bio-proteomics-data-import

name	bio-proteomics-data-import
description	Loads mass-spectrometry data into Python/R and strips the search engine's bookkeeping before any number is trusted -- removes decoys (REV__/Reverse), contaminants (CON__/Potential contaminant), Only-identified-by-site groups, and resolves semicolon razor/leading protein-ID ambiguity in MaxQuant proteinGroups.txt, DIA-NN report.parquet, and mzML/mzXML. Distinguishes Intensity (raw) vs LFQ intensity (MaxLFQ) vs iBAQ, treats a MaxQuant zero as missing (NaN, not log2(-inf)), and inherits the acquisition mode's missingness contract (DDA MNAR vs DIA MCAR). Use when starting an analysis from raw spectra or a search engine output. Downstream normalization and stats are differential-abundance; reporter-ion/MaxLFQ quant is quantification; protein grouping is protein-inference.
tool_type	mixed
primary_tool	pyOpenMS

Version Compatibility

Reference examples tested with: pyOpenMS 3.1+, pandas 2.2+, numpy 1.26+, MSnbase 2.28+

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

• Python: pip show <package> then help(module.function) to check signatures
• R: packageVersion('<pkg>') then ?function_name to verify parameters

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

Mass Spectrometry Data Import -- Inheriting the Acquisition Contract and Stripping the Bookkeeping

"Load my mass spec data into Python" -> Parse spectra or a search-engine table AND immediately enforce two contracts -- which quant column carries real biology, and which rows are search-engine bookkeeping that must be deleted -- because the same proteinGroups.txt yields different conclusions depending on the column read and the rows kept.

• Python: pyopenms.MzMLFile().load(path, exp) for raw spectra; pandas.read_csv(sep='\t') for MaxQuant; pandas.read_parquet for DIA-NN
• R: Spectra::Spectra() / QFeatures::readQFeatures() for raw and quantified data (MSnbase still works but is in maintenance mode)

Scope: this skill owns reading spectra/search outputs into memory, deleting decoy/contaminant/site-only rows, picking the correct quant column, and characterizing missingness. Format conversion (RAW -> mzML) -> peptide-identification. MaxLFQ/TMT reporter quant computation -> quantification. Protein-group parsimony -> protein-inference. Normalization and imputation -> differential-abundance and expression-matrix/normalization. OUT OF SCOPE: statistical testing, batch correction, and the actual imputation step (this skill only diagnoses the missingness so the right imputer is chosen later).

The Single Most Important Modern Insight -- Import Is Where Two Contracts Are Read and Enforced

1. A "data import" is never just file parsing -- it is the moment the acquisition mode's quantitative contract and its missingness structure are inherited. DDA selects the top-N most intense precursors per cycle, and which precursors get picked is partly stochastic and abundance-biased, so the same low-abundance peptide is sampled in run A and missed in run B; this manufactures structured, left-censored MNAR missingness. DIA fragments every precursor in every window every cycle, so its (fewer) missing values are closer to MCAR. The catastrophic error this prevents: imputing a DDA matrix with a mean/KNN method that assumes MCAR, which biases low-abundance proteins upward and manufactures false hits. The mode is born at acquisition and inherited at import; the missingness diagnosis made here dictates which imputation is even legitimate downstream.

2. The search engine's bookkeeping must be stripped before any number is trusted. A proteinGroups.txt carries decoy rows (Reverse == '+', REV__ prefix in the ID) from the target-decoy FDR machinery, contaminant rows (Potential contaminant == '+', CON__ prefix), and Only-identified-by-site rows (the protein has no unmodified-peptide evidence, only a modified site). Keeping any of these leaks non-biological signal into the intensity matrix and inflates IDs. The catastrophic error: reporting differential abundance on a matrix where decoy or keratin rows survived.

3. The same proteinGroups.txt yields different biology from different columns, and a zero is not a measurement. Intensity is raw summed precursor signal (not normalized, not comparable across samples for ratios). LFQ intensity is MaxLFQ-normalized and is the column for between-sample comparison. iBAQ is intensity divided by the number of observable tryptic peptides -- a within-sample molar proxy, not a between-sample quant. MaxQuant writes 0 for "not quantified", so log2(0) = -inf; replace 0 -> NaN before any transform. The catastrophic error: log2-transforming raw Intensity (or iBAQ) and reading the ratios as biology.

Tool Taxonomy

Tool / method	Citation	Mechanism / role	When
pyOpenMS MzMLFile().load	Chambers 2012 (ProteoWizard lineage)	Loads mzML/mzXML into an MSExperiment in memory; iterate spectra by MS level	Programmatic access to raw peaks, precursor m/z, isolation windows
pandas read_csv/read_parquet	--	Tabular ingest of MaxQuant TSV and DIA-NN parquet	All search-engine output tables
DIA-NN report	Demichev 2020	Long-format precursor table; report.parquet is the default (1.9+) and the only default (2.0)	DIA quant; pivot on PG.MaxLFQ after q-filtering
MaxQuant txt/ outputs	Cox 2014 (MaxLFQ)	proteinGroups.txt (group level), evidence.txt (per-PSM)	DDA label-free / TMT search results
Spectra + QFeatures (R)	--	Current Bioconductor raw + quantified-feature containers; readQFeatures, aggregateFeatures	R pipelines; preferred over MSnbase going forward
MSnbase readMSData (R)	--	On-disk raw reading; maintenance mode (route OUT to Spectra/QFeatures)	Legacy R code only
ThermoRawFileParser / msconvert	Hulstaert 2020 / Chambers 2012	RAW -> mzML conversion (route OUT)	File conversion is peptide-identification

Decision Tree by Scenario

Scenario	Recommended	Why
MaxQuant DDA label-free, between-sample comparison	Read LFQ intensity columns from proteinGroups.txt	MaxLFQ-normalized; the only MaxQuant column valid for cross-sample ratios
MaxQuant, absolute/molar abundance within one sample	Read iBAQ columns	iBAQ is a within-sample molar proxy; do not use across samples
Need raw uncorrected signal for a custom normalization	Read Intensity columns, normalize yourself	Intensity is raw summed precursor area, not comparable as-is
DIA-NN output (1.9 or 2.0)	pd.read_parquet('report.parquet'), filter q-values, pivot PG.MaxLFQ	2.0 dropped the TSV default; q-filter before pivot or low-confidence rows leak in
Raw spectra, need peaks/precursor/isolation window	pyOpenMS MzMLFile().load	Programmatic peak and isolation-window access for QC and co-isolation reasoning
R-based pipeline, quantified features	QFeatures readQFeatures + aggregateFeatures	Current Bioconductor; MSnbase is maintenance-only
Data came from DDA, planning imputation	Diagnose missingness as MNAR -> route to left-censored imputation	DDA top-N sampling makes missingness abundance-dependent
Data came from DIA, planning imputation	Treat missingness as closer to MCAR	DIA samples every precursor every cycle

Default when uncertain: read LFQ intensity (MaxQuant) or PG.MaxLFQ after q-filtering (DIA-NN), strip Reverse/contaminant/site-only rows, set 0 -> NaN, then diagnose missingness before choosing an imputer.

Loading mzML/mzXML with pyOpenMS

Goal: Parse raw spectra into memory for QC, peak access, and isolation-window reasoning.

Approach: Load into an MSExperiment (filled in place), iterate by MS level; get_peaks() returns a tuple of (mz, intensity) numpy arrays, and getPrecursors() returns a list.

from pyopenms import MSExperiment, MzMLFile

exp = MSExperiment()
MzMLFile().load('sample.mzML', exp)  # fills exp in place; returns None

for spectrum in exp:
    if spectrum.getMSLevel() == 1:
        mz, intensity = spectrum.get_peaks()  # tuple of two numpy arrays
    elif spectrum.getMSLevel() == 2:
        precursor = spectrum.getPrecursors()[0]  # getPrecursors returns a list
        precursor_mz = precursor.getMZ()
        window = precursor.getIsolationWindowLowerOffset() + precursor.getIsolationWindowUpperOffset()

Loading and Cleaning MaxQuant proteinGroups.txt

Goal: Get a trustworthy log2 intensity matrix with bookkeeping rows removed and missing values represented as NaN.

Approach: Strip Reverse/contaminant/site-only rows, resolve the semicolon protein-ID list to a leading ID, pick LFQ intensity columns, set 0 -> NaN, then log2-transform.

import pandas as pd
import numpy as np

pg = pd.read_csv('proteinGroups.txt', sep='\t', low_memory=False)  # mixed-type cols

# Flag columns hold '+' or empty string; all three are proteinGroups-only bookkeeping
mask = (pg.get('Reverse', '') != '+') & (pg.get('Potential contaminant', '') != '+') & (pg.get('Only identified by site', '') != '+')
pg = pg[mask].copy()

# Protein IDs / Majority protein IDs / Gene names are SEMICOLON lists; take the first (leading/razor) entry
pg['leading_protein'] = pg['Protein IDs'].str.split(';').str[0]
pg['leading_gene'] = pg['Gene names'].where(pg['Gene names'].notna(), '').str.split(';').str[0]

lfq_cols = [c for c in pg.columns if c.startswith('LFQ intensity ')]  # MaxLFQ-normalized, between-sample comparable
matrix = pg[['leading_protein', 'leading_gene'] + lfq_cols].copy()
matrix[lfq_cols] = matrix[lfq_cols].replace(0, np.nan)  # MaxQuant writes 0 for missing; log2(0) = -inf
matrix[lfq_cols] = np.log2(matrix[lfq_cols])

Loading DIA-NN report.parquet

Goal: Reshape the long DIA-NN report into a confident protein-by-run matrix.

Approach: Read the parquet (default since 1.9, only default in 2.0), filter precursor- AND protein-group q-values to 1% FDR BEFORE pivoting on PG.MaxLFQ.

import pandas as pd

report = pd.read_parquet('report.parquet')  # report.tsv dropped as default in DIA-NN 2.0
report = report[(report['Q.Value'] <= 0.01) & (report['PG.Q.Value'] <= 0.01)]  # 1% FDR before quant

matrix = report.pivot_table(index='Protein.Group', columns='Run', values='PG.MaxLFQ', aggfunc='first')

Diagnosing the Missingness Contract

Goal: Quantify the missing-value pattern so the legitimate imputation class can be chosen downstream.

Approach: Count NaN per protein and per sample; relate the pattern to acquisition mode (DDA -> structured MNAR; DIA -> closer to MCAR). A correlation between missingness and mean abundance is the MNAR signature.

import numpy as np

def assess_missingness(matrix, sample_cols):
    miss_per_protein = matrix[sample_cols].isna().sum(axis=1)
    miss_per_sample = matrix[sample_cols].isna().sum(axis=0)
    total_pct = 100 * matrix[sample_cols].isna().sum().sum() / matrix[sample_cols].size
    mean_abund = matrix[sample_cols].mean(axis=1)  # negative corr with missingness => MNAR / left-censored
    mnar_corr = mean_abund.corr(miss_per_protein)
    return {'per_protein': miss_per_protein, 'per_sample': miss_per_sample, 'total_pct': total_pct, 'abundance_missing_corr': mnar_corr}

Per-Method Failure Modes

MaxQuant wrong quant column

Trigger: Reading Intensity (raw) or iBAQ when between-sample ratios are intended. Mechanism: Intensity is un-normalized summed precursor signal; iBAQ is a within-sample molar proxy. Neither is comparable across samples the way LFQ intensity is. Symptom: Ratios track total loaded protein / sample depth rather than biology; fold changes shift when one sample's loading changes. Fix: Use LFQ intensity for cross-sample comparison; if computing custom normalization use Intensity and normalize explicitly (expression-matrix/normalization).

Zero treated as a measurement

Trigger: np.log2 applied directly to a MaxQuant matrix still containing 0. Mechanism: MaxQuant encodes "not quantified" as 0; log2(0) = -inf, which then propagates into means and tests. Symptom: -inf values, NaN means, proteins silently dropped or skewed. Fix: replace(0, np.nan) before any transform; then diagnose missingness.

Bookkeeping rows survive

Trigger: Loading proteinGroups.txt without filtering Reverse / Potential contaminant / Only identified by site. Mechanism: Decoys exist only for FDR estimation; contaminants are keratin/trypsin/BSA, not the sample; site-only groups have no unmodified-peptide quant evidence. Only identified by site exists only in proteinGroups.txt. Symptom: Inflated protein counts; a "hit" that is a decoy or keratin. Fix: Filter all three flag columns; cross-check with REV__/CON__ ID prefixes when joining to peptide tables. Caveat: do not delete CON__ rows blindly if a contaminant (e.g. keratin) is the protein of interest.

Razor / leading protein-ID ambiguity ignored

Trigger: Treating Protein IDs or Gene names as an atomic single value. Mechanism: These are semicolon-delimited lists; the first entry is the leading (razor) protein for the group, and Gene names can be blank while protein IDs are present. Symptom: Merges fail, NaN gene labels, ambiguous identity downstream. Fix: Split on ; and take the first entry; guard Gene names with .notna(). Group parsimony details -> protein-inference.

Stale DIA-NN parsing

Trigger: Reading report.tsv on DIA-NN 2.0, or pivoting before q-filtering. Mechanism: 2.0 defaults to (and only defaults to) report.parquet; pivoting unfiltered rows includes precursors above 1% FDR. Symptom: FileNotFoundError on report.tsv; or low-confidence quant inflating the matrix. Fix: pd.read_parquet('report.parquet'); filter Q.Value <= 0.01 & PG.Q.Value <= 0.01 before pivoting PG.MaxLFQ.

MNAR imputed as MCAR

Trigger: Mean/median/KNN imputation on a DDA matrix. Mechanism: DDA missingness is abundance-dependent (left-censored); MCAR imputers fill missing low values with the central tendency, biasing them upward. Symptom: Low-abundance proteins gain false high values; spurious differential hits. Fix: Diagnose the abundance-missingness correlation here; route DDA to left-censored imputation (downshifted-Gaussian / QRILC / MinProb) in differential-abundance; DIA tolerates standard imputers.

Quantitative Thresholds

Threshold	Source	Rationale
DIA-NN import filter Q.Value <= 0.01 AND PG.Q.Value <= 0.01	Demichev 2020; target-decoy convention	Precursor- and protein-group-level 1% FDR enforced before any quant value is used
Peptide/protein FDR 1% (q <= 0.01)	Target-decoy convention	Standard ID confidence at both peptide and protein levels
MaxQuant zero -> NaN	MaxQuant output convention	0 encodes "not quantified"; log2(0) = -inf corrupts every transform
Min peptides per protein for quant >= 2	Community quant practice	Single-peptide ("one-hit-wonder") proteins are ID/quant-unreliable
Valid-value filter >= 50-70% per group	Modeling choice (document per study)	Caps imputation burden; the exact cutoff is a study decision, not a universal constant
Take FIRST semicolon entry as leading protein/gene	MaxQuant proteinGroups convention	The leading/razor protein is the group identifier; trailing entries are shared-peptide members

Common Errors

Error / symptom	Cause	Solution
-inf values after log2	Zeros not converted to NaN	df.replace(0, np.nan) before np.log2
FileNotFoundError: report.tsv (DIA-NN 2.0)	TSV no longer the default output	pd.read_parquet('report.parquet')
KeyError: 'Only identified by site'	That column exists ONLY in proteinGroups.txt	Use df.get('Only identified by site', '') or guard the column lookup
Mixed-type / DtypeWarning on MaxQuant load	Wide TSV with mixed column types	pd.read_csv(..., low_memory=False)
NaN gene labels break a merge	Gene names is a semicolon list, sometimes blank	.where(notna(), '').str.split(';').str[0]
Ratios track loading not biology	Read Intensity (raw) instead of LFQ intensity	Use LFQ intensity for between-sample comparison
get_peaks() unpacking error	Expecting a 2D array	It returns a tuple (mz, intensity) of two numpy arrays

References

• Cox J, Hein MY, Luber CA, Paron I, Nagaraj N, Mann M. 2014. Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol Cell Proteomics 13(9):2513-2526.
• Demichev V, Messner CB, Vernardis SI, Lilley KS, Ralser M. 2020. DIA-NN: neural networks and interference correction enable deep proteome coverage in high throughput. Nat Methods 17(1):41-44.
• Chambers MC, Maclean B, Burke R, et al. 2012. A cross-platform toolkit for mass spectrometry and proteomics. Nat Biotechnol 30(10):918-920.
• Hulstaert N, Shofstahl J, Sachsenberg T, et al. 2020. ThermoRawFileParser: modular, scalable, and cross-platform RAW file conversion. J Proteome Res 19(1):537-542.

Related Skills

• peptide-identification - search raw spectra and convert vendor RAW to mzML
• quantification - compute MaxLFQ and TMT reporter-ion quantities from imported data
• protein-inference - resolve protein-group parsimony and razor assignment
• differential-abundance - normalize, impute (per the missingness diagnosis), and test
• proteomics-qc - assess run-level identification and quant quality
• dia-analysis - run DIA-NN to produce the report this skill imports
• expression-matrix/normalization - general intensity-matrix normalization patterns
• workflows/proteomics-pipeline - end-to-end pipeline that begins with this import step