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Related Experiment Videos

Minimizing resolution of isotopically coded peptides in comparative proteomics.

Roujian Zhang1, Fred E Regnier

  • 1Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, USA.

Journal of Proteome Research
|March 20, 2003
PubMed
Summary

Stable isotope labeling in proteomics is crucial for quantifying concentration changes. This study demonstrates that while 2H8-ICAT labeling allows for peptide resolution, 13C-succinate labeling does not effectively resolve peptide isoforms during chromatography.

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Area of Science:

  • Proteomics
  • Analytical Chemistry
  • Biochemistry

Background:

  • Stable isotopes are essential for quantitative proteomics.
  • Isotopically labeled peptides are used to measure concentration changes.
  • Chromatographic separation of labeled peptides can be affected by isotope effects.

Purpose of the Study:

  • To evaluate the resolution of isotopically coded peptides during chromatographic separation.
  • To investigate methods for minimizing isotope effects in peptide analysis.
  • To compare the effectiveness of different heavy isotope derivatizing agents.

Main Methods:

  • Utilized reversed-phase chromatography for peptide separation.
  • Employed heavy isotope derivatizing agents: 2H8-ICAT reagent and 13C4-succinic anhydride.

Related Experiment Videos

  • Analyzed tryptic digests of bovine serum albumin (BSA) differentially labeled with isotopes.

    • Main Results:

    • Resolution exceeding 0.5 was achieved for 20% of peptides labeled with 2H8-ICAT, particularly doubly labeled cysteine-containing peptides.
    • Only 23% of singly ICAT-labeled peptides showed resolution greater than 0.4.
    • Peptides labeled with 13C- and 12C-succinate showed no significant resolution (within +/- 0.01).

    Conclusions:

    • 2H8-ICAT labeling enables the resolution of some peptide isoforms during chromatography.
    • 13C-succinate labeling does not provide sufficient resolution for differentiating peptide isoforms.
    • Isotope ratio analysis for 13C/12C coding can be performed early in the elution profile from mass spectrometry.