = ['ModifiedSequence', 'PrecursorCharge', 'FragmentType',
                  'FragmentSeriesNumber', 'FragmentCharge']  # full key; charges matter

def merge_libraries(libs):
    combined = pd.concat(libs, ignore_index=True)
    combined['precursor_total'] = combined.groupby(
        ['ModifiedSequence', 'PrecursorCharge'])['LibraryIntensity'].transform('sum')
    combined = combined.sort_values('precursor_total', ascending=False)
    combined = combined.drop_duplicates(subset=TRANSITION_KEY).drop(columns='precursor_total')
    return combined

def library_stats(lib):
    n_prec = lib.groupby(['ModifiedSequence', 'PrecursorCharge']).ngroups
    return {'precursors': n_prec, 'proteins': lib['ProteinId'].nunique(),
            'transitions_per_precursor': round(len(lib) / n_prec, 1)}

Per-Method Failure Modes

Predicted RT/CCS used without calibration

Trigger: A predicted library is searched directly, RT column straight from the model. Mechanism: Predicted iRT/CCS are arbitrary model units; real RT depends on column/gradient/temperature. Symptom: Drastic ID loss with no error; peak groups extracted at the wrong time. Fix: Fit iRT/CiRT anchors (R^2 > 0.95) or run a GPF-DIA empirical correction (Searle 2020) before searching.

NCE mismatch

Trigger: A fixed collision energy (often 30) reused across instruments/methods. Mechanism: Intensity predictors are NCE-conditioned; wrong NCE shifts predicted relative intensities. Symptom: Quiet sensitivity loss; fewer confident peak groups than expected. Fix: Scan candidate NCE values, predict, and pick the one maximizing spectral contrast against real spectra.

Missing decoys for OpenSWATH

Trigger: A target-only library handed to OpenSWATH. Mechanism: Peptide-centric scoring needs a decoy null; OpenSWATH does not invent one. Symptom: FDR cannot be estimated or is meaningless. Fix: Run OpenSwathDecoyGenerator to append decoys; do NOT also supply decoys to DIA-NN/Spectronaut, which generate their own.

Modification mismatch between library and data

Trigger: Library lacks the sample's variable mods, or carries too many. Mechanism: A library without phospho/ox cannot find those peptidoforms; too many variable mods explode the search space and inflate FDR. Symptom: Missing modified peptides, or inflated IDs. Fix: Match library modifications to the biology; route PTM-resolved design to ptm-analysis.

Naive merge dropping transitions

Trigger: Dedup keyed on (sequence, fragment-type, fragment-number) only. Mechanism: Distinct transitions can share those three fields but differ in precursor or fragment charge. Symptom: Quietly thinner transition lists; weaker peak-group scoring. Fix: Key dedup on (modified-sequence, precursor-charge, fragment-type, fragment-number, fragment-charge).

Quantitative Thresholds

Common Errors

References

• Gillet LC, Navarro P, Tate S, et al. Targeted data extraction of the MS/MS spectra generated by data-independent acquisition: a new concept for consistent and accurate proteome analysis. Mol Cell Proteomics 2012;11(6):O111.016717.
• Searle BC, Pino LK, Egertson JD, et al. Chromatogram libraries improve peptide detection and quantification by data independent acquisition mass spectrometry. Nat Commun 2018;9:5128.
• Gessulat S, Schmidt T, Zolg DP, et al. Prosit: proteome-wide prediction of peptide tandem mass spectra by deep learning. Nat Methods 2019;16(6):509-518.
• Searle BC, Swearingen KE, Barnes CA, et al. Generating high quality libraries for DIA MS with empirically corrected peptide predictions. Nat Commun 2020;11:1548.
• Bouwmeester R, Gabriels R, Hulstaert N, Martens L, Degroeve S. DeepLC can predict retention times for peptides that carry as-yet unseen modifications. Nat Methods 2021;18(11):1363-1369.
• Zeng WF, Zhou XX, Willems S, et al. AlphaPeptDeep: a modular deep learning framework to predict peptide properties for proteomics. Nat Commun 2022;13:7238.

Related Skills

• dia-analysis - Run the DIA search against the library and choose the q-value context
• peptide-identification - Generate the DDA identifications a DDA library is built from
• ptm-analysis - Design PTM-resolved and modified-peptide libraries
• quantification - Summarize and roll up the search output to protein abundances