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<p>Shotgun proteomics deciphers biological regulation by quantifying dynamic changes in proteins and peptides, providing insights into phenotypic states inaccessible through genomic or transcriptomic analysis. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) enables large-scale protein identification and quantitation. Central to this methodology is the chromatogram, which is a temporal representation of analyte separation during LC. Understanding the chromatogram and peak shape characteristics for interpretation, particularly extracted ion chromatograms (XIC), is essential for accurate quantitative proteomics. This Back2Basics article provides a foundational guide to chromatographic principles for newcomers, with a focused examination of the extracted ion chromatogram (XIC) and its central role in quantification workflows for practicing proteomics researchers. We explain how XICs are used in label-free and isotope-labeled quantitation, describe common chromatographic peak anomalies and troubleshooting strategies, and outline the critical role of retention time alignment in multi-run analysis. Key concepts are illustrated with analytical schematics to bridge theoretical fundamentals with contemporary best practices. Key concepts are illustrated with analytical schematics to bridge theoretical fundamentals with contemporary best practices.</p>

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Back2Basics: interpreting chromatograms in LC-MS based proteomics

  • 期刊论文

  • 发表日期：

    2025年12月8日

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  • Prabhakar Babele,
  • Sanchit Singhania,
  • Amit Kumar Yadav

摘要

Shotgun proteomics deciphers biological regulation by quantifying dynamic changes in proteins and peptides, providing insights into phenotypic states inaccessible through genomic or transcriptomic analysis. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) enables large-scale protein identification and quantitation. Central to this methodology is the chromatogram, which is a temporal representation of analyte separation during LC. Understanding the chromatogram and peak shape characteristics for interpretation, particularly extracted ion chromatograms (XIC), is essential for accurate quantitative proteomics. This Back2Basics article provides a foundational guide to chromatographic principles for newcomers, with a focused examination of the extracted ion chromatogram (XIC) and its central role in quantification workflows for practicing proteomics researchers. We explain how XICs are used in label-free and isotope-labeled quantitation, describe common chromatographic peak anomalies and troubleshooting strategies, and outline the critical role of retention time alignment in multi-run analysis. Key concepts are illustrated with analytical schematics to bridge theoretical fundamentals with contemporary best practices. Key concepts are illustrated with analytical schematics to bridge theoretical fundamentals with contemporary best practices.