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Protein identification by in-gel digestion, high-performance liquid chromatography, and mass spectrometry: peptide analysis by complementary ionization techniques

Medzihradszky, Katalin F.; Leffler, Hakon LU ; Baldwin, Michael A. and Burlingame, A L (2001) In Journal of the American Society for Mass Spectrometry 12(2). p.215-221
Abstract
A biologically active protein fraction was isolated from rabbit intestine, purified by one-dimensional SDS-PAGE and stained with Coomassie Brilliant Blue. A predominant band of approximately 110-130 kDa was excised and digested in-gel with trypsin. The resulting peptides were extracted then separated by microbore reversed-phase high-performance liquid chromatography (HPLC). Mass spectrometric data from one HPLC fraction obtained by two different ionization techniques proved to be complementary. Matrix-assisted laser desorption/ionization (MALDI) showed nine peptide masses, which by post source decay analysis and database searching were attributed to two proteins. Nanoflow electrospray analysis performed on a hybrid tandem mass spectrometer... (More)
A biologically active protein fraction was isolated from rabbit intestine, purified by one-dimensional SDS-PAGE and stained with Coomassie Brilliant Blue. A predominant band of approximately 110-130 kDa was excised and digested in-gel with trypsin. The resulting peptides were extracted then separated by microbore reversed-phase high-performance liquid chromatography (HPLC). Mass spectrometric data from one HPLC fraction obtained by two different ionization techniques proved to be complementary. Matrix-assisted laser desorption/ionization (MALDI) showed nine peptide masses, which by post source decay analysis and database searching were attributed to two proteins. Nanoflow electrospray analysis performed on a hybrid tandem mass spectrometer of quadrupole-quadrupole-orthogonal acceleration time-of-flight (QqTOF) geometry detected six additional peptide components. On the basis of the additional peptides and superior quality collision-induced dissociation spectra typical of this instrument type, two further proteins were identified. The resolution afforded by the QqTOF instrument permitted charge state determination for the fragment ions while preserving the high detection sensitivity that was essential in obtaining the composition of this mixture of proteins. (Less)
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author
publishing date
type
Contribution to journal
publication status
published
subject
in
Journal of the American Society for Mass Spectrometry
volume
12
issue
2
pages
215 - 221
publisher
Elsevier
external identifiers
  • pmid:11212006
  • scopus:0035564727
ISSN
1879-1123
DOI
10.1016/S1044-0305(00)00214-2
language
English
LU publication?
no
id
e1f7fec3-5bc4-43d2-8e42-f9b091665b63 (old id 1123042)
date added to LUP
2008-07-04 15:54:45
date last changed
2018-05-29 11:23:55
@article{e1f7fec3-5bc4-43d2-8e42-f9b091665b63,
  abstract     = {A biologically active protein fraction was isolated from rabbit intestine, purified by one-dimensional SDS-PAGE and stained with Coomassie Brilliant Blue. A predominant band of approximately 110-130 kDa was excised and digested in-gel with trypsin. The resulting peptides were extracted then separated by microbore reversed-phase high-performance liquid chromatography (HPLC). Mass spectrometric data from one HPLC fraction obtained by two different ionization techniques proved to be complementary. Matrix-assisted laser desorption/ionization (MALDI) showed nine peptide masses, which by post source decay analysis and database searching were attributed to two proteins. Nanoflow electrospray analysis performed on a hybrid tandem mass spectrometer of quadrupole-quadrupole-orthogonal acceleration time-of-flight (QqTOF) geometry detected six additional peptide components. On the basis of the additional peptides and superior quality collision-induced dissociation spectra typical of this instrument type, two further proteins were identified. The resolution afforded by the QqTOF instrument permitted charge state determination for the fragment ions while preserving the high detection sensitivity that was essential in obtaining the composition of this mixture of proteins.},
  author       = {Medzihradszky, Katalin F. and Leffler, Hakon and Baldwin, Michael A. and Burlingame, A L},
  issn         = {1879-1123},
  language     = {eng},
  number       = {2},
  pages        = {215--221},
  publisher    = {Elsevier},
  series       = {Journal of the American Society for Mass Spectrometry},
  title        = {Protein identification by in-gel digestion, high-performance liquid chromatography, and mass spectrometry: peptide analysis by complementary ionization techniques},
  url          = {http://dx.doi.org/10.1016/S1044-0305(00)00214-2},
  volume       = {12},
  year         = {2001},
}