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Updated: Dec 23, 2025
Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification
Published on: November 15, 2017
Selective Maleylation-Directed Isobaric Peptide Termini Labeling for Accurate Proteome Quantification
Xiaobo Tian, Marcel P de Vries1, Susan W J Visscher
1Department of Pediatrics, University Medical Center Groningen, University of Groningen, Hanzeplein 1, Groningen 9713 GZ, The Netherlands.
A new selective maleylation-directed isobaric peptide termini labeling (SMD-IPTL) method enhances multiplexing capacity for quantitative proteomics. This approach offers more accurate protein quantification by utilizing full fragment-ion series, overcoming limitations of traditional methods.
Area of Science:
- Proteomics
- Quantitative Mass Spectrometry
- Biochemistry
Background:
- Traditional isobaric labeling methods (e.g., TMT, iTRAQ) in quantitative proteomics are limited by reporter ion accuracy.
- Existing Isobaric Peptide Termini Labeling (IPTL) methods offer improved accuracy but have limited multiplexing capacity (typically 3-plex).
- Accurate protein quantification is crucial for understanding complex biological systems and disease mechanisms.
Purpose of the Study:
- To develop a novel Isobaric Peptide Termini Labeling (IPTL) approach with enhanced multiplexing capacity for quantitative proteomics.
- To address the limitations of existing methods by improving accuracy and expanding multiplexing capabilities.
- To validate the new method using complex biological samples.
Main Methods:
- Developed Selective Maleylation-Directed Isobaric Peptide Termini Labeling (SMD-IPTL) for LysC digested peptides.
- SMD-IPTL involves a one-pot, three-step reaction: selective N-terminal maleylation, C-terminal Lys labeling with isotopically labeled acetyl-alanine, and N-terminal labeling via thiol Michael addition.
- Utilized LC-MS/MS with a narrow precursor isolation window (0.8 Th, -0.2 Th offset) for analysis of 4-plex labeled samples.
Main Results:
- SMD-IPTL demonstrated a 4-plex multiplexing capacity, with potential for higher levels using commercially available labeled amino acids.
- The method accurately quantified proteins across a 10-fold mixing range of bovine serum albumin (BSA) in a yeast proteome background.
- Accurate ratio measurements were achieved, with an average standard deviation of peptide ratios of 0.34 for BSA spiked at 1:2:5:10 ratios.
Conclusions:
- SMD-IPTL provides a robust and accurate method for quantitative proteomics, overcoming the multiplexing limitations of previous IPTL approaches.
- The use of full fragment-ion series in SMD-IPTL avoids ratio distortion issues inherent in reporter-ion-based methods.
- This enhanced labeling strategy holds significant potential for high-multiplex quantitative proteomics applications.
