TY - JOUR

T1 - Collisional activation by MALDI tandem time-of-flight mass spectrometry induces intramolecular migration of amide hydrogens in protonated peptides

AU - Jørgensen, Thomas J D

AU - Bache, Nicolai

AU - Roepstorff, Peter

AU - Gårdsvoll, Henrik

AU - Ploug, Michael

PY - 2005/12

Y1 - 2005/12

N2 - Considerable controversy exists in the literature as to the occurrence of intramolecular migration of amide hydrogens upon collisional activation of protonated peptides and proteins. This phenomenon has important implications for the application of CID as an experimental tool to obtain site-specific information about the incorporation of deuterium into peptides and proteins in solution. Using a unique set of peptides with their carboxyl-terminal half labeled with deuterium we have shown unambiguously that hydrogen (1H/2H) scrambling is such a dominating factor during low energy collisional activation of doubly protonated peptides that the original regioselective deuterium pattern of these peptides is completely erased (Jørgensen, T. J. D., Gårdsvoll, H., Ploug, M., and Roepstorff, P. (2005) Intramolecular migration of amide hydrogens in protonated peptides upon collisional activation. J. Am. Chem. Soc.127, 2785-2793). Taking further advantage of this unique test system we have now investigated the influence of the charge state and collision energy on the occurrence of scrambling in protonated peptides. Our MALDI tandem time-of-flight experiments clearly demonstrate that complete positional randomization among all exchangeable sites (i.e. all N- and O-linked hydrogens) also occurs upon high energy collisional activation of singly protonated peptides. This intense proton/deuteron traffic precludes the use of MALDI tandem time-of-flight mass spectrometry to obtain reliable information on the specific incorporation pattern of deuterons obtained during exchange experiments in solution.

AB - Considerable controversy exists in the literature as to the occurrence of intramolecular migration of amide hydrogens upon collisional activation of protonated peptides and proteins. This phenomenon has important implications for the application of CID as an experimental tool to obtain site-specific information about the incorporation of deuterium into peptides and proteins in solution. Using a unique set of peptides with their carboxyl-terminal half labeled with deuterium we have shown unambiguously that hydrogen (1H/2H) scrambling is such a dominating factor during low energy collisional activation of doubly protonated peptides that the original regioselective deuterium pattern of these peptides is completely erased (Jørgensen, T. J. D., Gårdsvoll, H., Ploug, M., and Roepstorff, P. (2005) Intramolecular migration of amide hydrogens in protonated peptides upon collisional activation. J. Am. Chem. Soc.127, 2785-2793). Taking further advantage of this unique test system we have now investigated the influence of the charge state and collision energy on the occurrence of scrambling in protonated peptides. Our MALDI tandem time-of-flight experiments clearly demonstrate that complete positional randomization among all exchangeable sites (i.e. all N- and O-linked hydrogens) also occurs upon high energy collisional activation of singly protonated peptides. This intense proton/deuteron traffic precludes the use of MALDI tandem time-of-flight mass spectrometry to obtain reliable information on the specific incorporation pattern of deuterons obtained during exchange experiments in solution.

KW - Amides

KW - Amino Acid Sequence

KW - Deuterium Oxide

KW - Models, Molecular

KW - Oligopeptides

KW - Peptide Fragments

KW - Protein Conformation

KW - Proteins

KW - Proteome

KW - Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1074/mcp.M500163-MCP200

DO - 10.1074/mcp.M500163-MCP200

M3 - Journal article

C2 - 16127176

VL - 4

SP - 1910

EP - 1919

JO - Molecular and Cellular Proteomics

JF - Molecular and Cellular Proteomics

SN - 1535-9476

IS - 12

ER -