Computational protocol: Proteins and lipids of glycosomal membranes fromLeishmania tarentolae andTrypanosoma brucei

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Protocol publication

[…] Glycosomes (corresponding to about 0.5 mg protein) were diluted 1:5 in TEDS (see 2.1), subjected to two freeze-thaw cycles, and centrifuged for 40 minutes at 140,000x g and 4°C. The resulting pellet was washed with 5 M urea for 1 h at 4°C to remove proteins that were not tightly associated with the glycosomal membranes. The glycosomal membranes were pelleted by centrifugation for 40 minutes at 140,000x g and 4°C. This 5 M urea wash-step was repeated once. The glycosomal membrane-enriched fraction was resuspended in denaturing Laemmli SDS-PAGE buffer and separated by SDS PAGE. In-gel trypsin digestion and nanoLC-MS/MS analysis of the obtained protein bands were performed as previously described . The obtained MS/MS spectra were analysed using MASCOT software and visualised in Scaffold. The comparison shown in was done using the 2012 version of the shotgun sequence of L. tarentolae ( http://tritrypdb.org/) ; only proteins for which at least 2 different peptides could be identified with >95% confidence were included. Leishmania proteins were first scanned for the presence of a PTS1 signal based on a published analysis for L. major and T. brucei and by manually examining the C-terminal sequences. Additional PTS1-containing proteins were identified using PTS1 Predictor , . Trans-membrane domains were identified using the TritrypDB annotation database. For potential glycosomal proteins with no known function and without an annotated trans-membrane domain, we also scanned for trans-membrane domains using the HMMTOP and SOSUI algorithms , . […]

Pipeline specifications

Software tools HMMTOP, SOSUI
Databases TriTrypDB
Application Membrane protein analysis
Organisms Leishmania tarentolae, Trypanosoma brucei
Diseases Cataract
Chemicals Fatty Acids, Phosphatidylcholines