Advanced

Membrane protein proteomics - Novel method for membrane protein identification and quantification

Bentz, Maria LU (2007)
Abstract (Swedish)
Popular Abstract in Swedish

Membranproteiner är tämligen svåra att digerera med enzym som trypsin. Mindre specifika enzym såsom elastase och pepsin har dock visat sig mer effektiva. Databassökningar med dessa icke-tryptiska peptider är mindre verkningsfulla pga avsaknaden av laddning vid peptidens N-terminal och C-terminal samt brist på sekvensspecificitet.



Vi beskriver en metod med specifik N-terminal inmärkning av icke-tryptiska peptider från membranproteiner. Inmärkingen underlättar databassökning och kan dessutom kan användas för relativ kvantifiering. Förhållandena för digerering med det icke-specifika enzymet Proteinase K har optimerats för att peptider med passande längd för MS fragmentering ska... (More)
Popular Abstract in Swedish

Membranproteiner är tämligen svåra att digerera med enzym som trypsin. Mindre specifika enzym såsom elastase och pepsin har dock visat sig mer effektiva. Databassökningar med dessa icke-tryptiska peptider är mindre verkningsfulla pga avsaknaden av laddning vid peptidens N-terminal och C-terminal samt brist på sekvensspecificitet.



Vi beskriver en metod med specifik N-terminal inmärkning av icke-tryptiska peptider från membranproteiner. Inmärkingen underlättar databassökning och kan dessutom kan användas för relativ kvantifiering. Förhållandena för digerering med det icke-specifika enzymet Proteinase K har optimerats för att peptider med passande längd för MS fragmentering ska erhållas. Vi visar hur effektiv metoden är genom att använda oss av plasmamembran från en leukemicellinje, resultatet är en kraftig ökning i antal membranproteiner som identifieras med små extra-membrandomäner jämfört med tidigare publicerade metoder.



Därefter utvecklades metoden ytterligare genom att göra den kvantitativ för membranproteiner. Detta exemplifierades genom analys av en membranfraktion från bakterien Bacillus subtilis som fått växa med eller utan närvaron av 0.5 % glukos. Vi tillämpade en isotopisk inmärkningsstrategi. Resultaten visar en god reproducerbarhet av de uträknade uppregleringarna/nedregleringarna av de identifierade peptiderna inom samma protein.



Metoden applicerades sedan på proteiner i plasmamembranet framrenade från cancerceller odlade med eller utan närvaro av syre. Målet var att hitta biomarkörer för vävnadshypoxi. (Less)
Abstract
Membrane proteins are fairly refractory to digestion especially by trypsin. Less specific proteases like elastase and pepsin are much more effective. However database searching using non-tryptic peptides is much less effective due to the lack of charge localisation at the N- and C-termini and the absence of sequence specificity. We describe a method for N-terminal specific labelling of peptides from non-tryptic digestions of membrane proteins which facilitates database searching and can be used for relative quantitation. The conditions for digestion using the non-specific enzyme Proteinase K have been optimised to obtain peptides of a suitable length for MS fragmentation. We show the effectiveness of the identification of membrane proteins... (More)
Membrane proteins are fairly refractory to digestion especially by trypsin. Less specific proteases like elastase and pepsin are much more effective. However database searching using non-tryptic peptides is much less effective due to the lack of charge localisation at the N- and C-termini and the absence of sequence specificity. We describe a method for N-terminal specific labelling of peptides from non-tryptic digestions of membrane proteins which facilitates database searching and can be used for relative quantitation. The conditions for digestion using the non-specific enzyme Proteinase K have been optimised to obtain peptides of a suitable length for MS fragmentation. We show the effectiveness of the identification of membrane proteins using a plasma membrane preparation from a leukaemia 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.



Our method was then further developed for relative quantitation of membrane proteins. We describe this by using membrane preparations from Bacillus subtilis grown with and without the presents of 0.5% glucose and an isotopic labelling strategy. The results show good reproducibility of the calculated fold changes of the identified peptides within the same protein.



The method was then applied on plasma membrane proteins from cancer cells grown with and without the presence of oxygen. The aim was to find biomarkers for tissue hypoxia. (Less)
Please use this url to cite or link to this publication:
author
opponent
  • Professor Nørregaard Jensen, Ole, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Biokemisk teknik, mebran, steroider, Lipider, membranes, steroids, Lipids, enzymologi, Proteiner, Membrane proteins, Proteomics, Proteinase K, N-terminal labelling, Relative Quantitation, Database search, Cancer, Cytology, oncology, cancerology, Cytologi, onkologi, cancer, enzymology, Proteins, Biochemical technology
pages
109 pages
publisher
Department of Immunotechnology, Lund University
defense location
Segerfalksalen, Wallenberg Neurocentrum, BMC, Sölvegatan 17, Lund
defense date
2007-12-07 10:15
ISBN
ISBN 978-91-628-7350-9
language
English
LU publication?
yes
id
bcfba546-9059-4ff9-b128-af33b8d89216 (old id 600519)
date added to LUP
2007-11-22 12:05:19
date last changed
2016-09-19 08:45:08
@misc{bcfba546-9059-4ff9-b128-af33b8d89216,
  abstract     = {Membrane proteins are fairly refractory to digestion especially by trypsin. Less specific proteases like elastase and pepsin are much more effective. However database searching using non-tryptic peptides is much less effective due to the lack of charge localisation at the N- and C-termini and the absence of sequence specificity. We describe a method for N-terminal specific labelling of peptides from non-tryptic digestions of membrane proteins which facilitates database searching and can be used for relative quantitation. The conditions for digestion using the non-specific enzyme Proteinase K have been optimised to obtain peptides of a suitable length for MS fragmentation. We show the effectiveness of the identification of membrane proteins using a plasma membrane preparation from a leukaemia 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.<br/><br>
<br/><br>
Our method was then further developed for relative quantitation of membrane proteins. We describe this by using membrane preparations from Bacillus subtilis grown with and without the presents of 0.5% glucose and an isotopic labelling strategy. The results show good reproducibility of the calculated fold changes of the identified peptides within the same protein.<br/><br>
<br/><br>
The method was then applied on plasma membrane proteins from cancer cells grown with and without the presence of oxygen. The aim was to find biomarkers for tissue hypoxia.},
  author       = {Bentz, Maria},
  isbn         = {ISBN 978-91-628-7350-9},
  keyword      = {Biokemisk teknik,mebran,steroider,Lipider,membranes,steroids,Lipids,enzymologi,Proteiner,Membrane proteins,Proteomics,Proteinase K,N-terminal labelling,Relative Quantitation,Database search,Cancer,Cytology,oncology,cancerology,Cytologi,onkologi,cancer,enzymology,Proteins,Biochemical technology},
  language     = {eng},
  pages        = {109},
  publisher    = {ARRAY(0x8865f00)},
  title        = {Membrane protein proteomics - Novel method for membrane protein identification and quantification},
  year         = {2007},
}