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Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water
Published on: February 20, 2013
Pitfalls in protein quantitation using acid-catalyzed O18 labeling: hydrolysis-driven deamidation
Shunhai Wang1, Cedric E Bobst, Igor A Kaltashov
1Department of Chemistry, University of Massachusetts-Amherst, Amherst, Massachusetts 01003, United States.
Analytical Chemistry
|August 9, 2011
Summary
Acid-catalyzed O(18) labeling for protein quantitation shows peptide-dependent results due to deamidation. Accounting for deamidation significantly improves quantitation consistency for peptides in proteomics research.
Area of Science:
- Proteomics
- Analytical Chemistry
- Biochemistry
Background:
- Proteolysis combined with O(18) labeling is a key technique for protein quantitation when molecular biology standards are unavailable.
- Acid-catalyzed O(18) labeling has been proposed as an improvement over enzymatic methods for protein quantitation due to enhanced isotopic cluster spacing.
- This method aims to reduce signal interference and improve the accuracy of peptide quantitation in complex biological samples.
Purpose of the Study:
- To investigate the reliability and consistency of acid-catalyzed O(18) labeling for protein quantitation.
- To identify factors contributing to variability in quantitation results obtained using this method.
- To propose a modification to improve the accuracy and robustness of acid-catalyzed O(18) labeling in proteomics.
Main Methods:
- Utilized acid-catalyzed O(18) labeling in conjunction with proteolysis for protein quantitation.
- Analyzed the impact of the acidic labeling environment on various peptide residues, including Asn, Gln, and carbamidomethylated Cys.
- Quantified proteins using mass spectrometry, comparing results with and without accounting for peptide modification.
Main Results:
- Acid-catalyzed O(18) labeling results were found to be highly peptide-dependent.
- Deamidation of Asn, Gln, and carbamidomethylated Cys residues occurred during the acidic labeling process, leading to signal loss for affected peptides.
- Incorporating deamidation into the quantitation strategy significantly improved consistency across different proteolytic fragments.
Conclusions:
- The effectiveness of acid-catalyzed O(18) labeling for protein quantitation is compromised by peptide deamidation.
- Accounting for deamidation is crucial for accurate and consistent protein quantitation using this method.
- This finding enhances the utility of O(18) labeling techniques in proteomics by addressing a key limitation.
