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Related Experiment Videos

Histidine-rich peptide selection and quantification in targeted proteomics.

Diya Ren1, Natalia A Penner, Benjamin E Slentz

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

Journal of Proteome Research
|March 5, 2004
PubMed
Summary

This study used copper (II) affinity chromatography (Cu(II)-IMAC) to identify yeast proteins that change during fermentation. Researchers found significant changes in histidine-rich proteins, some over 4-fold, using a novel labeling and elution strategy.

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Area of Science:

  • Proteomics
  • Biochemistry
  • Analytical Chemistry

Background:

  • Understanding yeast fermentation dynamics is crucial for optimizing industrial processes.
  • Identifying specific protein changes during fermentation provides insights into cellular responses.
  • Existing methods for quantitative proteomics can be complex and time-consuming.

Purpose of the Study:

  • To develop and apply a streamlined method for quantitative proteomic analysis of yeast extracts.
  • To identify and quantify changes in histidine-rich proteins during yeast fermentation.
  • To compare protein expression profiles at different fermentation time points.

Main Methods:

  • Utilized agarose-based immobilized copper (II) affinity chromatography (Cu(II)-IMAC) coupled with reversed-phase chromatography.

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  • Employed N-terminal acylation of tryptic peptides with N-acetoxysuccinamide for global labeling and quantification.
  • Implemented an imidazole elution procedure for selective enrichment of histidine-rich peptides.
  • Applied an inverse labeling strategy for enhanced reliability in quantitative determinations.

    • Main Results:

    • Successfully simplified yeast protein extracts, enabling efficient mass spectral analysis.
    • Quantitatively compared protein profiles from yeast extracts at 2.5 and 10 hours of fermentation.
    • Identified significant concentration changes (over 4-fold) in several histidine-rich proteins during fermentation.
    • Observed that these regulated proteins exhibit diverse molecular weights and isoelectric points (pI).

    Conclusions:

    • The combined Cu(II)-IMAC and N-terminal acylation method is effective for quantitative proteomic analysis of yeast.
    • Histidine-rich proteins play a significant role in yeast fermentation dynamics.
    • The developed strategy allows for reliable identification and quantification of protein up- and down-regulation.