Computational protocol: Intracellular expression of Tat alters mitochondrial functions in T cells: a potential mechanism to understand mitochondrial damage during HIV-1 replication

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Protocol publication

[…] Proteome analysis was performed as previously described []. Briefly, 200 μg of trypsin-digested proteins were loaded into the LC–MS/MS system and analyzed using a C-18 reversed phase nano-column (Thermo-Fisher, San Jose, CA, USA). Real-time ionization and peptide fragmentation was performed on an orbital ion trap mass spectrometer (LTQ Orbitrap XL, Thermo Fisher Scientific, San Jose, CA, USA) []. For peptide database searching, tandem mass spectra were analysed using Sequest (Thermo Fisher Scientific; version 1.3.0.339) and X! Tandem (http://www.thegpm.org; version CYCLONE). Sequest and X! Tandem were searched with a fragment ion mass tolerance of 30 PPM and a parent ion tolerance of 15 PPM. Scaffold 3.0 (Proteome Software Inc., Portland, OR, USA) was used to validate peptide identifications. Only peptide established at a probability greater than 95 % and with XCorr-score values above 1.75 were accepted. Proteins related to mitochondria and cytoskeleton that changed at least ±2.0-fold in Jurkat-Tat101 were selected and subjected to analysis with STRING 9.0 database (http://string-db.org/) []. […]

Pipeline specifications

Software tools Comet, X! Tandem
Application MS-based untargeted proteomics
Organisms Human immunodeficiency virus 1, Homo sapiens
Diseases HIV Infections, Mitochondrial Diseases
Chemicals Adenosine Triphosphate