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name: bio-proteomics-quantification description: Protein quantification from mass spectrometry data including label-free (LFQ, intensity-based), isobaric labeling (TMT, iTRAQ), and metabolic labeling (SILAC) approaches. Use when extracting protein abundances from MS data for differential analysis. tool_type: mixed primary_tool: MSstats measurable_outcome: Execute skill workflow successfully with valid output within 15 minutes. allowed-tools:

• read_file
• run_shell_command

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Protein Quantification

Label-Free Quantification (LFQ)

Intensity-Based (MaxLFQ Algorithm)

import pandas as pd
import numpy as np

def maxlfq_normalize(intensities):
    '''Simplified MaxLFQ normalization'''
    log_int = np.log2(intensities.replace(0, np.nan))

    # Median centering per sample
    sample_medians = log_int.median(axis=0)
    global_median = sample_medians.median()
    normalized = log_int - sample_medians + global_median

    return normalized

Spectral Counting

def spectral_count_normalize(counts, total_spectra):
    '''Normalized spectral abundance factor (NSAF)'''
    # Divide by protein length, then by total
    nsaf = counts / total_spectra
    return nsaf / nsaf.sum()

TMT/iTRAQ Quantification

library(MSnbase)

# Load reporter ion data
tmt_data <- readMSnSet('tmt_data.txt')

# Normalize with reference channel
tmt_normalized <- normalize(tmt_data, method = 'center.median')

# Summarize to protein level
protein_data <- combineFeatures(tmt_normalized, groupBy = fData(tmt_data)$protein,
                                 fun = 'median')

Python TMT Processing

def extract_tmt_intensities(spectrum, reporter_mz, tolerance=0.003):
    '''Extract TMT reporter ion intensities'''
    mz, intensity = spectrum.get_peaks()
    tmt_intensities = {}

    for channel, target_mz in reporter_mz.items():
        mask = np.abs(mz - target_mz) < tolerance
        if mask.any():
            tmt_intensities[channel] = intensity[mask].max()
        else:
            tmt_intensities[channel] = 0

    return tmt_intensities

TMT_10PLEX = {'126': 126.127726, '127N': 127.124761, '127C': 127.131081,
              '128N': 128.128116, '128C': 128.134436, '129N': 129.131471,
              '129C': 129.137790, '130N': 130.134825, '130C': 130.141145,
              '131': 131.138180}

SILAC Quantification

def calculate_silac_ratio(heavy_intensity, light_intensity):
    '''Calculate SILAC H/L ratio'''
    if light_intensity > 0 and heavy_intensity > 0:
        return np.log2(heavy_intensity / light_intensity)
    return np.nan

# Typical mass shifts
SILAC_SHIFTS = {
    'Arg10': 10.008269,  # 13C6 15N4 Arginine
    'Lys8': 8.014199,    # 13C6 15N2 Lysine
    'Arg6': 6.020129,    # 13C6 Arginine
    'Lys6': 6.020129     # 13C6 Lysine
}

MSstats Workflow (R)

library(MSstats)

# Prepare input from MaxQuant
maxquant_input <- MaxQtoMSstatsFormat(
    evidence = read.table('evidence.txt', sep = '\t', header = TRUE),
    proteinGroups = read.table('proteinGroups.txt', sep = '\t', header = TRUE),
    annotation = read.csv('annotation.csv')
)

# Process and normalize
processed <- dataProcess(maxquant_input, normalization = 'equalizeMedians',
                         summaryMethod = 'TMP', censoredInt = 'NA')

# Protein-level summary
protein_summary <- quantification(processed)

Related Skills

• data-import - Load MS data before quantification
• differential-abundance - Statistical testing after quantification
• expression-matrix/counts-ingest - Similar matrix handling

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