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Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT)
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Aliphatic dipeptide tags for multi-2-plex protein quantification.

Min-Soo Suh1, Jongcheol Seo, T D Thangadurai

  • 1Bio-Nanotechnology Center, Department of Chemistry, Pohang University of Science and Technology, San31 Hyoja-dong Nam-gu, Pohang, Kyungbuk 790-784, Korea.

The Analyst
|March 4, 2011
PubMed
Summary

Mass-balanced (1)H/(2)H-isotope dipeptide tags (MBIT) enable multiplexed protein quantification. These aliphatic tags offer improved LC retention and signal abundance with increasing chain length for accurate protein analysis.

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Published on: November 15, 2017

Area of Science:

  • Proteomics
  • Analytical Chemistry
  • Biochemistry

Background:

  • Mass-balanced (1)H/(2)H-isotope dipeptide tags (MBIT) are essential for multiplexed protein quantification.
  • Aliphatic MBITs, based on N-acetyl-Xxx-Ala dipeptides with varying alkyl chain lengths (C(2)-C(8)), offer a versatile platform for quantitative proteomics.

Purpose of the Study:

  • To diversify aliphatic MBITs for enhanced multiplexed protein quantification.
  • To characterize the synthesis, reactivity, and mass spectrometry performance of C(2)-C(8) aliphatic MBITs.

Main Methods:

  • Solid-phase synthesis and olefin metathesis were employed to synthesize C(2)-C(8) aliphatic MBITs.
  • Matrix-assisted laser desorption ionization and electrospray ionization tandem mass spectrometry were used to characterize the tags.
  • Reverse-phase liquid chromatography (LC) was utilized to assess peptide co-migration and tag performance.

Main Results:

  • Aliphatic MBITs were successfully synthesized and demonstrated reactivity towards peptide primary amines.
  • Tandem mass spectrometry revealed 2-plex quantitation signals and similar sequence ion abundances for MBIT-linked peptides.
  • Increasing alkyl chain length (C(2)-C(8)) resulted in stepwise increases in LC retention time and quantitation signal abundance.
  • Quantitation linearity was maintained within the 15-250 fmol range.
  • Multiplexed quantification of yeast heat shock proteins was achieved using three different C(6)-C(8) tags.

Conclusions:

  • Aliphatic MBITs provide a robust method for multiplexed protein quantification.
  • The systematic variation of alkyl chain length offers tunable LC retention and signal intensity.
  • These tags demonstrate excellent linearity and multiplexing capability for quantitative proteomics applications.