Intramolecular migration of amide hydrogens in protonated peptides upon collisional activation

TY - JOUR

T1 - Intramolecular migration of amide hydrogens in protonated peptides upon collisional activation

AU - Jørgensen, Thomas J D

AU - Gårdsvoll, Henrik

AU - Ploug, Michael

AU - Roepstorff, Peter

PY - 2005/3/2

Y1 - 2005/3/2

N2 - Presently different opinions exist as to the degree of scrambling of amide hydrogens in gaseous protonated peptides and proteins upon collisional activation in tandem mass spectrometry experiments. This unsettled controversy is not trivial, since only a very low degree of scrambling is tolerable if collision-induced dissociation (CID) should provide reliable site-specific information from (1)H/(2)H exchange experiments. We have explored a series of unique, regioselectively deuterium-labeled peptides as model systems to probe for intramolecular amide hydrogen migration under low-energy collisional activation in an orthogonal quadrupole time-of-flight electrospray ionization (Q-TOF ESI) mass spectrometer. These peptides contain a C-terminal receptor-binding sequence and an N-terminal nonbinding region. When the peptides form a receptor complex, the amide hydrogens of the interacting sequences are protected against exchange with the solvent, while the amide hydrogens of the nonbinding sequences exchange rapidly with the solvent. We have utilized such long-lived complexes to generate peptides labeled with deuterium in either the binding or nonbinding region, and the expected regioselectivity of this labeling was confirmed after pepsin proteolysis. CID of such deuterated peptides, [M + 2H](2+), yielded fragment ions (b- and y-ions) having a deuterium content that resemble the theoretical values calculated for 100% scrambling. Thus, complete randomization of all hydrogen atoms attached to nitrogen and oxygen occurs in the gaseous peptide ion prior to its dissociation.

AB - Presently different opinions exist as to the degree of scrambling of amide hydrogens in gaseous protonated peptides and proteins upon collisional activation in tandem mass spectrometry experiments. This unsettled controversy is not trivial, since only a very low degree of scrambling is tolerable if collision-induced dissociation (CID) should provide reliable site-specific information from (1)H/(2)H exchange experiments. We have explored a series of unique, regioselectively deuterium-labeled peptides as model systems to probe for intramolecular amide hydrogen migration under low-energy collisional activation in an orthogonal quadrupole time-of-flight electrospray ionization (Q-TOF ESI) mass spectrometer. These peptides contain a C-terminal receptor-binding sequence and an N-terminal nonbinding region. When the peptides form a receptor complex, the amide hydrogens of the interacting sequences are protected against exchange with the solvent, while the amide hydrogens of the nonbinding sequences exchange rapidly with the solvent. We have utilized such long-lived complexes to generate peptides labeled with deuterium in either the binding or nonbinding region, and the expected regioselectivity of this labeling was confirmed after pepsin proteolysis. CID of such deuterated peptides, [M + 2H](2+), yielded fragment ions (b- and y-ions) having a deuterium content that resemble the theoretical values calculated for 100% scrambling. Thus, complete randomization of all hydrogen atoms attached to nitrogen and oxygen occurs in the gaseous peptide ion prior to its dissociation.

KW - Amides

KW - Deuterium Exchange Measurement

KW - Hydrogen

KW - Mass Spectrometry

KW - Peptides

KW - Receptors, Cell Surface

KW - Receptors, Urokinase Plasminogen Activator

KW - Journal Article

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

U2 - 10.1021/ja043789c

DO - 10.1021/ja043789c

M3 - Journal article

C2 - 15725037

SN - 0002-7863

VL - 127

SP - 2785

EP - 2793

JO - Journal of the American Chemical Society

JF - Journal of the American Chemical Society

IS - 8

ER -