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

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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.

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Subject classification (UKÄ) – MANDATORY

  • Immunology in the medical area

Keywords

  • database, search, N-terminal labeling, proteinase K, membrane proteins, pepsin
Original languageEnglish
Pages (from-to)659-665
JournalJournal of Proteome Research
Volume7
Issue number2
Publication statusPublished - 2008
Publication categoryResearch
Peer-reviewedYes