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Determination of amide hydrogen exchange by mass spectrometry: A new tool for protein structure elucidation

Determination of amide hydrogen exchange by mass spectrometry: A new tool for protein structure... A new method based on protein fragmentation and directly coupled microbore high‐performance liquid chromatography–fast atom bombardment mass spectrometry (HPLC‐FABMS) is described for determining the rates at which peptide amide hydrogens in proteins undergo isotopic exchange. Horse heart cytochrome c was incubated in D2O as a function of time and temperature to effect isotopic exchange, transferred into slow exchange conditions (pH 2–3, 0 °C), and fragmented with pepsin. The number of peptide amide deuterons present in the proteolytic peptides was deduced from their molecular weights, which were determined following analysis of the digest by HPLC‐FABMS. The present results demonstrate that the exchange rates of amide hydrogens in cytochrome c range from very rapid (k > 140 h−1) to very slow (k < 0.002 h−1). The deuterium content of specific segments of the protein was determined as a function of incubation temperature and used to indicate participation of these segments in conformational changes associated with heating of cytochrome c. For the present HPLC‐FABMS system, approximately 5 nmol of protein were used for each determination. Results of this investigation indicate that the combination of protein fragmentation and HPLC‐FABMS is relatively free of constraints associated with other analytical methods used for this purpose and may be a general method for determining hydrogen exchange rates in specific segments of proteins. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Protein Science Wiley

Determination of amide hydrogen exchange by mass spectrometry: A new tool for protein structure elucidation

Zhang, Zhongqi; Smith, David L.
Protein Science , Volume 2 (4) – Apr 1, 1993
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References (37)

Publisher
Wiley
Copyright
Copyright © 1993 The Protein Society
ISSN
0961-8368
eISSN
1469-896X
DOI
10.1002/pro.5560020404
pmid
8390883
Publisher site
See Article on Publisher Site

Abstract

A new method based on protein fragmentation and directly coupled microbore high‐performance liquid chromatography–fast atom bombardment mass spectrometry (HPLC‐FABMS) is described for determining the rates at which peptide amide hydrogens in proteins undergo isotopic exchange. Horse heart cytochrome c was incubated in D2O as a function of time and temperature to effect isotopic exchange, transferred into slow exchange conditions (pH 2–3, 0 °C), and fragmented with pepsin. The number of peptide amide deuterons present in the proteolytic peptides was deduced from their molecular weights, which were determined following analysis of the digest by HPLC‐FABMS. The present results demonstrate that the exchange rates of amide hydrogens in cytochrome c range from very rapid (k > 140 h−1) to very slow (k < 0.002 h−1). The deuterium content of specific segments of the protein was determined as a function of incubation temperature and used to indicate participation of these segments in conformational changes associated with heating of cytochrome c. For the present HPLC‐FABMS system, approximately 5 nmol of protein were used for each determination. Results of this investigation indicate that the combination of protein fragmentation and HPLC‐FABMS is relatively free of constraints associated with other analytical methods used for this purpose and may be a general method for determining hydrogen exchange rates in specific segments of proteins.

Journal

Protein Science – Wiley

Published: Apr 1, 1993

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