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

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Membrane protein identification: N-terminal labeling of nontryptic membrane protein peptides facilitates database searching. / Bentz, Maria; Wårell, Kristofer; Levander, Fredrik; James, Peter.

In: Journal of Proteome Research, Vol. 7, No. 2, 2008, p. 659-665.

Research output: Contribution to journalArticle

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TY - JOUR

T1 - Membrane protein identification: N-terminal labeling of nontryptic membrane protein peptides facilitates database searching

AU - Bentz, Maria

AU - Wårell, Kristofer

AU - Levander, Fredrik

AU - James, Peter

PY - 2008

Y1 - 2008

N2 - 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.

AB - 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.

KW - database

KW - search

KW - N-terminal labeling

KW - proteinase K

KW - membrane proteins

KW - pepsin

U2 - 10.1021/pr070545t

DO - 10.1021/pr070545t

M3 - Article

VL - 7

SP - 659

EP - 665

JO - Journal of Proteome Research

JF - Journal of Proteome Research

SN - 1535-3893

IS - 2

ER -