title: 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.
authors.affiliation: Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, USA. xudong@wam.umd.edu
authors.forename: Xudong
authors.initials: X
authors.lastname: Yao
authors.forename: Carlos
authors.initials: C
authors.lastname: Afonso
authors.forename: Catherine
authors.initials: C
authors.lastname: Fenselau
mesh_terms: D000818:Animals; D001059:Apoproteins / Q000032:analysis / Q000737:chemistry; D002384:Catalysis; D002918:Chymotrypsin / Q000032:analysis / Q000737:chemistry; D017357:Cyclotrons; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D007700:Kinetics; D009211:Myoglobin / Q000032:analysis / Q000737:chemistry; D010104:Oxygen Radioisotopes; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry*; D040901:Proteomics / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D001059:Apoproteins; D009211:Myoglobin; D010104:Oxygen Radioisotopes; D010446:Peptide Fragments; C010857:apomyoglobin; D010450:Endopeptidases; D002918:Chymotrypsin