Protein identification with a single accurate mass of a cysteine- containing peptide and constrained database searching

David R. Goodlett, James E. Bruce, Gordon A. Anderson, Beate Rist, Ljiljana Pasa-Tolic, Oliver Fiehn, Richard D. Smith, Ruedi Aebersold

Research output: Contribution to journalArticle

128 Citations (Scopus)

Abstract

A method for rapid and unambiguous identification of proteins by sequence database searching using the accurate mass of a single peptide and specific sequence constraints is described. Peptide masses were measured using electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry to an accuracy of 1 ppm. The presence of a cysteine residue within a peptide sequence was used as a database searching constraint to reduce the number of potential database hits. Cysteine-containing peptides were detected within a mixture of peptides by incorporating chlorine into a general alkylating reagent specific for cysteine residues. Secondary search constraints included the specificity of the protease used for protein digestion and the molecular mass of the protein estimated by gel electrophoresis. The natural isotopic distribution of chlorine encoded the cysteine-containing peptide with a distinctive isotopic pattern that allowed automatic screening of mass spectra. The method is demonstrated for a peptide standard and unknown proteins from a yeast lysate using all 6118 possible yeast open reading frames as a database. As judged by calculation of codon bias, low-abundance proteins were identified from the yeast lysate using this new method but not by traditional methods such as tandem mass spectrometry via data-dependent acquisition or mass mapping.

Original languageEnglish (US)
Pages (from-to)1112-1118
Number of pages7
JournalAnalytical Chemistry
Volume72
Issue number6
DOIs
StatePublished - Mar 15 2000
Externally publishedYes

Fingerprint

Cysteine
Peptides
Proteins
Yeast
Chlorine
Mass spectrometry
Cyclotron resonance
Electrospray ionization
Molecular mass
Electrophoresis
Fourier transforms
Screening
Peptide Hydrolases
Gels
Ions

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Goodlett, D. R., Bruce, J. E., Anderson, G. A., Rist, B., Pasa-Tolic, L., Fiehn, O., ... Aebersold, R. (2000). Protein identification with a single accurate mass of a cysteine- containing peptide and constrained database searching. Analytical Chemistry, 72(6), 1112-1118. https://doi.org/10.1021/ac9913210

Protein identification with a single accurate mass of a cysteine- containing peptide and constrained database searching. / Goodlett, David R.; Bruce, James E.; Anderson, Gordon A.; Rist, Beate; Pasa-Tolic, Ljiljana; Fiehn, Oliver; Smith, Richard D.; Aebersold, Ruedi.

In: Analytical Chemistry, Vol. 72, No. 6, 15.03.2000, p. 1112-1118.

Research output: Contribution to journalArticle

Goodlett, DR, Bruce, JE, Anderson, GA, Rist, B, Pasa-Tolic, L, Fiehn, O, Smith, RD & Aebersold, R 2000, 'Protein identification with a single accurate mass of a cysteine- containing peptide and constrained database searching', Analytical Chemistry, vol. 72, no. 6, pp. 1112-1118. https://doi.org/10.1021/ac9913210
Goodlett, David R. ; Bruce, James E. ; Anderson, Gordon A. ; Rist, Beate ; Pasa-Tolic, Ljiljana ; Fiehn, Oliver ; Smith, Richard D. ; Aebersold, Ruedi. / Protein identification with a single accurate mass of a cysteine- containing peptide and constrained database searching. In: Analytical Chemistry. 2000 ; Vol. 72, No. 6. pp. 1112-1118.
@article{e53b726367f9434d9faa9fc424e6848d,
title = "Protein identification with a single accurate mass of a cysteine- containing peptide and constrained database searching",
abstract = "A method for rapid and unambiguous identification of proteins by sequence database searching using the accurate mass of a single peptide and specific sequence constraints is described. Peptide masses were measured using electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry to an accuracy of 1 ppm. The presence of a cysteine residue within a peptide sequence was used as a database searching constraint to reduce the number of potential database hits. Cysteine-containing peptides were detected within a mixture of peptides by incorporating chlorine into a general alkylating reagent specific for cysteine residues. Secondary search constraints included the specificity of the protease used for protein digestion and the molecular mass of the protein estimated by gel electrophoresis. The natural isotopic distribution of chlorine encoded the cysteine-containing peptide with a distinctive isotopic pattern that allowed automatic screening of mass spectra. The method is demonstrated for a peptide standard and unknown proteins from a yeast lysate using all 6118 possible yeast open reading frames as a database. As judged by calculation of codon bias, low-abundance proteins were identified from the yeast lysate using this new method but not by traditional methods such as tandem mass spectrometry via data-dependent acquisition or mass mapping.",
author = "Goodlett, {David R.} and Bruce, {James E.} and Anderson, {Gordon A.} and Beate Rist and Ljiljana Pasa-Tolic and Oliver Fiehn and Smith, {Richard D.} and Ruedi Aebersold",
year = "2000",
month = "3",
day = "15",
doi = "10.1021/ac9913210",
language = "English (US)",
volume = "72",
pages = "1112--1118",
journal = "Analytical Chemistry",
issn = "0003-2700",
publisher = "American Chemical Society",
number = "6",

}

TY - JOUR

T1 - Protein identification with a single accurate mass of a cysteine- containing peptide and constrained database searching

AU - Goodlett, David R.

AU - Bruce, James E.

AU - Anderson, Gordon A.

AU - Rist, Beate

AU - Pasa-Tolic, Ljiljana

AU - Fiehn, Oliver

AU - Smith, Richard D.

AU - Aebersold, Ruedi

PY - 2000/3/15

Y1 - 2000/3/15

N2 - A method for rapid and unambiguous identification of proteins by sequence database searching using the accurate mass of a single peptide and specific sequence constraints is described. Peptide masses were measured using electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry to an accuracy of 1 ppm. The presence of a cysteine residue within a peptide sequence was used as a database searching constraint to reduce the number of potential database hits. Cysteine-containing peptides were detected within a mixture of peptides by incorporating chlorine into a general alkylating reagent specific for cysteine residues. Secondary search constraints included the specificity of the protease used for protein digestion and the molecular mass of the protein estimated by gel electrophoresis. The natural isotopic distribution of chlorine encoded the cysteine-containing peptide with a distinctive isotopic pattern that allowed automatic screening of mass spectra. The method is demonstrated for a peptide standard and unknown proteins from a yeast lysate using all 6118 possible yeast open reading frames as a database. As judged by calculation of codon bias, low-abundance proteins were identified from the yeast lysate using this new method but not by traditional methods such as tandem mass spectrometry via data-dependent acquisition or mass mapping.

AB - A method for rapid and unambiguous identification of proteins by sequence database searching using the accurate mass of a single peptide and specific sequence constraints is described. Peptide masses were measured using electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry to an accuracy of 1 ppm. The presence of a cysteine residue within a peptide sequence was used as a database searching constraint to reduce the number of potential database hits. Cysteine-containing peptides were detected within a mixture of peptides by incorporating chlorine into a general alkylating reagent specific for cysteine residues. Secondary search constraints included the specificity of the protease used for protein digestion and the molecular mass of the protein estimated by gel electrophoresis. The natural isotopic distribution of chlorine encoded the cysteine-containing peptide with a distinctive isotopic pattern that allowed automatic screening of mass spectra. The method is demonstrated for a peptide standard and unknown proteins from a yeast lysate using all 6118 possible yeast open reading frames as a database. As judged by calculation of codon bias, low-abundance proteins were identified from the yeast lysate using this new method but not by traditional methods such as tandem mass spectrometry via data-dependent acquisition or mass mapping.

UR - http://www.scopus.com/inward/record.url?scp=0034653646&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0034653646&partnerID=8YFLogxK

U2 - 10.1021/ac9913210

DO - 10.1021/ac9913210

M3 - Article

VL - 72

SP - 1112

EP - 1118

JO - Analytical Chemistry

JF - Analytical Chemistry

SN - 0003-2700

IS - 6

ER -