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An SDS-PAGE based proteomic approach for N-terminome profiling.

Prashant Kaushal1, Yumi Kwon, Shinyeong Ju

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This study introduces a novel N-termini enrichment strategy using SDS-PAGE and negative selection. The method enables deep and quantitative N-terminome profiling even from limited protein samples.

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Area of Science:

  • Proteomics
  • Biochemistry
  • Analytical Chemistry

Background:

  • Current N-termini enrichment methods face challenges with sample quantity and complex processing.
  • Limitations include difficulties in solubilization, separation, and visualization of protein termini.

Purpose of the Study:

  • To develop a simple, reproducible N-termini enrichment strategy using negative selection.
  • To enable deep and quantitative N-terminome analysis from limited sample amounts.

Main Methods:

  • In-gel protein labeling with d6-acetic anhydride.
  • Post-digestion negative selection of N-terminal peptides using NHS-activated agarose beads.
  • Coupling with mass spectrometry for protein identification.

Main Results:

  • Successfully enriched N-termini from as low as 10 ng of bovine serum albumin.
  • Validated for complex protein mixtures, including neo-N-termini generated by Glu-C protease.
  • Demonstrated effectiveness for deep N-terminome profiling in human cell lysates.
  • Identified protease substrates in MMP2-perturbed HCT8 cell secretome.

Conclusions:

  • The proposed method offers an effective strategy for N-termini detection and analysis.
  • Enables deep and quantitative N-terminome profiling from limited biological samples.
  • Applicable to single protein analysis and complex biological systems.