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

Author:
  • Maria Bentz
  • Kristofer Wårell
  • Fredrik Levander
  • Peter James
Publishing year: 2008
Language: English
Pages: 659-665
Publication/Series: Journal of Proteome Research
Volume: 7
Issue: 2
Document type: Journal article
Publisher: The American Chemical Society

Abstract english

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.

Keywords

  • Immunology in the medical area
  • database
  • search
  • N-terminal labeling
  • proteinase K
  • membrane proteins
  • pepsin

Other

Published
  • ISSN: 1535-3893
Peter James
E-mail: peter.james [at] immun.lth.se

Professor

Department of Immunotechnology

+46 46 222 14 96

+46 70 247 79 60

MV406411E1

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