Computational protocol: Comparative host protein interactions with HTLV-1 p30 and HTLV-2 p28: insights into difference in pathobiology of human retroviruses

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Protocol publication

[…] Capillary-liquid chromatography-tandem mass spectrometry (Capillary-LC/MS/MS) for global protein identification was performed on a Thermo Finnigan LTQ Orbitrap mass spectrometer equipped with a microspray source (Michrom Bioresources Inc, Auburn, CA) operated in positive ion mode. Samples were separated on a capillary column (0.2X150 mm Magic C18AQ 3 μ 200A, Michrom Bioresources Inc, Auburn, CA) using an UltiMateTM 3000 HPLC system from LC-Packings A Dionex Co. (Sunnyvale, CA). The scan sequence of the mass spectrometer was based on the data dependant TopTenTM method. The resolution of a full scan was set at 30000 to achieve a high mass accuracy MS determination.The RAW data files collected on the mass spectrometer were converted to mzXML and MGF files by use of MassMatrix data conversion tools (version 1.3, http://www.massmatrix.net/download). The resulting MGF files were searched using Mascot Daemon by Matrix Science version 2.2.2 (Boston, MA) and the database searched against the full SwissProt database version 57.5 (471472 sequences; 167326533 residues) or NCBI database version 20091013 (9873339 sequences; 3367482728 residues). Considered modifications (variable) were methionine oxidation and the presence of carbamidomethyl cysteine. Three missed cleavages for the enzyme were permitted with a peptide tolerance of 1.2 Da, and the MS/MS ion tolerance was 0.8 Da. Mock transfected (empty pTriEx4-Neo) cell lysates treated similarly served as negative control. Search results were compiled and visualized using the Scaffold 3 sofware. Unweighted spectrum count and percent coverage provided semi-quantitative data analyses. Protein identifications were assigned using PeptideProphet. Proteins with 80% confidence were accepted with a minimum of one peptide displaying 95% threshold confidence level. […]

Pipeline specifications

Software tools Mascot Server, PeptideProphet
Application MS-based untargeted proteomics
Organisms Human T-lymphotropic virus 2, Human T-cell leukemia virus type I, Homo sapiens
Diseases Spinal Cord Diseases, Virus Diseases, Leukemia, T-Cell, HTLV-II Infections, Paraparesis, Tropical Spastic, Genetic Diseases, Inborn
Chemicals Amino Acids