Dissection of Proteolytic ¹⁸O Labeling:  Endoprotease-Catalyzed ¹⁶O-to-¹⁸O Exchange of Truncated Peptide Substrates

Abstract

Proteolytic labeling in H2(18)O has been recently revived as a versatile method for proteomics research. To understand the molecular basis of the labeling process, we have dissected the process into two separate events: cleavage of the peptide amide bonds and exchange of the terminal carboxyl oxygens. It was demonstrated that both carboxyl oxygens can be catalytically labeled, independent of the cleavage step. Reaction kinetics of the tryptic 16O-to-18O exchange of YGGFMR, YGGFMK, and the tryptic digest of apomyoglobin were studied by matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry. A larger KM for the Lys-peptide (4400 +/- 700 microM), when compared to that of the Arg-peptide (KM 1300 +/- 300 microM), was mainly responsible for the slower reaction with YGGFMK (kcat/KM 0.64 +/- 0.14 microM(-1)min(-1)) compared to YGGFMR (kcat/KM 2.6 +/- 0.9 microM(-1)min(-1)). Multiplexed kinetic studies showed that endoprotease-catalyzed oxygen exchange is a general phenomenon, allowing homogeneous 18O2-coding of a variety of peptides. It was demonstrated for the first time that chymotrypsin 18O2-codes peptides during proteolysis. On the basis of the analyses reported here, we propose that proteolytic 18O labeling can be advantageously decoupled from protein digestion, and endoproteases can be used in a separate step to 18O2-code peptides for comparative studies after proteolysis has taken place.

Trypsin catalyzed 16O-to-18O exchange for comparative proteomics: Tandem mass spectrometry comparison using MALDI-TOF, ESI-QTOF, and ESI-ion trap mass spectrometers

“…Alternative mass spectrometry-based approaches in conjunction with gel-free protein/peptide separation have been developed in recent years using various stable isotope labeling techniques. Common to all these techniques is the incorporation, biosynthetically or chemically, of a labeling moiety having either a natural isotope distribution of hydrogen, carbon, oxygen, or nitrogen (light form) or being enriched with heavy isotopes like deuterium, 13 C, 18 O, or 15 N, respectively. By mixing equal amounts of a control sample possessing for instance the light form of the label with a heavy-labeled case sample, differentially labeled peptides are detected by mass spectrometric methods and their intensities serve as a means for direct relative protein quantification.…”

“…Enzymatic labeling of proteins/peptides has been proposed to compare protein quantities in two counterpart proteomes mainly because, compared to chemical labeling methods, the enzymatic labeling is highly specific and is almost universally applicable [15]. Through its ability to universally label almost all the carboxyl termini, enzymatic labeling is an ideal choice to quantitatively study mixtures of low molecular weight proteins.…”

Simultaneous quantification and identification using 18O labeling with an ion trap mass spectrometer and the analysis software application “ZoomQuant”

“…The calculated efficiency for the myoglobin tryptic peptide GLSDEWQQVLNVWGK is 49%. The peptide dependence of EMOS has been reported to be linked to its efficiency as a pseudosubstrate for trypsin [8,9,12]. This underscores the necessity of using mass spectral information of sufficiently high resolution to include the singly 18 O labeled species in the isotopic ratio calculation.…”

“…Every peptide in a peptide mixture is labeled through the enzyme mediated oxygen substitution (EMOS) [8] of serine proteases, with the exception of carboxy-terminal peptides [9]. This produces a more representative mixture of the biological sample, and an enhanced possibility for protein detection and identification in a protein profiling experiment.…”

“…There have been recent efforts to use 18 O stable isotope labeling with ion trap mass spectrometers, however medium to high resolution instruments that use time of flight (TOF) or Fourier transform ion cyclotron resonance (FT-ICR) have been the primary instruments used for quantification with 18 O labeling [8,9,11,14].…”