Quantitative protein analysis using enzymatic [¹⁸O]water labeling

Abstract

This unit describes the stepwise procedure for differential oxygen isotope labeling of peptides and mass spectrometric quantification of relative protein levels in comparative proteomic experiments. The [¹⁸O] labeling of peptides happens at the peptide C-terminus and is achieved via the enzymatic oxygen exchange of tryptic peptides via catalysis of immobilized trypsin. Experimental considerations in effective incorporation and stabilization of the oxygen labels are discussed. Methods for mass spectrometric quantification of peptides with differential [¹⁶O] and [¹⁸O] isotopes are presented.

Keywords: 18O-labeling; back-exchange; enzymatic oxygen labeling; immobilized trypsin; quantitative proteomics.

Figure 23.4.1

Schematic representation of the enzyme-catalyzed ¹⁸O-labeling strategy for comparative proteomics with digestion and labeling steps decoupled.

Figure 23.4.2

Electrospray mass spectrum of a pair of doubly charged peptides, unlabeled (I₀) and labeled (I₄), and the theoretical molecular ion profile of the unlabeled peptide. (A) Observed isotopic distribution of ¹⁸O and ¹⁶O mixed samples. (B) Theoretical isotopic distribution without ¹⁸O labeling.

Figure 23.4.3

Schematic representation of the mechanism of double ¹⁸O incorporation with digestion and labeling in a single step.

Figure 23.4.4

Electrospray mass spectrum of two peptides, which were deglycosylated and digested in [¹⁸O]water in parallel with the [¹⁶O] counterparts. A triply charged peptide that did not contain a glycosylation site, and a singly charged peptide that had been glycosylated are detected with the respective 4 and 6 Da mass increments. (Reynolds et al., 2002) Reprinted with permission.

Figure 23.4.5

Electrospray mass spectrum of doubly charged N-terminally acetylated peptide ASGVAVSDGVIK from human cofilin isolated from MCF-7 human breast cancer cell lines. The unlabeled peptide was isolated from drug-susceptible cells, while the double ¹⁸O labeled peptide was isolated from doxorubicin-resistant cells. The ratio of cofilin in the two cell lines was calculated as resistant/susceptible: 1.2 ± 0.2.