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Membrane protein identification: N-terminal labeling of nontryptic membrane protein peptides facilitates database searching

Bentz, Maria LU ; Wårell, Kristofer LU ; Levander, Fredrik LU and James, Peter LU (2008) In Journal of Proteome Research 7(2). p.659-665
Abstract
Membrane proteins are fairly refractory to digestion especially by trypsin, and less specific proteases, such as elastase and pepsin, are much more effective. However, database searching using nontryptic peptides is much less effective because of the lack of charge localization at the N and C termini and the absence of sequence specificity. We describe a method for N-terminal-specific labeling of peptides from nontryptic digestions of membrane proteins, which facilitates Mascot database searching and can be used for relative quantitation. The conditions for digestion have been optimized to obtain peptides of a suitable length for mass spectrometry (MS) fragmentation. We show the effectiveness of the method using a plasma membrane... (More)
Membrane proteins are fairly refractory to digestion especially by trypsin, and less specific proteases, such as elastase and pepsin, are much more effective. However, database searching using nontryptic peptides is much less effective because of the lack of charge localization at the N and C termini and the absence of sequence specificity. We describe a method for N-terminal-specific labeling of peptides from nontryptic digestions of membrane proteins, which facilitates Mascot database searching and can be used for relative quantitation. The conditions for digestion have been optimized to obtain peptides of a suitable length for mass spectrometry (MS) fragmentation. We show the effectiveness of the method using a plasma membrane preparation from a leukemia cell line and demonstrate a large increase in the number of membrane proteins, with small extra-membranar domains being identified in comparison to previous published methods. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
database, search, N-terminal labeling, proteinase K, membrane proteins, pepsin
in
Journal of Proteome Research
volume
7
issue
2
pages
659 - 665
publisher
The American Chemical Society
external identifiers
  • wos:000252864400019
  • scopus:39749139655
ISSN
1535-3893
DOI
10.1021/pr070545t
language
English
LU publication?
yes
id
a6da1a45-161c-4b60-ad9f-65e7a33436c1 (old id 1198600)
date added to LUP
2008-09-10 15:48:25
date last changed
2017-01-01 04:57:19
@article{a6da1a45-161c-4b60-ad9f-65e7a33436c1,
  abstract     = {Membrane proteins are fairly refractory to digestion especially by trypsin, and less specific proteases, such as elastase and pepsin, are much more effective. However, database searching using nontryptic peptides is much less effective because of the lack of charge localization at the N and C termini and the absence of sequence specificity. We describe a method for N-terminal-specific labeling of peptides from nontryptic digestions of membrane proteins, which facilitates Mascot database searching and can be used for relative quantitation. The conditions for digestion have been optimized to obtain peptides of a suitable length for mass spectrometry (MS) fragmentation. We show the effectiveness of the method using a plasma membrane preparation from a leukemia cell line and demonstrate a large increase in the number of membrane proteins, with small extra-membranar domains being identified in comparison to previous published methods.},
  author       = {Bentz, Maria and Wårell, Kristofer and Levander, Fredrik and James, Peter},
  issn         = {1535-3893},
  keyword      = {database,search,N-terminal labeling,proteinase K,membrane proteins,pepsin},
  language     = {eng},
  number       = {2},
  pages        = {659--665},
  publisher    = {The American Chemical Society},
  series       = {Journal of Proteome Research},
  title        = {Membrane protein identification: N-terminal labeling of nontryptic membrane protein peptides facilitates database searching},
  url          = {http://dx.doi.org/10.1021/pr070545t},
  volume       = {7},
  year         = {2008},
}