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, 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