Advantages of isotopic depletion of proteins for hydrogen/deuterium exchange experiments monitored by mass spectrometry

George M. Bou-Assaf, Jean E. Chamoun, Mark Emmett, Piotr G. Fajer, Alan G. Marshall

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

Solution-phase hydrogen/deuterium exchange (HDX) monitored by mass spectrometry is an excellent tool to study protein?protein interactions and conformational changes in biological systems, especially when traditional methods such as X-ray crystallography or nuclear magnetic resonance are not feasible. Peak overlap among the dozens of proteolytic fragments (including those from autolysis of the protease) can be severe, due to high protein molecular weight(s) and the broad isotopic distributions due to multiple deuterations of many peptides. In addition, different subunits of a protein complex can yield isomeric proteolytic fragments. Here, we show that depletion of 13C and/or 15N for one or more protein subunits of a complex can greatly simplify the mass spectra, increase the signal-to-noise ratio of the depleted fragment ions, and remove ambiguity in assignment of the m/z values to the correct isomeric peptides. Specifically, it becomes possible to monitor the exchange progress for two isobaric fragments originating from two or more different subunits within the complex, without having to resort to tandem mass spectrometry techniques that can lead to deuterium scrambling in the gas phase. Finally, because the isotopic distribution for a small to medium-size peptide is essentially just the monoisotopic species ( 12Cc 1Hh 14N n 16Oo 32Ss), it is not necessary to deconvolve the natural abundance distribution for each partially deuterated peptide during HDX data reduction.

Original languageEnglish (US)
Pages (from-to)3293-3299
Number of pages7
JournalAnalytical Chemistry
Volume82
Issue number8
DOIs
StatePublished - Apr 15 2010
Externally publishedYes

Fingerprint

Deuterium
Mass spectrometry
Hydrogen
Ion exchange
Peptides
Proteins
Experiments
X ray crystallography
Protein Subunits
Biological systems
Data reduction
Signal to noise ratio
Peptide Hydrolases
Gases
Molecular weight
Nuclear magnetic resonance
Ions

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Advantages of isotopic depletion of proteins for hydrogen/deuterium exchange experiments monitored by mass spectrometry. / Bou-Assaf, George M.; Chamoun, Jean E.; Emmett, Mark; Fajer, Piotr G.; Marshall, Alan G.

In: Analytical Chemistry, Vol. 82, No. 8, 15.04.2010, p. 3293-3299.

Research output: Contribution to journalArticle

Bou-Assaf, George M. ; Chamoun, Jean E. ; Emmett, Mark ; Fajer, Piotr G. ; Marshall, Alan G. / Advantages of isotopic depletion of proteins for hydrogen/deuterium exchange experiments monitored by mass spectrometry. In: Analytical Chemistry. 2010 ; Vol. 82, No. 8. pp. 3293-3299.
@article{1ca484b02d8f4494b05ee689ccfa8b1a,
title = "Advantages of isotopic depletion of proteins for hydrogen/deuterium exchange experiments monitored by mass spectrometry",
abstract = "Solution-phase hydrogen/deuterium exchange (HDX) monitored by mass spectrometry is an excellent tool to study protein?protein interactions and conformational changes in biological systems, especially when traditional methods such as X-ray crystallography or nuclear magnetic resonance are not feasible. Peak overlap among the dozens of proteolytic fragments (including those from autolysis of the protease) can be severe, due to high protein molecular weight(s) and the broad isotopic distributions due to multiple deuterations of many peptides. In addition, different subunits of a protein complex can yield isomeric proteolytic fragments. Here, we show that depletion of 13C and/or 15N for one or more protein subunits of a complex can greatly simplify the mass spectra, increase the signal-to-noise ratio of the depleted fragment ions, and remove ambiguity in assignment of the m/z values to the correct isomeric peptides. Specifically, it becomes possible to monitor the exchange progress for two isobaric fragments originating from two or more different subunits within the complex, without having to resort to tandem mass spectrometry techniques that can lead to deuterium scrambling in the gas phase. Finally, because the isotopic distribution for a small to medium-size peptide is essentially just the monoisotopic species ( 12Cc 1Hh 14N n 16Oo 32Ss), it is not necessary to deconvolve the natural abundance distribution for each partially deuterated peptide during HDX data reduction.",
author = "Bou-Assaf, {George M.} and Chamoun, {Jean E.} and Mark Emmett and Fajer, {Piotr G.} and Marshall, {Alan G.}",
year = "2010",
month = "4",
day = "15",
doi = "10.1021/ac100079z",
language = "English (US)",
volume = "82",
pages = "3293--3299",
journal = "Analytical Chemistry",
issn = "0003-2700",
publisher = "American Chemical Society",
number = "8",

}

TY - JOUR

T1 - Advantages of isotopic depletion of proteins for hydrogen/deuterium exchange experiments monitored by mass spectrometry

AU - Bou-Assaf, George M.

AU - Chamoun, Jean E.

AU - Emmett, Mark

AU - Fajer, Piotr G.

AU - Marshall, Alan G.

PY - 2010/4/15

Y1 - 2010/4/15

N2 - Solution-phase hydrogen/deuterium exchange (HDX) monitored by mass spectrometry is an excellent tool to study protein?protein interactions and conformational changes in biological systems, especially when traditional methods such as X-ray crystallography or nuclear magnetic resonance are not feasible. Peak overlap among the dozens of proteolytic fragments (including those from autolysis of the protease) can be severe, due to high protein molecular weight(s) and the broad isotopic distributions due to multiple deuterations of many peptides. In addition, different subunits of a protein complex can yield isomeric proteolytic fragments. Here, we show that depletion of 13C and/or 15N for one or more protein subunits of a complex can greatly simplify the mass spectra, increase the signal-to-noise ratio of the depleted fragment ions, and remove ambiguity in assignment of the m/z values to the correct isomeric peptides. Specifically, it becomes possible to monitor the exchange progress for two isobaric fragments originating from two or more different subunits within the complex, without having to resort to tandem mass spectrometry techniques that can lead to deuterium scrambling in the gas phase. Finally, because the isotopic distribution for a small to medium-size peptide is essentially just the monoisotopic species ( 12Cc 1Hh 14N n 16Oo 32Ss), it is not necessary to deconvolve the natural abundance distribution for each partially deuterated peptide during HDX data reduction.

AB - Solution-phase hydrogen/deuterium exchange (HDX) monitored by mass spectrometry is an excellent tool to study protein?protein interactions and conformational changes in biological systems, especially when traditional methods such as X-ray crystallography or nuclear magnetic resonance are not feasible. Peak overlap among the dozens of proteolytic fragments (including those from autolysis of the protease) can be severe, due to high protein molecular weight(s) and the broad isotopic distributions due to multiple deuterations of many peptides. In addition, different subunits of a protein complex can yield isomeric proteolytic fragments. Here, we show that depletion of 13C and/or 15N for one or more protein subunits of a complex can greatly simplify the mass spectra, increase the signal-to-noise ratio of the depleted fragment ions, and remove ambiguity in assignment of the m/z values to the correct isomeric peptides. Specifically, it becomes possible to monitor the exchange progress for two isobaric fragments originating from two or more different subunits within the complex, without having to resort to tandem mass spectrometry techniques that can lead to deuterium scrambling in the gas phase. Finally, because the isotopic distribution for a small to medium-size peptide is essentially just the monoisotopic species ( 12Cc 1Hh 14N n 16Oo 32Ss), it is not necessary to deconvolve the natural abundance distribution for each partially deuterated peptide during HDX data reduction.

UR - http://www.scopus.com/inward/record.url?scp=77951039991&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=77951039991&partnerID=8YFLogxK

U2 - 10.1021/ac100079z

DO - 10.1021/ac100079z

M3 - Article

VL - 82

SP - 3293

EP - 3299

JO - Analytical Chemistry

JF - Analytical Chemistry

SN - 0003-2700

IS - 8

ER -