Nanosecond laser-induced photochemical oxidation method for protein surface mapping with mass spectrometry

Thin Thin Aye, Low Teck Yew, Siu Kwan Sze

Research output: Contribution to journalArticle

85 Citations (Scopus)

Abstract

We have developed an ultrafast pulse method for protein surface footprinting by laser-induced protein surface oxidations. This method makes use of a pulse UV laser that produces, in nanoseconds, a high concentration of hydroxyl (OH) free radicals by photodissociation of a hydrogen peroxide (H 2O2) solution. The OH radicals oxidize amino acid residues located on the protein surface to produce stable covalent modifications. The oxidized protein is then analyzed by mass spectrometry to map the oxidized amino acid residues. Ubiquitin and apomyoglobin were used as model proteins in this study. Our results show that a single laser pulse can produce extensive protein surface oxidations. We found that monooxidized ubiquitins were more susceptible to further oxidations by subsequent laser irradiation, as compared to nonoxidized ones. This is due to the conformational changes of proteins by oxidation that increases the solvent-accessible surface area. Therefore, it is crucial to perform this experiment with a single pulse of laser so as to avoid oxidation of proteins after conformation of the protein changes. Subsequently, to obtain a high frequency and coverage of the oxidation sites while keeping the number of laser shots to one, we further optimized the laser power and concentration of hydrogen peroxide as well as the concentration of protein. This ultrafast OH radical generation method allows for rapid and accurate detection of surface residues, enabling mapping of the solvent-accessible regions of a protein in its native state.

Original languageEnglish
Pages (from-to)5814-5822
Number of pages9
JournalAnalytical Chemistry
Volume77
Issue number18
DOIs
Publication statusPublished - 15 Sep 2005
Externally publishedYes

Fingerprint

Mass spectrometry
Membrane Proteins
Oxidation
Lasers
Laser pulses
Hydroxyl Radical
Proteins
Hydrogen Peroxide
Ubiquitins
Photodissociation
Amino Acids
Laser beam effects
Ubiquitin
Free Radicals
Conformations
Experiments

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Nanosecond laser-induced photochemical oxidation method for protein surface mapping with mass spectrometry. / Aye, Thin Thin; Teck Yew, Low; Sze, Siu Kwan.

In: Analytical Chemistry, Vol. 77, No. 18, 15.09.2005, p. 5814-5822.

Research output: Contribution to journalArticle

@article{6d4ebbd4aad74da7ad9aa73467e8906a,
title = "Nanosecond laser-induced photochemical oxidation method for protein surface mapping with mass spectrometry",
abstract = "We have developed an ultrafast pulse method for protein surface footprinting by laser-induced protein surface oxidations. This method makes use of a pulse UV laser that produces, in nanoseconds, a high concentration of hydroxyl (OH) free radicals by photodissociation of a hydrogen peroxide (H 2O2) solution. The OH radicals oxidize amino acid residues located on the protein surface to produce stable covalent modifications. The oxidized protein is then analyzed by mass spectrometry to map the oxidized amino acid residues. Ubiquitin and apomyoglobin were used as model proteins in this study. Our results show that a single laser pulse can produce extensive protein surface oxidations. We found that monooxidized ubiquitins were more susceptible to further oxidations by subsequent laser irradiation, as compared to nonoxidized ones. This is due to the conformational changes of proteins by oxidation that increases the solvent-accessible surface area. Therefore, it is crucial to perform this experiment with a single pulse of laser so as to avoid oxidation of proteins after conformation of the protein changes. Subsequently, to obtain a high frequency and coverage of the oxidation sites while keeping the number of laser shots to one, we further optimized the laser power and concentration of hydrogen peroxide as well as the concentration of protein. This ultrafast OH radical generation method allows for rapid and accurate detection of surface residues, enabling mapping of the solvent-accessible regions of a protein in its native state.",
author = "Aye, {Thin Thin} and {Teck Yew}, Low and Sze, {Siu Kwan}",
year = "2005",
month = "9",
day = "15",
doi = "10.1021/ac050353m",
language = "English",
volume = "77",
pages = "5814--5822",
journal = "Analytical Chemistry",
issn = "0003-2700",
publisher = "American Chemical Society",
number = "18",

}

TY - JOUR

T1 - Nanosecond laser-induced photochemical oxidation method for protein surface mapping with mass spectrometry

AU - Aye, Thin Thin

AU - Teck Yew, Low

AU - Sze, Siu Kwan

PY - 2005/9/15

Y1 - 2005/9/15

N2 - We have developed an ultrafast pulse method for protein surface footprinting by laser-induced protein surface oxidations. This method makes use of a pulse UV laser that produces, in nanoseconds, a high concentration of hydroxyl (OH) free radicals by photodissociation of a hydrogen peroxide (H 2O2) solution. The OH radicals oxidize amino acid residues located on the protein surface to produce stable covalent modifications. The oxidized protein is then analyzed by mass spectrometry to map the oxidized amino acid residues. Ubiquitin and apomyoglobin were used as model proteins in this study. Our results show that a single laser pulse can produce extensive protein surface oxidations. We found that monooxidized ubiquitins were more susceptible to further oxidations by subsequent laser irradiation, as compared to nonoxidized ones. This is due to the conformational changes of proteins by oxidation that increases the solvent-accessible surface area. Therefore, it is crucial to perform this experiment with a single pulse of laser so as to avoid oxidation of proteins after conformation of the protein changes. Subsequently, to obtain a high frequency and coverage of the oxidation sites while keeping the number of laser shots to one, we further optimized the laser power and concentration of hydrogen peroxide as well as the concentration of protein. This ultrafast OH radical generation method allows for rapid and accurate detection of surface residues, enabling mapping of the solvent-accessible regions of a protein in its native state.

AB - We have developed an ultrafast pulse method for protein surface footprinting by laser-induced protein surface oxidations. This method makes use of a pulse UV laser that produces, in nanoseconds, a high concentration of hydroxyl (OH) free radicals by photodissociation of a hydrogen peroxide (H 2O2) solution. The OH radicals oxidize amino acid residues located on the protein surface to produce stable covalent modifications. The oxidized protein is then analyzed by mass spectrometry to map the oxidized amino acid residues. Ubiquitin and apomyoglobin were used as model proteins in this study. Our results show that a single laser pulse can produce extensive protein surface oxidations. We found that monooxidized ubiquitins were more susceptible to further oxidations by subsequent laser irradiation, as compared to nonoxidized ones. This is due to the conformational changes of proteins by oxidation that increases the solvent-accessible surface area. Therefore, it is crucial to perform this experiment with a single pulse of laser so as to avoid oxidation of proteins after conformation of the protein changes. Subsequently, to obtain a high frequency and coverage of the oxidation sites while keeping the number of laser shots to one, we further optimized the laser power and concentration of hydrogen peroxide as well as the concentration of protein. This ultrafast OH radical generation method allows for rapid and accurate detection of surface residues, enabling mapping of the solvent-accessible regions of a protein in its native state.

UR - http://www.scopus.com/inward/record.url?scp=24944566705&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=24944566705&partnerID=8YFLogxK

U2 - 10.1021/ac050353m

DO - 10.1021/ac050353m

M3 - Article

C2 - 16159110

AN - SCOPUS:24944566705

VL - 77

SP - 5814

EP - 5822

JO - Analytical Chemistry

JF - Analytical Chemistry

SN - 0003-2700

IS - 18

ER -