SpringerProtocols: Abstract: Proteolytic Labeling With 18O for Comparative Proteomics Studies: Preparation of 18O-Labeled Peptides and the 18O/16O Peptide Mixture

Proteolytic Labeling With 18O for Comparative Proteomics Studies: Preparation of 18O-Labeled Peptides and the 18O/16O Peptide Mixture

By: Catherine Fenselau², Xudong Yao³

Abstract

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The method reported here uses proteolytic catalysis to introduce two ¹⁸O atoms into the carboxyl termini of peptides in mixtures, and is intended to be part of the work-flow in comparative proteomics strategies. Proteins are first cleaved with trypsin in water, and subsequently the peptide products are dried and labeled by incubation with trypsin in ¹⁸O-enriched water. One important aspect of this two-step procedure is that peptides, and not proteins, are dried and redissolved in H₂ ¹⁸O for the labeling reaction. Incorporation can exceed 95% if it is carried out in water that is sufficiently enriched with H₂ ¹⁸O. The byproduct of the reaction is water. The use of catalytic enzyme immobilized on beads facilitates its removal and termination of the exchange. In differential proteomic studies, heavy isotope-labeled peptides are combined with peptides carrying ¹⁶O for isotope ratio measurements by mass spectrometry.

Affiliation(s): (2) Department of Chemistry and Biochemistry, University of Maryland, College Park, MD
(3) Department of Chemistry, University of Connecticut, Storrs, CT

Book Title: Quantitative Proteomics by Mass Spectrometry

Series: Methods in Molecular Biology | Volume: 359 | Pub. Date: Feb-05-2007 | Page Range: 135-142 | DOI: 10.1007/978-1-59745-255-7_9

Subject: Biochemistry

Key Words: Comparative proteomics - isotope labeling - proteolytic catalysis - 18O/16O labeling - mass spectrometry - differential analysis - non-gel proteomics - peptide analysis - isotope ratios - immobilized trypsin - serine proteases

References

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