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Related Experiment Video

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Sample multiplexing with cysteine-selective approaches: cysDML and cPILOT.

Liqing Gu1, Adam R Evans, Renã A S Robinson

  • 1Department of Chemistry, University of Pittsburgh, Pittsburgh, PA, 15260, USA.

Journal of the American Society for Mass Spectrometry
|January 16, 2015
PubMed
Summary
This summary is machine-generated.

Two new cysteine-selective proteomics methods, cysteine-selective dimethyl labeling (cysDML) and cysteine-selective combined precursor isotopic labeling and isobaric tagging (cPILOT), enhance protein detection. These approaches identified over 2000 unique proteins in Alzheimer

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Area of Science:

  • Proteomics
  • Biochemistry
  • Analytical Chemistry

Background:

  • Cysteine-selective proteomics enhances the detection of low-abundance proteins in complex mixtures.
  • Quantitative proteomics can be advanced by coupling cysteinyl-peptide enrichment with isotopic labeling and isobaric tagging methods.

Purpose of the Study:

  • To introduce and validate two novel cysteine-selective proteomics approaches: cysteine-selective dimethyl labeling (cysDML) and cysteine-selective combined precursor isotopic labeling and isobaric tagging (cPILOT).
  • To assess the applicability of cysDML and cPILOT for quantitative proteomic analysis in biological samples, specifically liver tissues from an Alzheimer's disease (AD) mouse model and wild-type (WT) controls.

Main Methods:

  • CysDML: A duplex quantification technique involving cysteinyl-peptide enrichment coupled with on-resin stable-isotope dimethyl labeling.
  • Cysteine-selective cPILOT: A 12-plex workflow incorporating cysteinyl-peptide enrichment, on-resin stable-isotope dimethyl labeling, and iodoTMT tagging on cysteine residues.

Main Results:

  • CysDML identified an average of 850 proteins (594 quantified); cPILOT identified 330 proteins (151 quantified).
  • A total of 2259 unique proteins were detected across both methods with significant overlap.
  • 65 statistically significant differentially expressed proteins were identified in AD mouse liver tissue compared to WT controls.

Conclusions:

  • CysDML and cPILOT are effective cysteine-selective proteomics approaches for quantitative analysis.
  • The study highlights the performance, advantages, and limitations of duplex versus 12-plex experimental designs in proteomics.
  • These methods provide valuable insights into the proteomic changes associated with Alzheimer's disease.